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The Effects of Acidic Conditions on Bone Quality and Function
Mikayla Kelsey Moody, PhD
University of Connecticut, 2024
Acidotic conditions within the body, such those developed in metabolic acidosis, can have an
adverse impact on the functionality and health of various organs and tissues, including bone.
Metabolic acidosis, which results in reduced blood pH and bicarbonate, has been shown to
cause bone loss via two avenues: physiochemical dissolution and osteoclast-mediated
resorption. Although some studies have shown an increase in osteoclast activity with a
corresponding decrease in osteoblast activity, resulting in disturbed bone remodeling, other
studies have shown that physiochemical dissolution is the predominate factor in bone
dissolution of acidotic mice. In this dissertation, various studies used graded dosing of
ammonium chloride to stimulate metabolic acidosis in skeletally mature (3 – 6-month-old) CD-1
mice to determine the effects of ammonium chloride administration on bone health of male and
female mice along with the impact of bicarbonates and bisphosphonates on the recovery of
acid-exposed bone. Graded dosing rather than a flat dose of ammonium chloride produced
metabolic acidosis over a span of fourteen days. Additionally, the graded model showed early
changes in the composition, structure, and mechanics of bone followed by a return to control
parameters. It is hypothesized that there is a protective mechanism against heightened
osteoclastic activity. This protective mechanism can be further illustrated in the NanoString RNA
genetic data obtained from male and female acidotic mice. Femurs from both sexes illustrated a
time-dependent upregulation in genes that impair osteoclast behavior and downregulation in
genes that increase this behavior. When administrating bisphosphonates to acidotic mice,
acidosis was worsened, most likely due to the lack of buffering ions being released from bone.
Compared to sodium bicarbonate, potassium bicarbonate was shown to increase bone loss,
potentially due to its increase in blood sodium levels. In conclusion, metabolic acidosis induced
Mikayla Kelsey Moody – University of Connecticut, 2024
by a graded dosing model of ammonium chloride produced sufficient changes in blood pH and
bicarbonate to mimic acidosis. However, further analysis and development of this model is
needed to obtain more drastic bone phenotypical changes.
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The Effects of Acidic Conditions on Bone Quality and Function
Mikayla Kelsey Moody
B.S., North Carolina State University, 2018
A Dissertation
Submitted in Partial Fulfillment of the
Requirements for the Degree of
Doctor of Philosophy
at the
University of Connecticut
2024
ii
Copyright by
Mikayla Kelsey Moody
2024
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Doctor of Philosophy Dissertation
The Effects of Acidic Conditions on Bone Quality and Function
Presented by
Mikayla Kelsey Moody, B.S.
Approved by
Major Advisor: Alix Deymier, PhD
Associate Advisor: Caroline Dealy, PhD
Associate Advisor: Yusuf Khan, PhD
University of Connecticut
2024
iv
Acknowledgements
There are many people I would like to thank, making it hard to figure out where to even begin.
However, without the immense amount of help I received during my PhD, I do not think I would
be where I am today, so I am forever grateful to the following people.
First, I would like to thank my advisor, Dr. Alix Deymier. I am so grateful that you found me
while I was applying to graduate school. I am glad that I chose your lab and learned about the
wonderful world of bones, apatite, and acid-base balance (or in our case, imbalance). Your
kindness and understanding nature really helped me thrive in the lab, even when it felt like things
were not going right with my research. Thank you for also letting me go to so many conferences
and inviting me to go to Germany to do synchrotron experiments. All those experiences were very
eye-opening and super wonderful. So, thank you for all the support you have shown me these
past five years. I would not have made it through this PhD without your guidance.
Next, I want to thank the rest of the Deymier Lab. I want to thank everyone in the lab who
supported me directly and indirectly. I first want to thank Anna Peterson for her guidance. Thank
you for letting me tag along with you in the mouse house when I was an early PhD student asking
a ton of questions. I learned so much for you, and my PhD experience would have been so much
harder if you hadn’t been there to guide me. Next, I want to thank Stephanie Wong. You were my
lab bestie, and I miss you so much since you have graduated, but I am so happy for you that you
are at Oak Ridge now. We had some very fun and stressful adventures in Europe, and I wouldn’t
have wanted to go on those trips with anyone else. I next want to thank Sobhan Katebifar for
being so funny and kind and being a great desk neighbor. I also want to mention how kind
Kennedy Drake and Katherine Arnold have been. I will miss you three! Also, I want to immensely
thank all the undergraduate students who worked with me and helped me gather and analyze
data, so thank you to Anthony D’Angio, Nayara Zainadine, Margaret Easson, Trey Doktorski,
Christina Fares, Tyler Grubelich, Michael Nogaj, Sydney Whittaker, and Abby Messina. You all
are lifesavers.
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I would like to thank my committee members — Dr. Caroline Dealy and Dr. Yusuf Khan.
Thank you for your constant support during my PhD. I would also like to thank Dr. Tannin Schmidt,
Dr. Ali Tamayol, Dr. Kshitiz, and Dr. Liisa Kuhn for their advice throughout my time at UConn
Health. Combined, all of you have made me a better researcher.
Thank you to the UConn Harriett Fellowship and GEM Fellowship for financially supporting
my PhD. Because of these fellowships, I was able to pursue my graduate degree, attend
conferences, and meet other wonderful fellows. Additionally, I specifically want to thank the GEM
Fellowship for providing me the opportunity to work at Oak Ridge National Laboratory the summer
before starting my PhD. The internship was unforgettable. Lastly, I want to thank Dr. Stuart
Duncan for giving me the opportunity to attend the Institute on Teaching and Learning my first
year of my PhD. It really showed me that I was in the right place and that I was capable of being
a PhD-level researcher.
Next, I would like to thank the Biomedical Engineering (BME) crew at UConn Health. You all
are the best. There are so many current and past graduate students within the BME department
that I would like to thank for their constant encouragement. It was fun to go on many adventures
together, such as meeting at Parkville Market for dinner, having a picnic at AW Stanley Park, and
doing high ropes courses in a furniture store in New Haven. I specifically would like to thank
Evelyn (my bestie), Jacob, Noah, Adam, Nikhil, Delaram, Farnoosh, Pejman, Mehdi, Fatemah,
Askhan, Mikaela, Steven, Yamin, Hui, Wenqiang, and Travis.
There are so many others at UConn Health. To Iris, thank you for being a great friend and
taking care of my cat on occasion as well. It is always fun to watch horror movies with you. I can’t
wait to see what you do after your PhD. To Renata at the MicroCT core, thank you for being
patient with me when I had a lot samples. To Zhifang at the Histology core, thank you for being
so kind and understanding when I needed to get samples done quickly. To Lauren in the Canalis
Lab, thanks for always answering my many emails and helping me figure out all the things.
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To my NC State buddies – Guinevere, Charlie, and Sara: thank you for being a huge support
in my life, even though we were physically apart for a majority of my PhD. It has been wonderful
to see how you three changed over the years since we graduated college. I am so proud of who
you all have become, and I can’t wait to continue being a part of each other’s lives.
I want to thank Aida Ghiaei and Dr. Stephany Santos for their endless support. Thank you for
believing in me when I didn’t. Thank you for showing me that I can make it through the PhD. Aida,
thank you for always motivating me. Stephany, thank you for always being a guiding light in my
life. You two have changed my life for the better.
I would like to thank Dr. Anne Marie LaChance. You are amazing and an inspiration. Thank
you for starting Queer Science at UConn and creating a beautiful LGBT+ community of STEM
nerds. Also, thank you for your guidance about being an engineering teaching faculty. I don’t think
I would have had the confidence to become a teaching faculty if it wasn’t for your support.
I want to thank the graduate community at the UConn Storrs campus. I was fortunate to be a
part of many organizations during my PhD. I was lucky to be picked as a part of the John Lof
Leadership Academy my first two years at UConn. Thank you to everyone in John Lof for being
so warm, welcoming, uplifting, and kind. That community was unlike any other and I miss it every
day. To SAGE and its members, thank you for taking a shot on me as president my third year.
SAGE is such a wonderful community, bringing all the graduate engineering students together. I
worked with and met so many wonderful people because of SAGE. Lastly, I want to thank my
SHPE familia, Grad SHPE. Jessica, Karla, Leana, and Evelyn, you are the best and literally made
me feel like I was at home. I could be my authentic half-Venezuelan, half-white self with you all
without any judgement. I couldn’t have asked for better people to have built an organization with.
I next want to thank my family for their undying support, even when they didn’t quite
understand what I was doing. Thank you for making such huge sacrifices so I was able to get to
where I am today. Mom, you are such a superstar and wonderful professor. I love you to the moon
and back. Dad, thank you for always being there for me and showing me endless support. I love
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you so much. Kaydence and Jentri, thank you for making me see that life doesn’t need to serious
all the time. You both are one of the reasons why I kept going with this PhD, so I could show you
that you can do anything if you put your mind to it. I hope you two never change and continue to
be silly as ever. To the rest of my family, thanks for checking in on me and showing me that you
cared during these past five years and more. I am forever grateful for your love.
Lastly, I would like to thank Lindsay – my wife, my love. You literally knew what I was going
through during my PhD because you were also doing a BME PhD at the exact time, starting at
the same time. You were always there for me when times were hard. When I cried, you were
always there to give me a hug. When I was angry about an experiment not working, you were
understanding but also willing to be angry with me. Thank you for being my rock, my person.
Thank you for talking to me about my research, even when you didn’t quite understand what I
was doing. We have been through a lot of trials and tribulations, but we made it through it all
together. I can’t wait to see what Milwaukee brings us.
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Table of Contents
Acknowledgements ............................................................................................................ iv
Chapter 1: Introduction ....................................................................................................... 1
1.1 Background and Significance ............................................................................................... 1
1.2 Chronic Metabolic Acidosis ................................................................................................... 3
1.3 Bone and Acid ....................................................................................................................... 5
1.4 Animal Models of Acidosis .................................................................................................... 6
1.5 Bicarbonate Supplementation for Treatment of Metabolic Acidosis .................................... 7
1.6 Specific Aims ......................................................................................................................... 8
1.7 Innovation and Relevance .................................................................................................. 10
Chapter 2: Temporal Response of Diet-induced Acidosis on the Murine Skeleton ....... 11
Chapter 2.1 Physiochemical dissolution governs early modifications in acid-exposed murine
bone with long-term recovery .................................................................................................... 11
2.1.1 Introduction ................................................................................................................................. 11
2.1.2 Materials and Methods ............................................................................................................... 11
2.1.3 Results ........................................................................................................................................ 21
2.1.4 Discussion ................................................................................................................................... 36
2.1.5 Conclusion .................................................................................................................................. 45
Chapter 2.2 Cellular contributions are not observed in murine femurs after 14 days of
metabolic acidosis induction utilizing NH4Cl diet ...................................................................... 46
2.2.1 Introduction ................................................................................................................................. 46
2.2.2 Methods and Materials ............................................................................................................... 46
2.2.3 Results ........................................................................................................................................ 49
2.2.4 Discussion ................................................................................................................................... 54
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Chapter 3: The Effects of Treatments on the Recovery of Acid-exposed Bone ............. 58
Chapter 3.1 Administration of alendronate exacerbates ammonium chloride-induced acidosis
in mice ....................................................................................................................................... 58
3.1.1 Introduction ................................................................................................................................. 58
3.1.2 Materials and Methods ............................................................................................................... 59
3.1.3 Results ........................................................................................................................................ 64
3.1.4 Discussion ................................................................................................................................... 73
3.1.5 Conclusion .................................................................................................................................. 75
Chapter 3.2 Potassium bicarbonate, not sodium bicarbonate, maintains acidosis-mediated
bone dissolution ........................................................................................................................ 77
3.2.1 Introduction ................................................................................................................................. 77
3.2.2 Materials and Methods ............................................................................................................... 77
3.2.3 Results ........................................................................................................................................ 84
3.2.4 Discussion ................................................................................................................................... 96
3.2.5 Limitations and future studies ................................................................................................... 101
3.2.6 Conclusions .............................................................................................................................. 102
Chapter 4: Sexual differences in potential protective mechanisms against acid-mediated
bone dissolution .............................................................................................................. 103
4.1 Introduction ........................................................................................................................ 103
4.2 Methods and Materials ...................................................................................................... 104
4.3 Results............................................................................................................................... 109
4.4 Discussion ......................................................................................................................... 133
4.5 Conclusion ......................................................................................................................... 139
Chapter 5: Conclusions and Future Directions .............................................................. 140
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5.1 Conclusions ....................................................................................................................... 140
5.2 Future Directions ............................................................................................................... 141
References...................................................................................................................... 144
Appendices ..................................................................................................................... 163
8.1 Weight, Urine pH, and Blood Gas Collection Protocol ..................................................... 163
8.2 Histology and Histomorphometry Femur Preparation Protocol ....................................... 170
8.3 Mouse Femur Three Point Bending Protocol ................................................................... 173
8.4 Moment of Inertia MicroCT Analysis with Bone Protocol ................................................. 185
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Chapter 1: Introduction
1.1 Background and Significance
Bone is a mineralized tissue that not only provides structural integrity and mobility to the
body, but also protects organs and provides support to various tissues. The fluids that the skeletal
system encounters help it maintain homeostasis. When the composition of these fluids becomes
pathological due to disease, such as metabolic acidosis, then it can become detrimental to bone
[1–3]. These diseases can lead to a decrease in blood pH and bicarbonate (HCO3-) levels, which
can prompt dissolution of bone to release buffering ions to increase the pH back to homeostatic
levels [3–5]. Some of the deleterious effects to bone health due to increased acidity include
increased osteolysis, fracture risk, and associated bone pain [6–9]. In addition, systemic acidemia
clinically decreases bone mineral density (BMD) and bone volume while increasing functional
limitations [10–13]. Together, these conditions significantly decrease quality of life and increase
mortality rates [14–16]. In addition, acidemia-promoted fractures affect thousands of individuals
in the US and millions of individuals globally each year [17,18] with enormous medical costs
[19,20]. Concerns about the effect of acidemia on bone health are only growing due to the
prevalence of acidic diets, the rising number of patients with chronic kidney disease and diabetes,
and an aging population, all correlated with a decreased physiological pH [21–23]. Although
acidemia can be treated via the administration of bicarbonate salts, the efficacy of these
treatments on the skeletal consequences of acid exposure remains variable and not well
understood [24,25]. Without effective treatments, acidosis continues to contribute to bone
degradation, significantly affecting population quality of life.
Understanding how acidosis induces bone degradation is critical for developing treatment
solutions. Two primary processes have been identified: physiochemical dissolution and cell-
mediated resorption [26–28]. Bone is composed of pH sensitive materials, including carbonate-
substituted calcium apatite, which acts as the body’s primary reservoir of buffering ions [3,29,30],
and collagen. Both components undergo dissolution and reorganization in response to slight
2
acidification, allowing for the release of buffering ions and changing the bone composition
[4,26,31–36]. Cellularly, bone is comprised of osteoclasts and osteoblasts, which resorb and
rebuild bone, respectively. Both in vitro and ex vivo extracellular acidification promote osteoclast
proliferation and activity, resulting in increased bone resorption and changes in bone structure
[28,37–40]. Although these cellular and physiochemical processes have been identified, their
relative contributions to bone structural and compositional degradation, and therefore their effects
on mechanical function, remain unknown.
Despite the prevalence and establishment of the negative effects of metabolic acidosis on
the skeletal system, there are limited studies of murine models that recapitulate the bone
phenotype of metabolic acidosis. Previously published models of metabolic acidosis insufficiently
modeled the biochemistry of the disease in adult mice, leaving our understanding of the functional
consequences of metabolic acidosis incomplete [41]. Moreover, mouse disease models are
preferable to rats in biomedical research as genetically engineered mice are readily available,
cost effective, and can be experimentally scaled. Our lab created a diet-induced murine model of
metabolic acidosis that was able to mimic the key clinical features observed in patients with
metabolic acidosis [4,42–45].
Clinical evidence indicates that acidemia compromises bone mechanics [10,13].
Decreased bone mass has been suggested as the cause; however, it is a poor predictor of
fracture risk [46–49]. Instead, it is necessary to examine how disease processes affect bone
quality [50,51]. We have shown that acidosis compromises bone quality, affecting mineral
composition and content, collagen structure, and bone architecture [4]. Similar changes in bone
quality, such as modifications to mineral and collagen content and chemistry, increased micro-
porosity, and changes in bone shape and size have been shown to negatively affect bone stiffness
and fracture mechanics [32,48,52–56]. As a result, studies examining the effects of disease on a
single characteristic of bone composition or structure often inaccurately predict the mechanical
consequences. We have developed a comprehensive view of the structural, compositional, and
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mechanical consequences of acidosis disease processes to more accurately understand how it
regulates bone mechanical function.
Current treatments of metabolic acidosis focus primarily on increasing serum pH via
administration of HCO3- containing compounds, such as sodium bicarbonate (NaHCO3) or
potassium bicarbonate (KHCO3) [25,57]. Although these are clinically effective in raising pH and
HCO3- levels, their effects on bone quality remain less clear [25]. In clinical studies, treating
patients with KHCO3 significantly decreased bone loss and calcium release compared to patients
treated with NaHCO3 [24]. The mechanism for this difference is unknown, although physiological,
cellular, and physiochemical processes have been suggested. Physiochemically, both sodium
and potassium easily substitute into bone mineral, significantly affecting bone composition [58].
Cellularly, osteoclast motility, number, and activity are all affected by changes in extracellular
K+/Na+ ratios, suggesting a possible effect on bone resorption and structure [59–63]. In other
models, the compositional and structural changes described above have all been associated with
changes in bone functional mechanics [52,56,64–66]. However, the complex four-way
relationship between treatment of cellular/physiochemical diseases processes, bone composition,
bone structure, and mechanical properties remains unexplored. We define the relationships
between NaHCO3 and KHCO3 and bone quality and function in order identify if one treatment may
be superior and in turn suggest improved treatment options.
1.2 Chronic Metabolic Acidosis
Blood pH is maintained within a small window of 7.35 to 7.45 [10,11,67,68]. If the blood
pH in humans goes below 7.35, acidemia develops. Acidosis occurs when the body is unable to
remove the accumulated acid. There are two primary types of acidosis — respiratory and
metabolic. In respiratory acidosis, the pulmonary system maintains a pH balance by expelling
carbon dioxide out of the body, reducing the creation of carbonic acid. However, when the body
does not remove the carbon dioxide quickly enough, the body becomes acidified [69]. On the
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other hand, the kidneys help regulate an acid-base balance by reabsorbing or generating
bicarbonate [70]. Metabolic acidosis occurs when the kidneys are no longer functioning properly,
either due to disease or damage, or there is too much acid in the body such that the kidneys are
not able to excrete it adequately. When this happens, the level of blood bicarbonate decreases
along with the blood pH [71]. Acidosis can also happen at various intensities and intervals. Acute
acidosis happens in shorter stretches of time (minutes to days), while chronic acidosis occurs
over days to months. Within this dissertation, chronic metabolic acidosis will be the main focus,
but acute and respiratory acidosis is important to consider when researching the effects of
acidosis on the body.
Metabolic acidosis comes in two forms — high anion gap and normal anion gap. The anion
gap describes the difference between the concentration of dissolved cations and the
concentration of dissolved anions in the blood [71]. The typical ions observed in the anion gap
are sodium (Na+), chlorine (Cl-), and bicarbonate (HCO3-). A high anion gap in metabolic acidosis
can develop when there is no change in sodium or chlorine, but a decrease in bicarbonate. On
the other hand, a normal anion gap could also be present in patients with metabolic acidosis
where they have an increase in chlorine and a decrease in bicarbonate levels [71,72]. Clinically,
it is important to note which type of metabolic acidosis a patient has in order to determine the root
cause.
Chronic metabolic acidosis can originate from ageing, chronic kidney disease, acidic diets,
diabetes, and many other sources. Patients with chronic kidney disease typically have metabolic
acidosis due to a decrease in nephron mass. Although the remaining healthy nephrons are able
to excrete out more acid, they are unable to offset the amount of acid accumulating within the
body [70]. Diabetic ketoacidosis occurs when there is an accumulation of ketones within the body
due to the breakdown of fat [73]. In this dissertation, we used diet-induced metabolic acidosis in
our murine models by providing the mice ammonium chloride in their drinking water. Currently,
the human diet is made of too many acidic foods, such as animal protein, and not enough
5
bicarbonate-rich foods, such as fruits and vegetables, which could led to low-grade chronic
metabolic acidosis [11,74]. The kidneys can counteract this increase in acid by increasing the
glomerular filtration rate [75], decreasing urinary excretion of citrate [76,77], and increasing the
urinary excretion of calcium [76]. If diet-induced acidosis is maintained through an animal protein
heavy diet, nephrolithiasis can develop [76,78–80]. Other complications can occur with metabolic
acidosis, such muscle protein degradation [81], insulin resistance [82], and hypertension [83].
Although the main focus for this dissertation is bone, it is also important to note how metabolic
acidosis affects other tissues and organs and how these impacts are interconnected with bone.
1.3 Bone and Acid
Clinically, metabolic acidosis is associated with increased skeletal defects [84,85].
Metabolic acidosis has been correlated with a decrease in bone formation [86], bone mineral
density [12], and bone volume, in addition an increase in the rate of fracture [13]. Many have
found that skeletal consequences of acidosis are caused by short-term physiochemical bone
dissolution and long-term cell-mediated bone resorption [3,27,87–89].
As stated previously, bone is composed of a mineral phase identified as carbonate-
substituted calcium apatite, containing 2–9% carbonate (CO32-) [30,90], making it an important
reservoir of buffering ions. Bone mineral is very sensitive to pH, leading it to quickly dissolve when
exposed to small decreases in pH [91]. The physiochemical dissolution of bone apatite leads to
a rapid, preferential release of HCO3-, which can serve to buffer acid [31].
If acidosis is maintained, the reduced pH affects the cellular response of bone. Due to
acidosis, osteoblasts, which are bone depositing cells, can decrease in number and function while
osteoclasts, which are bone resorbing cells, can increase in number and function [39,40,69,92–
95]. Studies have shown an increase in RANKL RNA in mouse calvariae exposed to acid [96],
where RANKL interacts with RANK on the surface of osteoclasts and results in increased activity
and formation [97]. Others have shown that there is an early overexpression of RUNX2 and type
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I collagen, but an underexpression of osterix and alkaline phosphatase in human mesenchymal
stem cells exposed to acidic media. Although these human mesenchymal stem cells were able to
undergo the early stages of osteoblastic differentiation, they were unable to keep up with the later
stages of differentiation, altering their ability to mineralize [98]. An imbalance in bone remodeling
could lead to continued release of HCO3-, which has presented in both human patients affected
by acidosis and animal models of metabolic acidosis [3,99–101]. Although the release of skeletal
bicarbonate aids in the restoration of pH homeostasis, it can result in significant bone loss [102].
In humans, this bone phenotype in response to metabolic acidosis may progress into the
development of osteopenia and osteoporosis [103,104]. Additionally, osteocytes play an
important role in bone remodeling by resorbing the lacunar canalicular network (LCN) [105]. When
there is a need for calcium release, such during lactation, the lacunar volume increases as the
surrounding mineral is removed [106–109]. Despite a lack of an established between acidosis
and osteocyte behavior, it is important to note that chronic levels of parathyroid hormone, which
is present in acidosis [110,111], stimulates bone remodeling, leading to bone loss [112].
1.4 Animal Models of Acidosis
Although current animal models of metabolic acidosis exist, they lack clinically relevant
time frames and the physiology of metabolic acidosis. Some in vivo models for metabolic acidosis
use invasive procedures, such as nephrectomies, that directly prevent bicarbonate production
[113,114]. Since these techniques are irreversible and require extensive recovery, they are not
adept to probe the initial effects of metabolic acidosis and can result in variability of renal function
[114]. Because metabolic acidosis is a systemic disease observed in aging patients [102],
neonates [115,116] and young animals [114] are not sufficient models for evaluating chronic
metabolic acidosis on the basis of nonequivalent tissue development and tissue mineralization.
The administration of dietary ammonium chloride (NH4Cl) has been widely used to induce
acidosis both in animal and clinical studies [41,117–121]. Although protocols exist for the
7
administration of acid through NH4Cl modified diets, it has been suggested that the route of
application will affect the buffering compensatory mechanisms. It has been shown that the
ingestion of NH4Cl through food is not as effective at inducing metabolic acidosis as is adding
NH4Cl to the drinking water [41]. Moreover, previous research has shown species-specific
differences in the response to NH4Cl loading in drinking water between mice and rats. Rats were
shown to have mild dehydration, while the mice did not, which results in a higher expression of
aldosterone in the rats. This aldosterone could have then led to a stimulation in urinary
mechanisms [41]. Additionally, most of these models fail to maintain acidosis for long periods of
time since they only use one dose of ammonium chloride, which led our lab to create a new model
using increasing stepwise doses of ammonium chloride.
1.5 Bicarbonate Supplementation for Treatment of Metabolic Acidosis
Treating metabolic acidosis has no clear answer due to its complexity in origin, such as
diet versus disease, and severity [57,122,123]. The administration of bicarbonates in the form of
sodium or potassium bicarbonate (NaHCO3 or KHCO3) is a common treatment for metabolic
acidosis [124–126]. Along with increasing blood pH and bicarbonate, sodium bicarbonate
supplementation has been shown to decrease the decline in kidney function in those with chronic
kidney disease [127]. However, very few studies have researched the effects of sodium
bicarbonate on acidosis-affected bone. Researchers found that sodium bicarbonate prevented
bone loss during the early stages of lactic acidosis [128] but it did not improve bone health during
the late stages of acidosis [129,130]. Conversely, sodium bicarbonate treatment improved bone
turnover and bone mineralization and reduced bone pain in patients with renal osteodystrophy
[131–133]. Because of these conflicting outcomes, it is important to determine how sodium
bicarbonate affects bone impacted by acid exposure.
Potassium bicarbonate could be an alternative treatment to sodium bicarbonate with
potential bone health effects. Studies have shown that potassium bicarbonate increases bone
8
formation markers, such as serum osteocalcin levels [134], and decreases bone resorption
markers, such as urinary N-terminal telopeptides of type I collagen (NTX) [125,135,136]. Unlike
sodium bicarbonate, potassium bicarbonate also decreases calcium excretion, which could
impact systemic calcium balance [134,135]. Nonetheless, no studies have currently researched
the direct effect of potassium bicarbonate on bone health during acidosis.
1.6 Specific Aims
Using multi-scale, materials science characterization techniques, we planned to elucidate
the impact of diet-induced metabolic acidosis on the composition, structure, and mechanics of
bone using a murine model. Additionally, we used biological assays and histological procedures
to determine whether bone alterations seen in this model were due to physiochemical or cellular
influences. Lastly, we determined if current treatments of metabolic acidosis recover acid-
mediated bone loss and if there are sexual differences in acid-exposed bone. In this dissertation,
I focused on these specific aims:
Specific Aim 1: Investigate the temporal response of diet-induced metabolic acidosis on the
murine skeleton.
Hypothesis: Acidic conditions will temporally impact the dissolution and remineralization of bone
via physiochemical processes. Moreover, acid exposure will deleteriously alter the structural and
compositional properties of bone in the short-term, leading to delayed compromised mechanical
behavior. I propose that we will observe differences between the traditional, flat-dose model and
our lab’s established graded-dose model. Within the graded-dose model, we will not observe any
changes in bone cell number and activity due to a lack of osteoclastic bone resorption.
• Sub Aim 1.1 (Chapter 2.1): Physiochemical dissolution governs early
modifications in acid-exposed murine bone with long-term recovery
9
• Sub Aim 1.2 (Chapter 2.2): Cellular contributions are not observed in murine
femurs after 14 days of metabolic acidosis induction utilizing graded NH4Cl diet
Aim 2: Evaluate the effects of treatments on the recovery of acid-exposed bone.
Hypothesis: Sodium bicarbonate treatments will not be enough to overcome compromised bone
properties due to acid-exposure, while potassium bicarbonate treatments will return the properties
of acid-exposed bone to normal levels. I also hypothesize that bisphosphonates will reduce bone
loss during diet-induced acidosis.
• Sub Aim 2.1 (Chapter 3.1): Administration of alendronate exacerbates
ammonium chloride-induced acidosis in mice
• Sub Aim 2.2 (Chapter 3.2): Potassium bicarbonate, not sodium bicarbonate,
maintains acidosis-mediated bone dissolution
Aim 3: Examine sexual dimorphism in bone tissue exposed to acid.
Hypothesis: Both male and female mice will undergo similar levels of acidosis from a NH4Cl diet;
however, acidotic conditions will disrupt skeletal functionality more in female mice than male mice.
Conclusively, this dissertation will provide insight into how metabolic acidosis affects bone
integrity and function; and eventually, into how comprised bone health under acidic conditions
could be restored. The graded-dose model of metabolic acidosis could be utilized to embody other
acid-base disorders and will help bring focus to a part of bone disease research that is often
overlooked.
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1.7 Innovation and Relevance
A novel in vivo model of acidosis
Acidosis studies have been limited due to the difficulty in inducing acidosis in mice. Current
models fail to maintain acidosis or recapitulate the clinically seen skeletal defects [41,137]. We
have developed a novel model of murine acidosis using graded administration of ammonium
chloride [4]. This murine model of acidosis maintains reduced pH up to 14 days and induces
significant changes in the skeletal system, providing us with a unique tool to investigate the in
vivo effects of acidosis.
Ionic effect of bicarbonate treatments on bone health
As previously mentioned, Na+ and K+ have shown to create different responses in bone both
cellularly and physiochemically; therefore, affecting bone structure and composition [24,25,59–
63]. However, studies have not shown how these ions, or bicarbonates containing these ions,
might affect the functionality of bone. This dissertation would be the first to consider the impact of
Na+, K+, and HCO3- on bone quality and function.
Sexual dimorphism of acid-exposed bone tissue
Studies have shown that there is sexual dimorphism under acid loading for cardiac tissue
[42] and renal ammonia metabolism [138]. Despite the NIH mandating the use of female subjects
and women in preclinical studies [139], findings from male-dominated studies are usually imparted
onto on women, leading to poor outcomes [140–143]. Because of this, it is important to see how
bone from female mice is impacted by diet-induced metabolic acidosis. This dissertation contains
the first study to analyze the impact of acid loading on murine bone in females.
11
Chapter 2: Temporal Response of Diet-induced Acidosis on the Murine Skeleton
Chapter 2.1 Physiochemical dissolution governs early modifications in acid-exposed
murine bone with long-term recovery
Subchapter 2.1 is a published manuscript with an altered introduction [43].
2.1.1 Introduction
Many models of metabolic acidosis (MA) utilize a flat-dose administration of ammonium
chloride (NH4Cl) [41,120,121,137,144–148]. Although these studies were able to successfully
induce acidosis in the short term (up to 3 days), acidosis usually was only maintained by day 7
[137,146]. In addition, a flat-dose model of NH4Cl has shown no changes in bone [137]. It is
uncertain whether this lack of effect on bone is due to low dosage or limited experimental time.
Our lab established a graded-dose murine model of NH4Cl in the drinking water, where NH4Cl is
increasingly administered step-wise throughout the experiment [4]. This model maintained a
lowered blood pH and bicarbonate after 14 days of administration and induced changes in bone
mineral composition and macroscale bone mechanics, adding to its value as a model to replicate
bone phenotypes seen in clinical metabolic acidosis. However, with our previous study, we only
looked at days 1 and 14 of acidosis induction. The temporal effects of this graded dosing on
murine bone are still unclear. For this project, we examined both the flat-dose and graded-dose
models in CD-1, male mice to determine whether bone has a dose- and time-dependent response
to diet-induced metabolic acidosis.
2.1.2 Materials and Methods
12
2.1.2.1 Induction of Metabolic Acidosis
All animal experimental procedures were approved by the Institutional Animal Care and
Use Committee at UConn Health Center and Columbia University and comply with the National
Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023,
revised 1978). 4-6-month-old male CD-1 mice (Charles River Laboratories, Worcester, MA) were
separated into 4 groups: flat-dose and graded and their respective controls. Within the flat-dose
and graded-dose groups, the mice were further divided into separate time points representing the
time of sacrifice after acidosis induction: day 1, day 7, and day 14 for flat-dose with an additional
day 3 for graded-dose. Control mice were housed in adjoining cages and had no changes in diet
for the extent of the experiment. Our previous studies showed that control mice exhibited no
changes over the course of 14 days; therefore, they were sacrificed throughout the experiment
but not necessarily on days equivalents to Days 1, 3, 7, and 14 [149]. Metabolic acidosis was
induced in flat-dose and graded-dose mice by replacing their drinking water with an aqueous
solution of ammonium chloride (NH4Cl) and 5% sucrose. For the flat-dose group, the NH4Cl dose
remained constant at 0.28 M for 14 days as per Nowik et al [41]. For the graded-dose group, the
NH4Cl dosing began at 0.2M and was increased by 0.1 M every 3 days for 14 days as per our
previously established graded model [149]. Water consumption and weights were measured
every 3 days for the graded-dose mice. To evaluate the temporal effects of acidosis on bone, the
mice were sacrificed at their assigned time point via CO2 asphyxiation, which was followed by
femur collection. The femurs were characterized for (1) mechanical, (2) structural, (3)
compositional, and (4) cellular properties (Fig. 2.1).
13
Figure 2.1. Arrangement of the methodological techniques done on each femur for the graded-dose and
flat-dose mice groups. The sample sizes are reflective of the number of samples that were analyzed and
graphed.
2.1.2.2 Blood and urine chemistries and assessment of acidosis
Confirmation of acidosis induction and evaluation of mouse health was determined by
blood analysis for both groups with additional urine analysis for the graded-dose mice. For all
mice, blood gas testing was performed on the day of sacrifice immediately prior to sacrifice. For
blood chemistries, 200-300 μL of blood was extracted from non-anesthetized mice via
14
submandibular puncture procedures [150]. To control any potential reduction of CO2, blood
samples were obtained and immediately processed using a Heska PoC Epoc blood-gas analyzer
(Loveland, CO, USA) to obtain values of blood pH, partial pressure of O2, partial pressure of CO2,
as well as ion concentrations of HCO3-, calcium, sodium, and chloride along with metabolic indices
including lactate and glucose. Urine pH was measured for the graded-dose group only using
colorimetric pH strips with a 0.5 pH unit resolution (Hydrion, Brooklyn, NY) starting on the day of
acidosis induction (day 0) and every 3 days thereafter for the duration of the experimental
timeframe.
2.1.2.3 Assessment of bone tissue composition
Raman spectroscopy was used to evaluate the composition of both the femoral surface
and interior using a Witec alpha 300 Raman Spectrometer (Witec, Ulm, Germany). Samples were
obtained and processed as previously described (n=3 mice/time point) [149]. For the external
surface measurements, the anterior surface of intact femurs was cleaned of all soft tissue,
including the periosteum, by manual clearance of tissue with a scalpel blade and buffing with fine
grained sandpaper. Five measurements were then taken across the bone in a medial-lateral
direction with a 50X objective at each of three locations along the bone: proximal, distal, and mid-
shaft, using a 785 nm laser with a laser power of 65mW or less, and an acquisition time of 2 x 30
seconds. After the surface measurements, the bones were mounted in Optimal Cutting
Temperature (OCT) medium and frozen at -80°C. The bones were then cryosectioned along the
coronal plane into 20 μm thick frozen sections using the tape method for mineralized tissues [151].
For the internal samples, six measurements on the cortical bone interior were made at each of
three locations: midshaft, proximal, and distal, where the latter two were located ~2 mm above
and below the midshaft location. Acquisition parameters were consistent with the surface
measurements. The spectra collected at each location were cropped to 200-1800 cm-1,
background corrected, and averaged into a single spectrum using Witec Project 5.1. Peak fitting
15
was performed using 8 peaks and assuming a Lorentzian shape function in the Witec Program
5.1 integrated fitting software (Fig. 2.2).
Figure 2.2. The steps of background removal and fitting for the Raman spectra.
The resultant areas of the fitted peaks were used to calculate bone composition values. The
ratio of the 960 Δcm-1 ν1 phosphate in apatite peak and the 1003 Δcm-1 phenylalanine collagen
16
peaks were used to determine the mineral:matrix ratio. The bone carbonate content is defined
as the area ratio of the 1070 Δcm-1 carbonate in apatite peak and the 960 Δcm-1 ν1 phosphate
(PO4) in apatite peak, known as the carbonate:phosphate (CO3:PO4) ratio. In addition, we fitted
peak ratios for CH2 wag and amide I (Table 2.1).
Table 2.1. The peaks used for fitting and their corresponding wavenumber ranges
2.1.2.4 Evaluation of whole-bone mechanics
3-point bend tests were used to evaluate the mechanical properties of the femurs.
Samples were obtained and processed as previously described [149] (n=10-15 mice/time point).
Femurs were placed into a Biomomentum Mach-1 3-point bend Mechanical testing rig
(Biomomentum, Laval, Canada) with the posterior side facing up to induce fracture on the anterior
side. The span across the beams was 8 mm and the femurs were loaded at a rate of 0.1 mm/s
until failure using a 25 kg load cell. Samples were immersed in phosphate buffered saline
maintained at body temperature (37oC) throughout testing to replicate in vivo conditions and
maintain hydration. Applied force was recorded from the load cell and platen displacement was
used as a measure of sample displacement. Measurements of bone cross-section, moment of
inertia, and centroid distance were measured using microcomputed tomography (μCT) as
17
described in the next section. Load vs. displacement curves were analyzed using custom
programs in MATLAB to determine structural mechanical properties, such as stiffness, maximum
load, and work to fracture. Stress vs. strain curves were calculated by normalizing the force and
displacement using the beam span length, the bone centroid distance, and the area moment of
inertia. Material mechanical properties were calculated from these plots to obtain modulus,
maximum stress, resilience, and toughness.
2.1.2.5 Structural analysis using microcomputed tomography
After mechanical testing, the femoral samples were evaluated by microcomputed
tomography (μCT) to obtain structural information for the region of fracture, the cortical midshaft,
and the distal trabecular bone. Samples (n=7-15 mice/time point) were obtained and processed
as previously described [149]. Bones were dehydrated using graded ethanol rinses and then
imaged with a Scanco 40 μCT system (μCT 40, Scanco Medical, Bruttisellen, Switzerland) located
in the UConn Health MicroCT Imaging Facility using a resolution of 16 μm. For the fracture region,
scans were collected over a region spanning +/-2 mm around the fracture site. From these scans,
the 30 μCT sections immediately adjacent to either side of the fracture site were isolated and
analyzed using the BoneJ toolbox of ImageJ [152] (U. S. National Institutes of Health, Bethesda,
Maryland, USA) to determine centroid distance and area moment of inertia of the bone at the
fracture site. The values for femur centroid distance and area moment of inertia were used to
normalize the mechanical data as described above. The trabecular parameters were measured
from μCT slices at the distal epiphysis of the femur distal to the growth plate and within the distal
shaft in a region 160 μm proximal from the distal growth plate. This region of interest was selected,
thresholded, and analyzed using Bruker CTAn software to obtain bone volume/total volume
(BV/TV), trabecular separation (Tb.Sp), trabecular thickness (Tb.Th), and trabecular number
(Tb.N). For cortical thickness (Ct.Th), the region of interest was composed of 100 slices located
18
~1 mm above the distal growth plate and was isolated and analyzed using Bruker CTAn software.
The cortical and trabecular segmentation procedures included thresholding, despeckling, ROI
shrink-wrapping, and morphological operations. Some samples that underwent mechanical
testing were omitted from μCT analysis either due to trabecular bone breakage within in the distal
epiphysis, the presence of a crack in the region of interest for the cortical bone, or the sample
was used for another analysis technique.
2.1.2.6 Bone dynamic histomorphometry
Five mice per experimental group received intraperitoneal injections of calcein (2.5mg/mL)
and alizarin complexone (3mg/mL) 7 and 2 days before sacrifice, respectively (Sigma-Aldrich, St.
Louis, MO). Because of this necessary time delay between injections, dynamic histomorphometry
was not performed on mice from the day 1 or day 3 time point. These fluorescent dyes attach to
the mineralizing bone surface thus acting as markers for bone remodeling or deposition occurring
between injection dates [149,151]. At the time of sacrifice, the femurs were immediately excised
and fixed in 4% paraformaldehyde (PFA) followed by sucrose fixation. Samples were then
mounted in OCT, frozen at -80oC, and cryosectioned into 7 μm thick sections along the coronal
plane using a Leica CM3050-S cryostat. The sections were analyzed using the OsteoMeasure
(OsteoMetrics, Atlanta, GA, USA) image analysis system to determine the double label surface
(dLS), single label surface (sLS), Mineral Apposition Rate (MAR) and Bone Formation Rate
(BFR). Due to animal loss and sample loss during processing, the sample sizes for this technique
decreased to about a n=2-3 mice/time point.
2.1.2.7 Tartrate-resistant acid phosphatase (TRAP) staining
Traditional non-fluorescent TRAP histological staining was also performed on femurs
(n=4-16 mice/time point). The femurs were dissected immediately after sacrifice and fixed in 4%
19
PFA at 4oC for 24 hours, followed by dehydration using graded ethanol rinses. The femurs
underwent demineralization using EDTA prior to traditional tartrate resistant acid phosphatase
(TRAP) staining and methyl green counterstaining (Sigma) as described previously [149]. TRAP+
cells in trabecular bone were analyzed using the OsteoMeasure (OsteoMetrics, Atlanta, GA, USA)
image analysis system to obtain osteoclast (Oc.N) number and osteoclast (Oc.S) surface relative
to measurements of bone surface (BS) [44]. Due to animal loss and sample loss during
processing, the sample sizes for this technique decreased from the original n=10-16 mice/time
point.
2.1.2.8 Statistical analysis
For quantitative outcomes, statistical analysis was done by using one-way ANOVAs with
the use of Minitab and GraphPad Prism version 9.2.0 software. A significance level of at least
0.05 was used for all tests. Comparisons between groups over time were made using one-way
ANOVAs and post-hoc Tukey’s tests. GraphPad Prism version 9.2.0 was used for data
visualization of raw data with a selection of statistical summaries. P values less than or equal to
0.1 were reported on graphs, while significance was established as p<0.05. Correlations were
calculated between blood gas, compositional, structural, and mechanical parameters to
determine any linear relationships (Table 2.2).
20
1
-1
0
Table 2.2. Correlation table for blood gas, mechanical, composition and structural data.
21
2.1.3 Results
2.1.3.1 Blood gas ion analysis showed diets induced different durations of MA
The progression of the acid-base dysregulation with either flat-dosing or graded-dosing of
NH4Cl was determined by monitoring altered blood-gas values over the course of two weeks
(Figs. 2.3 – 3.5). Mice on the graded-dose diet regimen (Fig. 2.3A) exhibited significantly reduced
blood HCO3- and pH at late time points (days 7 and 14) compared to its control, indicating that
long term acidosis was successfully induced. At day 1, HCO3- levels were also reduced to 14.17
mmol/L compared to a control level of 19.75 mmol/L; however, there was only a trending decrease
in the blood pH (Figs. 2.3B-C). This is followed by a return to baseline levels for both pH and
HCO3- at day 3 (Fig. 2.3C). There was a strong correlation between the blood pH and the HCO3-
level (C=0.88).
22
Figure 2.3. Graded dosing of ammonium chloride creates a time-dependent expression of chronic
metabolic acidosis in mice. (A) A schematic illustrating the experimental design for graded dosing as well
as urine and blood collection. For each timepoint, blood collected was analyzed for (B) pH, (C)
bicarbonate (HCO3-), (D) calcium (Ca2+), (E) sodium (Na+), and (F) chlorine (Cl-). (G) A schematic
showing the effects of an ammonium chloride (NH4Cl) liquid diet has on the vascular and skeletal
systems. One-way ANOVAs and post-hoc Tukey’s tests were used. P values less than or equal to 0.1 are
reported.
23
Figure 2.4. Blood gas metrics for the flat-dose model. For each timepoint, blood collected was
analyzed for (A) pH, (B) bicarbonate (HCO3-), (C) calcium (Ca2+), (D) sodium (Na+), (E) chlorine (Cl-), (F)
partial pressure of oxygen (pO2), (G) partial pressure of carbon dioxide (pCO2), (H) lactate, and (I)
glucose.
C
on
tr
o
l
Day 1
Day 3
Day 7
Day 14
0
25
50
75
100
pO2 (mmHg)
0.0959
0.0688
C
o
n
tr o
l
Day 1
Day 7
Day 14
6.5
7.0
7.5
8.0
Blood pH
<0.0001
<0.0001
0.0006
Contr
o
l
Day 1
Day 7
Day 14
110
120
130
140
Cl- (mmol/L)
<0.0001
0.0387
0.0089
0.0061
0.0144
C
on
tr
o
l
Day 1
Day 7
Day 14
0
100
200
300
400
Glucose (mg/dL)
C
o
n
tr o
l
Day 1
Day 3
Day 7
Day 14
20
30
40
50
60
pCO2 (mmHg)
C
on
tr
ol
Day 1
Day 7
Day 14
0
10
20
30
cHCO3- (mmol/L)
0.0318
0.0427
C
o
n
tr
ol
Day 1
Day 7
Day 14
0
50
100
150
200
250
pO2 (mmHg)
Co
ntr
o
l
Day 1
Day 3
Day 7
Day 14
0
5
10
15
20
25
Lactate (mmol/L)
0.0714
0.0097
0.0042
Co
ntr
ol
Day 1
Day 7
Day 14
0.5
1.0
1.5
Ca
2+
(mmol/L)
0.0001
0.0005
0.0441
Co
n
tr
ol
Day 1
Day 7
Day 14
0
20
40
60
pCO
2
(mmHg)
Co
n
tr
o
l
Day 1
Day 3
Day 7
Day 14
100
150
200
250
300
350
Glucose (mg/dL)
C
o
ntr
o
l
Day 1
Day 7
Day 14
140
150
160
170
Na+ (mmol/L)
<0.0001
0.0004
0.0005
0.0509
Contr ol
Day 1
Day 7
Day 14
0
5
10
15
20
Lactate (mmol/L)
0.0007
24
Figure 2.5. Other blood gas metrics for the graded-dose model. (A) Partial pressure of oxygen (pO2),
(B) partial pressure of carbon dioxide (pCO2), (C) lactate levels, and (D) glucose levels.
The flat-dose mice group exhibited a significant decrease in pH after 1 day of NH4Cl-
dosing compared to its control (Fig. 2.4A). However, following day 1 of acidemia, the pH returned
and were maintained at levels similar to the control throughout the remaining experiment. The
flat-dose group showed no change in blood HCO3- levels compared to its control (10.82 mmol/L)
at any time point, but HCO3- levels increased to 12.29 mmol/L and 11.92 mmol/L for days 7 and
14 compared to day 1 (7.431 mmol/L) (Fig. 2.4B).
Blood ion levels were also modified in response to the dosing and duration of NH4Cl
administration. Blood calcium (Ca2) and sodium (Na+) were monitored as elevations in these ions
are associated with changes in skeletal tissue [153,154]. In the graded-dose group, Ca2+ and Na+
levels significantly rose at all time points except day 3 (Fig. 2.3D-E). The flat-dose group had
significant increases in blood Ca2+ at days 1 (1.226 mmol/L) and 14 (1.203 mmol/L), with a return
to normal levels at day 7 (1.148 mmol/L), compared to the control (1.099 mmol/L) (Fig. 2.4C).
Additionally, Na+ increased at days 1 (153 mmol/L) and 7 (151.7 mmol/L) compared to its control
group (148.7 mmol/L).
To establish consumption of ammonium chloride (NH4Cl) and its effect on the health of
the animals, we also evaluated the chlorine (Cl-) concentration in the blood in addition to murine
weight and the volume of liquid consumed by the mice during standardized water changes. As
anticipated with ammonium chloride supplementation, blood Cl- levels were significantly
increased for all time points compared to the controls for both flat (119.8 mmol/L to 124.2 mmol/L)
Control
Day 1
Day 3
Day 7
Day 14
0
25
50
75
100
pO2 (mmHg)
0.0959
0.0688
Control
Day 1
Day 7
Day 14
6.5
7.0
7.5
8.0
Blood pH
<0.0001
<0.0001
0.0006
Control
Day 1
Day 7
Day 14
110
120
130
140
Cl- (mmol/L)
<0.0001
0.0387
0.0089
0.0061
0.0144
Control
Day 1
Day 7
Day 14
0
100
200
300
400
Glucose (mg/dL)
Control
Day 1
Day 3
Day 7
Day 14
20
30
40
50
60
pCO2 (mmHg)
Control
Day 1
Day 7
Day 14
0
10
20
30
cHCO3- (mmol/L)
0.0318
0.0427
Control
Day 1
Day 7
Day 14
0
50
100
150
200
250
pO2 (mmHg)
Control
Day 1
Day 3
Day 7
Day 14
0
5
10
15
20
25
Lactate (mmol/L)
0.0714
0.0097
0.0042
Control
Day 1
Day 7
Day 14
0.5
1.0
1.5
Ca2+ (mmol/L)
0.0001
0.0005
0.0441
Control
Day 1
Day 7
Day 14
0
20
40
60
pCO2 (mmHg)
Control
Day 1
Day 3
Day 7
Day 14
100
150
200
250
300
350
Glucose (mg/dL)
Control
Day 1
Day 7
Day 14
140
150
160
170
Na+ (mmol/L)
<0.0001
0.0004
0.0005
0.0509
Control
Day 1
Day 7
Day 14
0
5
10
15
20
Lactate (mmol/L)
0.0007
A
B
C
D
25
and graded-dose (114.6 mmol/L to 130.1 mmol/L) groups (Figs. 2.3F and 2.4E). The graded-dose
mice also began to significantly lose weight starting on day 12 (38.82 g) compared to day 0 (42.78
g) and drank less liquid at higher concentrations of ammonium chloride (4.852 mL at 0.6M
compared to 8.574 mL at 0.2M) (Fig. 2.6A-B). However, weight loss was not large enough to
perturb quality of life nor justify sacrifice due to poor health.
Figure 2.6. Diet-induced acidosis decreased weight, NH4Cl solution intake, and urine pH. Other
measurements taken during experimentation were (A) weight, (B) consumption of ammonium chloride
(NH4Cl) solution per mice per day, and (C) urine pH. One-way ANOVAs and post-hoc Tukey’s tests were
used. P values less than or equal to 0.1 are reported.
Control
Day 0
Day 1
Day 3
Day 6
Day 7
Day 9
Day 12
Day 14
25
30
35
40
45
50
55
Weight (g)
A
0.0016
<0.0001
0.0954
0.0446
0.0035
0.0335
0.0387
0.0046
0.2M 0.3M 0.4M 0.5M 0.6M
0
10
20
30
NH4Cl Concentration
Consumption per mouse per day (mL)
B
0.0102
0.0014
0.0560
0.0122
Control
Day 0
Day 1
Day 3
Day 6
Day 7
Day 9
Day 12
Day 14
5
6
7
8
Urine pH
C
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
26
The progression of the acid-base imbalance was also assessed by urine pH
measurements. Urine pH of the graded-dose mice remained significantly low at all time points
(pH 5.912 by day 14) compared to its control and day 0 groups (pH 6.895 and 6.793, respectively)
(Fig. 2.6C).
2.1.3.2 Assessment of internal and external bone tissue composition in response to acid-loading
Raman spectroscopy was used to assess compositional changes on the exterior surface
(Fig. 2.7A) and internal cross-sections (Fig. 2.8A) of bone tissue. In the graded-dose group, we
observed that systemic acid-loading significantly altered bone tissue composition. For the bone
interior, the CO3:PO4 ratio and 960 cm-1 peak width lowered at days 1 and 3 while the 1450 cm-1
peak width rose at these same time points (Fig. 2.8C, D & G). These indicate a decrease in bone
carbonate content [53], and increase in bone mineral crystallinity [155,156], and an increase in
collagen structural disorder [155], respectively . In addition, the mineral:matrix ratio significantly
decreased at days 1, 3, and 14 (Fig. 2.8E). For the bone exterior, CO3:PO4 decreased, and the
1670/1640 ratio—a collagen denaturation marker [157]—increased at day 14 compared to control
but showed no change in the mineral:matrix ratio at any time point (Fig. 2.7).
27
Figure 2.7. Exterior composition of murine bone exposed to acid experienced a slight reduction in
carbonate (CO3) and an increase in collagen denaturation. (A) A schematic showing where spectra
were taken on the exterior of whole bone, as indicated by the red dots. Raman spectra were then used to
calculate (B) 1450 peak width, (C) carbonate: phosphate ratio (CO3:PO4), (D) mineral:matrix, (E)
1670/1604 ratio, and (F) 960 peak width. One-way ANOVAs and post-hoc Tukey’s tests were used. P
values less than or equal to 0.1 are reported.
28
Figure 2.8. Interior composition of murine bone exposed to acid experienced a reduction in
carbonate (CO3) and an increase in collagen denaturation at early timepoints. (A) A schematic
showing where spectra were taken on sectioned bone slices, as indicated by the red dots. (B) Raman
spectra were then used to calculate (C) 1450 peak width, (D) carbonate: phosphate ratio (CO3:PO4), (E)
29
mineral:matrix, (F) 1670/1604 ratio, and (G) 960 peak width. One-way ANOVAs and post-hoc Tukey’s
tests were used. P values less than or equal to 0.1 are reported.
The bone interior for the flat-dose model exhibited a significant decrease in CO3:PO4 and
a trending decrease in the mineral:matrix ratio at day 1 compared to the control, followed by a
return to control levels at days 7 and 14 (Fig. 2.9B-C). However, these results were not mirrored
on the bone exterior where the only significant changes were a decrease in the width of the 1450
cm-1 peak at day 1 and a trending increase in CO3:PO4 at day 14 compared to control (Fig. 2.9F-
G). This model exhibited no changes internally and externally for the 1670/1640 ratio nor with the
960 cm-1 peak width (Fig. 2.9I-J).
30
Figure 2.9. Interior and exterior composition of femur in flat-dose model varies from graded-dose
model. Interior Raman spectra were then used to calculate (A) 1450 peak width, (B) carbonate:
phosphate ratio (CO3:PO4), (C) mineral:matrix, (D) 1670/1604 ratio, and (E) 960 peak width. Exterior
Raman spectra were then used to calculate (F) 1450 peak width, (G) carbonate: phosphate ratio
(CO3:PO4), (H) mineral:matrix, (I) 1670/1604 ratio, and (J) 960 peak width. One-way ANOVAs and post-
hoc Tukey’s tests were used. P values less than or equal to 0.1 are reported.
31
2.1.3.3 Administration of NH4Cl influences whole bone femur mechanics
Three-point bending of the femur (Fig. 2.10A-B) was used to evaluate whether acid-
loading influences whole bone mechanics. In the graded-dose group, toughness increased from
day 1 (5.264 MPa) to day 14 (7.060 MPa) (Fig. 2.10E). Additionally, resilience increased from
between days 1 and 14 and between days 3 and 14 in the graded-dose group (Fig. 2.10F). The
other structural and material mechanical properties, such as maximum load and modulus, were
not altered in this model (Fig. 2.10C-D). The flat-dose group did not exhibit any changes in
material, such modulus, nor structural, such as maximum load, mechanical properties at any time
point evaluated compared to its control group (Fig. 2.11).
Figure 2.10. Mechanical properties recovered after 14 days of acidosis induction. (A) The set-up from
3-point bend testing for femoral samples. The red arrow indicates the direction of the top point during the
test. (B) Stress vs. strain curves were used to calculate various mechanical parameters, including (C)
maximum load, (2) modulus, (E) toughness, and (F) resilience. One-way ANOVAs and post-hoc Tukey’s
tests were used. P values less than or equal to 0.1 are reported.
32
Figure 2.11. Flat-dose model had no changes in mechanics. (A) 30 microcomputed tomography
images sections to the left and right of the fracture were analyzed to obtain centroid distance and moment
of inertia data to calculate stress vs. strain curves. These curves were then used to calculate (B)
maximum load, (C) modulus, (D) toughness, and (E) resilience. One-way ANOVAs and post-hoc Tukey’s
tests were used. P values less than or equal to 0.1 are reported.
2.1.3.4 Structural analysis revealed early changes in trabecular bone
μCT was employed to examine acid-dosing effects on the structure and geometry of the
trabecular bone in the femoral epiphysis (Fig. 2.12A) and distal shaft (Fig. 2.12D) as well as the
midshaft cortical bone (Fig. 2.12G). Bone parameters were altered in response to gradation and
duration of acid-loading in the trabecular bone. The graded-dose diet altered the bone structure.
The femurs exhibited a trending decrease in epiphyseal trabecular bone volume (BV/TV) for day
1 compared to its control (Fig. 2.12C). The Tb.N significantly decreased at day 3 in the graded-
dose group (Fig. 2.12C). Neither the diaphyseal trabecular bone nor the cortical indices showed
no measurable changes in the graded-dose model of MA. The flat-dose group did not exhibit any
significant changes in the cortical or epiphyseal trabecular structural metrics at any time point
(Fig. 2.13). However, the distal shaft increased in bone volume (BV/TV) and trabecular number
33
(Tb.N) as well as decreased in trabecular separation (Tb.Sp) at the early timepoints compared to
its respective control group (Fig. 2.13D).
Figure 2.12. Slight reduction in trabecular bone in condyles of early acid-exposed bone. (A) A
schematic showing the Region of Interest (ROI) of trabecular analysis inside the condyles. (B) Images of
condyles. (C) Distal condyle trabecular bone parameters calculated during analysis. (D) A schematic
showing the ROI of trabecular analysis within the distal shaft. (E) Images of distal shaft. (F) Distal shaft
trabecular bone parameters calculated during analysis. (G) A schematic showing the ROI of cortical
analysis in the shaft. (H) Images of shaft. (I) Shaft cortical bone parameters calculated during analysis.
One-way ANOVAs and post-hoc Tukey’s tests were used. P values less than or equal to 0.1 are reported.
34
Figure 2.13. Slight increase in trabecular bone in distal shaft of flat-dose bone. (A) A schematic
showing the Region of Interest (ROI) of trabecular analysis inside the condyles. (B) Distal condyle
trabecular bone parameters calculated during analysis. (C) A schematic showing the ROI of trabecular
analysis within the distal shaft. (D) Distal shaft trabecular bone parameters calculated during analysis. (E)
A schematic showing the ROI of cortical analysis in the shaft. (F) Shaft cortical bone parameter calculated
during analysis. One-way ANOVAs and post-hoc Tukey’s tests were used. P values less than or equal to
0.1 are reported.
2.1.3.5 Lack of cellular bone resorption in ammonium chloride MA models
Mineral apposition and erosion were determined by bone calcein/alizarin dynamic
histomorphometry (Fig. 2.14A). For all femurs, the single labeled surfaces, double labeled
surfaces, mineral apposition rate, and bone formation rate did not significantly change compared
to their respective controls (Figs. 2.14B-E & 2.15A-D). We also assessed how the dosing and
35
duration of NH4Cl administration influenced femur osteoclast (OC) activity by traditional TRAP
histological staining and quantification of TRAP+-area and cells (Fig. 2.14F). However, the TRAP-
surface, cell number, or eroded surface in the femur did not significantly change for the flat-dose
nor the graded-dose groups (Figs. 2.14G-J & 2.15E-H).
Figure 2.14. No cellular changes in acid-exposed bone. (A) Images of bone stained with calcein
(green) and alizarin complexone (red). These images were used to calculate at (B) single layer surface
per bone surface (sLS/BS), (C) double layer surface per bone surface (dLS/BS), (D) mineral apposition
rate (MAR), and (E) bone formation rate per bone surface (BFR/BS). (F) Images of TRAP stained fixed
36
and paraffin-embedded femoral sections (black triangles pointing towards TRAP+-cells), which were used
to measure (G) osteoclast surface per bone surface (Oc.S/BS), (H) osteoclast number per total area
(N.Oc/T.Area), (I) osteoclast number per bone perimeter (N.Oc/B.Pm), and (J) eroded surface per bone
surface (ES/BS). One-way ANOVAs and post-hoc Tukey’s tests were used. P values less than or equal to
0.1 are reported.
Figure 2.15. No cellular changes in acid-exposed bone. Dynamic histomorphometry was done in order
to obtain data on (A) single layer surface per bone surface (sLS/BS), (B) double layer surface per bone
surface (dLS/BS), (C) mineral apposition rate (MAR), and (D) bone formation rate per bone surface
(BFR/BS). TRAP histological staining was done to obtain (E) osteoclast surface per bone surface
(Oc.S/BS), (F) osteoclast number per total area (N.Oc/T.Area), (G) osteoclast number per bone perimeter
(N.Oc/B.Pm), and (H) eroded surface per bone surface (ES/BS). One-way ANOVAs and post-hoc Tukey’s
tests were used. P values less than or equal to 0.1 are reported.
2.1.4 Discussion
Clinically, metabolic acidosis is characterized by a decrease in serum pH and HCO3-,
which can cause bone dissolution and lead to reduced BMD and increased rates of fracture [1–
3,158]. With increased incidence of acidosis, it is becoming essential to understand the process
by which acidosis induces bone damage in vivo. Current murine models of metabolic acidosis
center on the either flat-dose or graded-dose administration of NH4Cl via the drinking water
[41,137,149]. However, the dose and time dependence of these models on bone quality remain
unclear. Acidosis-induced changes in bone appear to be caused by either physiochemical or cell-
37
mediated processes, but the relative effects of these processes on bone quality and function as
a function of time are unknown. Here we apply a multi-technique approach to understanding the
temporal consequences of acidosis on bone quality and function in both the flat-dose and graded-
dose MA models.
2.1.4.1 Dosing regimen of acid-loading is critical for long-term MA induction
The ability of the flat-dose and graded-dose murine models of MA to induce and maintain
acidosis up to 14 weeks was determined using blood and urine measures. The graded-dose
model maintained the decreased blood pH out to days 7 and 14. This onset of acidemia, defined
as reduce blood pH, was accompanied by a concurrent decrease in HCO3- at days 1, 7, and 14,
indicating that the model can induce chronic metabolic acidosis in CD-1 male adult mice up to 14
days. This more closely mimics clinical metabolic acidosis in humans [67,159] and supports the
relevance of the graded-dose model [149]. For the flat-dose model, in which 0.28 M NH4Cl was
continuously administered [41,137,146], we found that blood pH only decreased at day 1 and
HCO3- levels were unaffected compared to controls. This indicates that the flat-dose model in CD-
1 mice was unable to induce acidosis and that only acidemia, a reduction in blood pH by not
HCO3-, was induced at day 1. Other studies have similarly shown an inability to maintain murine
metabolic acidosis beyond a few days using a flat-dose model [146], but not to the same extent
as we see here. This discrepancy may result from the different mouse strains used in this study
(CD-1) and others (C57Bl/6J). Overall, these results indicate that the graded-dose model is
appropriate as a long-term model of metabolic acidosis in CD-1 male adult mice, while the flat-
dose model fails to induce acidosis.
Interestingly, with the inclusion of additional time points in the current study, we saw blood
pH and HCO3- return to baseline levels at day 3 in the graded-dose model, despite the decrease
on days 1, 7, and 14. Although this result was unexpected, patients with chronic kidney disease
and concomitant metabolic acidosis commonly experience eubicarbonatemic metabolic acidosis
38
(EMA), where their blood pH and HCO3- levels are normal but they exhibit ongoing H+ ion retention
[153,160,161]. To determine whether the graded-dose mice exhibit EMA or if this return to
baseline at day 3 is caused by other variables, we assessed consumption and urine-based
factors. We found that consumption volume of the drinking water remained constant for the NH4Cl
doses during this time period (0.2 and 0.3 M). This, in addition to the elevated blood Cl- levels,
indicate that changes in NH4Cl intake were not the reason for the lack of acidosis at day 3. Urine
pH of the graded-dose mice remained acidic and unchanged throughout the 14 days compared
to the control. This suggests that acid-wasting via urinary excretion was unaltered and thus fails
to explain the day 3 blood gas results [162]. Having excluded NH4Cl consumption and acid
wasting as possible factors, the pH and HCO3- increase at day 3 points to the development of
EMA due to activation of compensatory buffering mechanisms. We predict that these mechanisms
center on dissolution of bone tissue and release of buffering ions, temporarily restoring blood pH
and HCO3- to normal values [161,163,164]. The increasing doses of NH4Cl in the graded-dose
model past day 3 overcome this compensatory response, allowing for continued clinical metabolic
acidosis at longer time points. Overall, we demonstrated that the graded-dose model is more
effective in exhibiting clinically translatable metabolic acidosis symptoms than the flat-dose model
in CD-1 male mice and that there are strong temporal responses to acidosis.
2.1.4.2 Chronic acidosis significantly affects bone matrix composition and structure
Having established that the models induce time-dependent acidemia or acidosis and that
these conditions may be inhibited by compensatory mechanisms, we were interested in
examining the effect of each model on bone dissolution and thus buffering ion release. Both the
flat-dose and graded-dose models exhibited an increase in blood Ca2+ and Na+ ions with an NH4Cl
challenge. The stronger correlation between blood pH and Ca2+ (C=-0.41) as compared to HCO3-
and Ca2+ (C=-0.28) suggests that the calcium release is primarily being controlled by changes in
pH. Since increased blood Ca2+ and Na+ levels are used as indicators of bone dissolution
39
[153,154], this points to reduced pH promoting bone dissolution. Examination of the effect of
possible acid-induced dissolution on the bone was performed using a multi-technique approach
to characterize structural and compositional modifications to the flat-dose and graded-dose
murine bones.
Modifications in bone composition are commonly observed in acidic microenvironments,
spanning acidic cancerous environments to biomineral exposure to acidic media [165,166].
Femurs from the graded-dose model exhibited significant compositional changes at early time
points. The mineral:matrix ratio decreased in the interior of the femurs at nearly all time points,
suggesting rapid onset of mineral dissolution upon induction of MA. There was a negative
correlation (C=-0.15) between the interior and exterior mineral matrix that could suggest a
preferential dissolution of the interior over the exterior bone. This reduction in mineral content is
also accompanied by an early decrease in the CO3:PO4 ratio of the remaining mineral at days 1
and 3. Acting as a strong buffer, carbonate removal from the bone mineral has been previously
reported in vivo, ex vivo, and in vitro in response to acidic environments [31,88,149,165].
Interestingly, its removal is associated with a narrowing in the width of the 960cm-1 peak,
indicating an increase in mineral crystallinity at days 1 and 3. A combination of decreased CO32-
and increased crystallinity indicates physiochemical mineral dissolution/recrystallization
[31,91,167]. In the case of increased remodeling, simultaneous decrease of both CO32- and
crystallinity would be expected [168,169]. This suggests that the mineral loss seen at days 1 and
3 is likely caused by physiochemical dissolution and reprecipitation mechanisms. This
improvement in bone mineral crystallinity at early timepoints will reduce its solubility, thus
inhibiting further dissolution of bone mineral as well as possibly acting as a negative feedback
loop to regulate bone mineral loss [170]. This early carbonate loss at days 1 and 3 may explain
the elevated pH and HCO3- seen at day 3, while the subsequent return to acidic pH afterwards
may be a consequence of reduced mineral solubility from day 3. In addition, the width of the 1450
cm-1 peak significantly widened at days 1 and 3, which is associated with reduced atomic ordering
40
of the CH2 bonds in the organic matrix. This suggests that the graded dosing causes degradation
or denaturation of the organic matrix [171]. This process is reversed at later time points possibly
due to increased fibrillogenesis in the presence of elevated Cl- ions [172,173]. Together, these
results indicate that there are significant interactions between acidic body fluids and bone
composition that vary with time and possibly act as a physiochemical feedback loop to inhibit
excessive bone loss, providing a key factor in understanding the body’s ability to regulate
acidosis.
Structurally, the trabecular bone exhibited a trending decrease in BV/TV at day 1 and a
significant decrease in Tb.N at day 3 compared to the control, suggesting bone loss at the early
time points. Although this rapid bone loss parallels the rapid decrease in mineral and increase in
collagen denaturation seen in the Raman data, this can seem like an overly significant result for
such a short time span. However, rapid bone loss is not uncommon, as seen in animal studies
looking at the effects of radiation [174], COVID-19 [175], and spinal cord injuries [176] on bone.
In addition, due to the high permeability of bone and high dissolution rate of bone mineral it is
possible that these physiochemical processes act quickly [177,178]. Contrarily, the diaphyseal
trabecular bone did not show any changes. The epiphyseal results were similar to studies looking
at acidosis in rats via NH4Cl administration, where bone resorption was higher in the epiphysis
than the diaphysis of the tibia [179]. However, our results were also different compared to studies
done in ovariectomized rats, which found decreased trabecular bone within the metaphysis and
a lack of change within the epiphysis [180,181]. The mechanistically different remodeling
behaviors present within the epiphysis and diaphysis between our present study and other studies
might be due to the trabecular structural differences between rats and mice as well sex-based
differences (female vs. male) [182].
In the flat-dose bones, we found that the CO3:PO4 ratio decreased in the bone interior at
day 1 suggesting a decrease in carbonate content in response to the acidemia which was
recovered once the pH returned to baseline. Structurally, the diaphyseal trabecular parameters
41
BV/TV and Tb.N increased at day 7, indicating that bone growth occurred even with acid
exposure. Taken together, this data suggests that the short-term acidemia induced by the flat-
dose model was not sufficient to cause any major bone loss or compositional changes compared
to controls. The lack of tissue loss during this time suggests that remodeling processes may not
have been impaired due to acidosis. In comparison, the lack of bone growth in the graded-dose
group points to acid-exposure potentially deterring bone formation. These results further iterate
that the graded-dose regimen mimics altered bone phenotypes seen in clinical metabolic acidosis
while the flat-dose model is not sufficient to mimic long-term MA. In addition, it is clear that there
a temporal response to the acid induction with physiochemical processes have rapid effects on
bone composition and structure.
2.1.4.3 Early changes in bone structure and composition during MA are not cell-mediated
Although structural and compositional data point to primarily physiochemical processes, it
was important to address both physiochemical dissolution and the possible cell-mediated
resorption [27]. Cellularly, in vitro studies have shown that acidic media leads to increased
osteoclastogenesis as well as osteoclast activity [37–39,183,184]. This has also been seen in
certain in vivo experiments [120]. However, our previous work using the graded-dose model
showed a decrease in osteoclast numbers after 14 days of acidosis compared to controls,
potentially due to an increase in blood HCO3- levels [149]. Thus, cellular characterization was
performed to understand the role that cellular activity may play in the measured compositional
and structural changes as a function of time.
As osteoclasts are the primary cells responsible for resorption, we focused on examining
the temporal effects of MA on the number and activity of TRAP+-cells. Histologically, we did not
see any significant change in osteoclast numbers at any time points via TRAP staining in either
the flat-dose or the graded-dose model in agreement with Meghji et al. [39] who observed no
change in osteoclast number but a marked increase in their activity. However, neither model in
42
this study exhibited an increase in osteoclast activity, as illustrated by the lack of change in eroded
surface. This lack of osteoclast response may be due an insufficient change in pH to promote
differentiation or activation. Previous studies showing an increase in osteoclast number and
activity, had significantly larger reductions in pH than those measured in this study, some reaching
a pH of 6.8 [28,39]. In addition, 14 days may not be sufficient to measure significant osteoclast
activation and longer timepoints should be investigated.
To further clarify the role that cellular remodeling might play on the structural and
compositional response of bone to MA, we performed dynamic histomorphometry on the samples.
Despite the small sample numbers, looking at single labeled surface, double labeled surface,
mineral apposition rate (MAR), and bone formation rate (BFR/BS), the flat-dose and graded-dose
groups showed no changes at any time points. This agrees with the lack of change in diaphyseal
trabecular bone, especially in the graded-dose model. However, the lack of remodeling presented
by the dynamic histomorphometry data suggests that there was no significant cellular contribution
to the metabolic acidosis response of bones in either the flat-dose or graded-dose models. This
was expected since we saw no changes in TRAP+-cell number or activity. Due to the lack of bone
formation after apparent bone loss indicated by Tb.N and BV/TV as well as bone dissolution of
Ca2+, Na+, and HCO3-, this further suggests that changes in the bone are likely dominated by
physiochemical processes at early timepoints of acidosis.
2.1.4.4 Bone toughness is slightly altered with acidosis
Three-point bend testing of the graded-dose bones showed no differences in any
mechanical parameters compared to control. However, resilience and toughness significantly
increase between days 1 and 3 and day 14. This difference between early and late acidosis time
points suggests that acidosis may cause an initial decrease in the tissue toughness/resilience
followed by an increase at 14 days. Due to the positioning of the bone in 3-point bend, such that
43
the crack propagates through the structurally unchanged cortical mid-shaft, we do not expect that
these changes in mechanics are caused by macro-scale structural modifications but by
compositional and small-scale structural changes instead. The change in toughness is most
significantly correlated to the measure of collagen disorder on the bone exterior (1450 width
C=0.42). Such changes in collagen order or structure have been associated with decreased tissue
toughness [53,157]. The mechanical changes between days 1 and 14 are mirrored by increased
carbonate content and decreased crystallinity at the bone surface as shown by the increase in
CO3:PO4 and 960 peak width between the two timepoints. These compositional changes point to
an initial stiffening of the apatite crystals at early timepoints followed by an increase in crystal
compliance at day 14 [32]. Stiffening of the mineral reinforcing phase at early timepoints could
result in decreased tissue toughness seen here due to reduced crack deflection and increased
reinforcement cracking [185,186]. This suggests that the tissue toughness is primarily controlled
by the quality of the collagen and mineral on the bone surface. Indeed, it is expected during 3pt
bending that a crack will form on the tensile surface of the cortical bone. If the collagen at that
bone surface exhibits a more disordered and less plastic organic matrix along with a stiffened
mineral reinforcement, crack propagation would be facilitated, and toughness reduced. The
decrease in tissue toughness in response to changes in bone composition after an acidic
challenge may explain the clinically observed increased fracture risk in patients with acidosis
[187].
Beyond the change in toughness, we saw no changes in tissue strength or stiffness, which
is in disagreement with our previous work that found a decrease in bone maximum load [149].
The decrease in maximum load in our previous study was caused by an associated decrease in
the cortical bone geometry not paralleled here. As the current study was modified to be a time
course study, we believe that the repeat in vivo blood draws in our initial study caused the acidosis
induction to have a more significant effect on the murine metabolism, and the increase of blood
loss led to the differing bone structure and subsequent change in mechanics reported in our prior
44
study. By eliminating the repeated blood draws and significant blood loss in our current study, we
expect that this study more clearly investigates the effect of acidosis on the skeletal system as
observed in non-surgical procedures and minimizes the compounded influence of altered
hematopoiesis [150]. 3-point bend testing indicated that flat-dose bones exhibited no change in
mechanical properties at any time points. Overall, these results show that compositional changes
induced by acidosis can induce significant time-dependent mechanical changes in the bone.
2.1.4.5 Limitations
Although this study was able to determine the temporal effects of a flat and graded dosing
NH4Cl regimens on the structural, compositional, and mechanical properties of murine bone, like
all animal studies it has some limitations. First, due to the age and skeletal maturity of the mice
and experimental time, we did not expect that there would be any changes in the bone properties
in the control group over the course of the experiment. Therefore, a single control group was
selected instead of providing a control group for every timepoint. Although the later could have
provided better comparisons between controls and acidosis, the number of mice needed to do so
would have been prohibitive especially considering the small, expected changes.
Secondly, even though the blood pH and HCO3- remained low after 14 days of MA
induction, the bones only experienced changes in the first three days. We propose that this could
be caused by a compositional feedback loop but there could also be other participating factors.
Although osteoblast activity has generally been shown to decrease with acidosis [28,40], no
osteoblastic measurements were made here. However, dynamic histomorphometry showed no
significant changes with acidosis suggesting that this may not be cellularly controlled. Despite
this, further biological examinations, such as RNA-Seq, could provide interesting broader
understanding of the cellular response.
In addition, in the graded dose model, the mice consumed lower volumes of the solutions
as the concentration of the NH4Cl increased. This limits the usefulness of the model at timepoints
45
beyond 14 days where consumption may be too low to reasonably maintain mouse health. This
could be overcome by either adding NH4Cl to food and drinking water or by switching to gavage
feeding of NH4Cl.
Lastly, the flat-dose and graded-dose models were performed at different times in different
institutions. Controls are appropriate to each model type therefore we do not expect any issues
within a single model. In addition, 4-6-month-old CD-1 male mice from the same supplier were
used for both models. However, differences in animal husbandry, sample preparation, and
experimental design could have led to some of the differences seen between the flat dose and
the graded dose models.
2.1.5 Conclusion
This study shows that unlike the flat-dose model, graded administration of NH4Cl temporally
affected the bone matrix composition and structure. At early time points, the bone exhibited a
decrease in mineral content as well as compositional changes to the mineral, including decreased
carbonate content and increased crystallinity. Concurrently, the collagen matrix exhibited a
decrease in atomic order, suggesting a certain extent of collagen degradation. These time-
dependent compositional changes were associated with alterations in tissue resilience and
toughness that could lead to the increased fracture risk seen clinically. Thus, our study indicates
that one must consider the temporal and dosage effects of exogenous acid on the material
properties of bone. We believe our graded-dose model will help lead to a better understanding of
the bone dissolution mechanisms of MA, and in the long-term, to the discovery of better
treatments.
46
Chapter 2.2 Cellular contributions are not observed in murine femurs after 14 days of
metabolic acidosis induction utilizing NH4Cl diet
2.2.1 Introduction
Metabolic acidosis, a condition that causes blood pH and bicarbonate levels to decrease,
can adversely alter bone. As mentioned in the Introduction, bone dissolution from acidosis falls
under two categories: physicochemical dissolution and cell-mediated resorption. In our previous
studies using a graded dosing of ammonium chloride, we found that there was no changes in
osteoclast and osteoblast number and activity, hypothesizing that physiochemical dissolution is
the main contributor to bone dissolution under acidotic conditions. To test this hypothesis, a study
using the graded dosing model of metabolic acidosis was conducted to detect any changes in (1)
serum crosslinked c-telopeptide of type I collagen (CTX), a bone resorption maker, as well as (2)
bone cell numbers and activity.
2.2.2 Methods and Materials
2.2.2.1 Acidosis Induction via Ammonium Chloride Graded-Dosing
This study used 4 – 5-month-old, wild-type, retired breeder male CD-1 mice (Charles River
Laboratory). The animal protocol for this study was approved by the Institutional Animal Care and
Use Committee at UConn Health. Mice were split up into four groups: Day 3 control, Day 3
acidosis, Day 14 control, and Day 14 acidosis (8-9 mice/group). These mice were then further
separated into subgroups of A and B. Mice in subgroups were sacrificed at their respective
timepoint (Day 3 or Day 14 of acidosis induction). For example, acidosis induction started for all
of subgroup B at the same time. After three days of acidosis induction, both Day 3 control and
Day 3 acidosis groups in subgroup B were sacrificed. Acidosis induction was implemented by
giving the mice an ammonium chloride (NH4Cl) solution for up to 14 days [43–45,149]. The
solution started at a concentration of 0.2 M NH4Cl supplemented with 5% sucrose. Every three
47
days, the NH4Cl solution increased by 0.1 M NH4Cl, ending at Day 14 with a solution of 0.6 M
NH4Cl + 5% sucrose. Control mice were given water from UConn Health’s Center for Comparative
Medicine every three day as well. Weights, urine pH, food consumption, and fluid consumption
were measured every three days corresponding with water and NH4Cl solution changes. Urine
pH was measured via manual expression using Hydrion pH Strips with increments of 0.5 pH units.
2.2.2.3 Blood Serum Analysis
On the day of sacrifice (Days 3 and 14), mice were individually euthanatized via carbon
dioxide asphyxiation, and about 1mL of blood was collected via intracardiac puncture (8-9
mice/group). Samples sat at room temperature for 30 – 60 minutes to allow them to clot. The
samples were then centrifuged at 2,000 x g for 10 minutes. The serum was removed and put in
new tubes. The samples were then centrifuged at 8,000 x g for 10 minutes to separate out any
additional red blood cells, and serum was placed in new tubes. Serum Crosslinked C-Telopeptide
of Type I Collagen (CTX) was then analyzed as a bone resorption marker via a CTX ELISA
(MBS2700259, MyBioSource.com).
2.2.2.3 Lacunar Analysis via Silver Nitrate Staining
Femurs were dissected immediately after intracardiac puncture and placed in 4% PFA in
1X PBS for three days (5 femurs/group). After the three days, the femurs were decalcified for 12
days in 14% EDTA. When decalcifying the samples, the 14% EDTA solution was changed every
other day. The femurs were then dehydrated via ethanol rinses (30%, 50%, and 70%). After
dehydration, samples stayed in 70% ethanol until they were paraffin embedded and sectioned
coronally by the Histology Core at UConn Health. Silver nitrate staining was done using the
protocol by Alliston et al [188]. The samples were then imaged at 40X on a Zeiss Axio Observer.
Four regions in the trabecular bone of the distal shaft and four regions in cortical bone within the
48
midshaft were selected and imaged. Areas and numbers of lacunae in each image was measured
using ImageJ.
2.2.2.4 Osteoclast and Osteoblast Analysis via Toluidine Blue Staining
The same samples used for silver nitrate staining were also used for toluidine blue staining
but using different sections (5 femurs/group). The samples were sectioned and stained at the
UConn Health Histology Core. The sections were then analyzed using the Osteomeasure
(Osteometrics) imaging system. The region of analysis started by moving 400 µm away from the
growth plate and then reaching a total area of 2.0 mm2. Standardized nomenclature and units set
by ASBMR were used [189].
2.2.2.5 Statistical Analysis
Prism 10 was used for statistical analysis and graphical visualization. Two-way ANOVAs
and mixed-effects analysis with post-hoc Tukey’s and Sidak’s multiple comparison tests were
used. QQ plots were used to determine normality. Significance was determined using a p-value
of less than 0.05. Data on graphs are shown as mean ± standard deviation.
49
2.2.3 Results
2.2.3.1 Decreases in Urine pH, but not weight, due to acid loading
To determine if mice were experiencing acid loading via NH4Cl administration and to
monitor their health, urine pH and weights were measured every three days as well as on the day
of sacrifice. At Day 0, there were no differences between the control and acidosis groups.
However, after three days of acidosis induction, the urine pH decreased in the acidosis groups
compared to the control for both timepoints (Day 3 and Day 14) (Figs. 2.16A-B). For the Day 14
mice, this decrease in urine pH continued for the rest of the experiment. Weight did not change
due to acidosis for each of conditions for either of the timepoints (Figs. 2.16C-D).
Figure 2.16. Urine pH decreased with acidic loading while weight remained unaffected. Urine pH was
measured for both (A) mice sacrificed at Day 3 and (B) mice sacrificed at Day 14. Weight was also
measured for both (A) mice sacrificed at Day 3 and (B) mice sacrificed at Day 14.
Day 0 Day 3
0
2
4
6
8
10
Urine pH
Day 3
Control Acidosis
<0.0001
Day 0
Day 3
Day 6
Day 9
Day 12
Day 14
0
2
4
6
8
10
Urine pH
Day 14
Control Acidosis
0.0004 0.0044 0.0050 0.0209 0.0002
Day 0 Day 3
0
20
40
60
Weight (g)
Day 0
Day 3
Day 6
Day 9
Day 12
Day 14
0
20
40
60
Weight (g)
AB
C D
50
2.2.3.2 Limited changes in eating and drinking habits of mice under acidic conditions
To observe any changes in the eating and drinking habits of the control and acidosis mice,
food and water consumption were measured every three days. For both the Day 3 and Day 14
groups, the amount of water consumed at the beginning time points (Day 0 to Day 3 and Day 3
to Day 6) increased with the acidosis groups compared to their respective control groups (Fig.
2.17A-B). There were no changes in food consumption between the control and acidosis groups
at any day interval (Fig. 2.17C-D).
Figure 2.17. Little to no changes in drinking and food consumption under acid loading. Change in
solution consumption was measured for both (A) mice sacrificed at Day 3 and (B) mice sacrificed at Day
14. Change in food consumption was also measured for both (A) mice sacrificed at Day 3 and (B) mice
sacrificed at Day 14.
Control
Acidosis
-40
-30
-20
-10
0
Day 3
D Drinking Liquid (mL)
0.0243
Day 3 - Day 0
Day 6 - Day 3
Day 9 - Day 6
Day 12 - Day 9
Day 14 - Day 12
-40
-30
-20
-10
0
D Drinking Liquid (mL)
Day 14
Control Acidosis
0.0204 0.0029
Control
Acidosis
-40
-30
-20
-10
0
D Food (g)
Day 3 - Day 0
Day 6 - Day 3
Day 9 - Day 6
Day 12 - Day 9
Day 14 - Day 12
-150
-100
-50
0
50
D Food (g)
AB
C D
51
2.2.3.3 Influence of acidosis on serum CTX levels
CTX levels in the serum did not significantly change due to acid loading for both Day 3
and Day 14 mice (Fig. 2.18).
Figure 2.18. Serum CTX levels did not change due to acidosis induction.
2.2.3.4 Impact of acidosis on lacunar area in cortical and trabecular bone
In the trabecular and cortical bone within the shaft, lacunar area was quantified and
averaged by the number of lacunae in each selected section. For the cortical bone (Fig. 2.19A-
D), lacunar area decreased between Day 3 control mice and Day 14 acidosis mice (Fig. 2.19E).
For the trabecular bone (Fig. 2.20A-D), lacunar area decreased between the Day 3 control mice
and the Day 14 control and acidosis mice. Additionally, lacunar area decreased between Day 3
acidosis mice and Day 14 acidosis mice (Fig. 2.20E).
2.2.3.5 Lack of changes in osteoblast and osteoclast number and activity due to acidosis
Toluidine blue staining was used to observe changes in osteoblasts and osteoclasts in the
femoral samples. Osteoclast perimeter (Oc.Pm), osteoclast number (N.Oc), and eroded perimeter
(E.Pm) respective to bone perimeter (B.Pm) and total area (T.Ar) were not altered due to acidosis
Day 3 Day 14
0
1000
2000
3000
4000
5000
CTX (ng/mL)
Control
Acidosis
52
(Fig. 2.21A-C). Osteoblast perimeter (Ob.Pm) and osteoblast number (N.Ob) respective to bone
perimeter (B.Pm) and total area (T.Ar) were not modified due to acidosis (Fig. 2.22A-C).
Figure 2.19. Lacunar area in cortical bone of acidotic mice. Silver nitrate strained sections of cortical
bone for (A) Day 3 control mice, (B) Day 3 acidosis mice, (C) Day 14 control mice, and (D) Day 14 acidosis
mice were chosen to determine (E) lacunar area.
Control Acidosis
Day 3
Day 14
Day 3 Day 14
0
20
40
60
80
100
Cortical
Lacunar Area (um
2
)
Control
Acidosis
0.0104
AB
CD
E
53
Figure 2.20. Lacunar area in trabecular bone of acidotic mice. Silver nitrate strained sections of
trabecular bone for (A) Day 3 control mice, (B) Day 3 acidosis mice, (C) Day 14 control mice, and (D) Day
14 acidosis mice were chosen to determine (E) lacunar area.
Control Acidosis
Day 3
Day 14
Day 3 Day 14
0
10
20
30
40
50
Trabecular
Lacunar Area (um
2
)
Control
Acidosis
0.0022
0.0004
0.0276
A
D
B
C
E
54
Figure 2.21. Lack of changes in osteoclastic changes due to acidosis. Toluidine blue stained femoral
sections were used to measure (A) osteoclast perimeter per bone perimeter (Oc.Pm/B.Pm), (B) number of
osteoclasts per total area (N.Oc/T.Ar), (C) number of osteoclasts per bone perimeter (N.Oc/B.Pm), and
(D) eroded perimeter per bone perimeter (E.Pm/B.Pm).
Figure 2.22. Lack of changes in osteoblastic changes due to acidosis. Toluidine blue stained femoral
sections were used to measure (A) osteoblast perimeter per bone perimeter (Ob.Pm/B.Pm), (B) number of
osteoblasts per total area (N.Ob/T.Ar), and (C) number of osteoblasts per bone perimeter (N.Ob/B.Pm).
2.2.4 Discussion
Since bone is negatively affected by acid loading [2,3,10,190,191], it is important to
determine the mechanisms behind these effects. Under acidic conditions, bone dissolution can
occur either physiochemically or through cellular resorption. To isolate the cellular impacts of
acidosis on bone tissue, acidosis was induced in male, CD-1 mice using an established model
that utilized graded dosing of ammonium chloride over a span of 14 days. Using a CTX ELISA
assay and various histological techniques, there were no biological changes found in the bone of
acidosis induction.
As mentioned in the Introduction, previous studies have shown that in the early stages of
acidosis, physiochemical dissolution most likely predominates [192–194] over cell-mediated
Day 3 Day 14
-0.5
0.0
0.5
1.0
1.5
2.0
Oc.Pm/B.Pm (%)
Day 3 Day 14
0
2
4
6
8
N.Oc/T.Ar (mm-2)
Day 3 Day 14
-0.5
0.0
0.5
1.0
1.5
2.0
N.Oc/B.Pm (µm-1)
Day 3 Day 14
0.0
0.5
1.0
1.5
E.Pm/B.Pm (%)
Control
Acidosis
AB DC
Day 3 Day 14
0
1
2
3
4
Ob.Pm/B.Pm (%)
Day 3 Day 14
0
10
20
30
40
N.Ob/T.Ar (mm-2)
Day 3 Day 14
0
2
4
6
8
N.Ob/B.Pm (µm-1)
Control
Acidosis
AB C
55
resorption. However, in the long-run, osteoblastic activity has been shown to decrease and
osteoclastic behavior was shown to increase due to acid loading. In our study, we did not find any
changes in osteoblast number, osteoclast number and activity, and lacunar area at early (Day 3)
or late (Day 14) time points due to acidosis induction. This contradicts what has been seen in
other studies. Using neonatal murine calvaria, acidic media has shown to decrease collagen
synthesis and alkaline phosphatase activity, both relating to osteoblast activity, while beta-
glucuronidase, which relates to osteoclast activity, increased [28]. Nonetheless, this study used
skeletally immature (4–6-day old mice), intramembranous (calvaria) bone that had been cultured
in acidic media for 48 hours while our study used skeletally mature (44–5-month-oldmice),
endochondral (femoral) bone. Zhang et al illustrated that bone repair is different between
intramembranous ossification and endochondral ossification in a osteoporotic setting [195].
Moreover, younger mice tend to have higher amounts of bone cells due to the fact that bone cells
have a limited lifespan [169]. Because of these differences, the osteoblasts and osteoclasts in the
younger mice model might have been more reactive to acidic conditions than the in vivo model
used in this study.
Lacunar area within the trabecular bone decreased over time in both the control and
acidosis groups over the 14 days of experimentation, but unexpectedly this result was not due to
acidosis. The administration of parathyroid hormone (PTH), which is elevated under acidotic
conditions [110,111], increases lacunar area by decreasing mineral and carbonate content at the
lacuna wall [196,197]. Additionally, lactation is another source of calcium release, which also
causes enlarged lacunae [198]. On the other hand, lacunae area tends to decrease with age,
which ageing increases the incidence of metabolic acidosis. Instead of an increase in osteocyte
behavior, potentially both the control and acidosis mice were undergoing osteocyte apoptosis due
to bone remodeling [199,200]. Further analysis into osteocyte behavior and the lacunar
canalicular network under acidic conditions need to be examined.
56
Lastly, an extended study using the graded dosing of ammonium chloride may be required
to observe longer and more drastic alterations to bone tissue when under metabolic acidosis
conditions. In various studies using this model, bone was either slightly altered or unchanged due
to acidosis [43,44,149]. The mice in this specific study also underwent acid loading, as made
evident by the decrease in urine pH; however, there were no changes in serum CTX, a marker
for bone resorption, with the acidosis mice compared to the control mice. A hypothesis for this
lack of phenotypical bone modification is that the body is reducing acid-mediated bone dissolution
through protective mechanisms. As seen in Chapter 2.1, the acid-exposed femurs
compositionally, structurally, and mechanically recovered after 14 days of acidosis induction.
Additionally, Chapter 4 presents genetic changes that reduced osteoclastic activity, further
illustrating the bone’s potential capacity to protect itself from sudden changes due to a decrease
in blood pH. However, a study using rats and NH4Cl administration via gavage found that after 6
and 10 weeks of acidosis induction, bone microarchitecture was impaired via a reduction in
trabecular bone [201]. Since our study is only two weeks in comparison, it might be necessary to
lengthen our study by a few more weeks in order to observe any cellular changes; and therefore,
mimic the phenotype of bone seen in patients with metabolic acidosis.
Limitations of this study include a lack of more biological assays. Originally, this study
contained PTH and procollagen type 1 N-terminal propeptide (P1NP) ELISA assays. Both these
assays were done on serum samples, but it was discovered that the dilution used for the P1NP
assay was too low. Additionally, the undiluted samples used in the PTH assay had too low of a
signal. After conducting these ELISA assays, there was not enough serum to conduct further
analyses. In future studies, it might be important to include more animals to get a sample size big
enough to conduct multiple ELISAs. Moreover, blood gas analysis was not done in these mice,
unlike what was usually done in previous studies, due to the worry that there might not have been
enough blood for the intracardiac punctures. For future studies, it would be useful to test if doing
submandibular bleeds beforehand affects the amount of blood collected during intracardiac
57
punctures. To better study how acidosis impacts the osteocyte lacunar canalicular network (LCN),
rhodamine staining could help better visualize the LCN three dimensionally rather than in two
dimensions using silver nitrate staining.
In conclusion, this study observed a lack of changes in bone cell (osteoblast, osteoclast,
and osteocyte) number and activity due to acidosis induction over a span of 14 days. This absence
may support our hypothesis that physiochemical dissolution is the main contributor to any bone
alterations during the two weeks of acid loading. However, since some of our studies have shown
little to no modifications in bone compositionally, structurally, and mechanically, an extended
study (months rather than weeks) may be needed in order to observe more extreme phenotypical
changes and cell-mediated resorption.
58
Chapter 3: The Effects of Treatments on the Recovery of Acid-exposed Bone
Chapter 3.1 Administration of alendronate exacerbates ammonium chloride-induced
acidosis in mice
This subchapter (Chapter 3.1) is a published manuscript [44].
3.1.1 Introduction
Bone disease is an extremely common comorbidity of chronic kidney disease (CKD) due
to abnormal calcium-phosphate metabolism, changes in calcitriol and parathyroid hormonal
levels, and metabolic acidosis [202]. This leads to increased fracture risk resulting in a 3-4 times
higher mortality rate than that of the non-CKD population [203]. Thus, there is a great interest in
treating CKD patients with osteoporosis medications, such as bisphosphonates (BPP), to
maintain bone volume and quality, thus increasing overall health and quality of life. However, the
prescription of BPP has been limited due to possible side effects, including increased kidney
damage, risk of osteonecrosis of the jaw, and atypical femur fractures [204–207]. However, recent
animal and human studies have shown that properly administered BPP does not appear to have
significant negative effects on kidney function and instead improves bone health [207,208].
Nonetheless, most of these studies fail to investigate the effects of BPP administration on patients
that present with metabolic acidosis alongside CKD.
With reduced kidney function, CKD patients are less able to excrete acid and regulate
serum bicarbonate HCO3-, leading to an accumulation of acid in the body. This reduction in body
pH is associated with cardiac and immune dysfunction as well as a decrease in bone mineral
density [10,42,84,102]. As the primary reservoir of buffering ions in the body, the skeleton
undergoes dissolution during acidosis resulting in the release of buffering phosphate (PO43-) and
bicarbonate (HCO32-) ions from the bone mineral. This process is mediated by a combination of
physiochemical processes, where acid simply dissolves the mineral, and cell-mediated
processes, where acid promotes osteoclastogenesis and cellular activity resulting in increased
59
bone resorption [209,43,38,210]. Thanks to this release of buffering ions, the pH can be restored
to more physiological levels, increasing patient health.
BPP acts primarily on cell-mediated processes by inhibiting osteoclast resorption, thus
reducing bone loss and maintaining bone mass [211]. However, in cases of acidosis, this reduced
bone dissolution may inhibit the release of buffering ions, exacerbating the acidosis and its
associated complications. It is therefore necessary to understand how treatment with BPP affects
acidosis and its consequences on bone health. In this study, we use a murine model of diet-
induced acidosis to isolate the effects of BPP treatment on pH dysregulation and bone health.
3.1.2 Materials and Methods
3.1.2.1 Induction of metabolic acidosis and administration of alendronate
4–5-month-old, wild-type, CD-1 male mice from Charles River Laboratory (Worcester,
MA) were used in this study. All animal procedures were approved by the University of
Connecticut Health Center (UConn Health) Institutional Animal Care and Use Committee
(IACUC) via written consent and abided by the standards set by the National Institutes of Health
[212]. The approved IACUC protocol number is AP-200306-1123. The mice were euthanized via
carbon dioxide (CO2) asphyxiation followed by cervical dislocation. No anesthesia methods
were used in this study. The mice were separated into three groups (N=8/experiment group):
control, acidosis, and acidosis + bisphosphonate (acidosis+BPP) (Fig 3.1). Alendronate sodium
trihydrate (7.5 µg/mL) (Sigma-Aldrich, #A4978) was administered through subcutaneous
injections every three days at a dosage of 75 µg/kg (Fig 4.1C). Injections started 3 days before
acidosis induction to ensure effectiveness. Acidosis was induced in the acidosis and
acidosis+BPP groups through a graded dosing of ammonium chloride (NH4Cl) in the drinking
water, starting at a concentration of 0.2 M NH4Cl + 5% sucrose and ending at 0.6 M NH4Cl + 5%
sucrose with increased increments of 0.1 M NH4Cl every three days (Fig 4.1B & 4.1C), as done
previously [43,209]. Like the acidosis and acidosis+BPP groups, the control group was also
60
housed for fourteen days in the vivarium while the experiment was being conducted (Fig 4.1A).
After 14 days of experimentation (acidosis induction), the mice were sacrificed and frozen at -
20°C.
Figure 3.1. Schematics for acidosis induction and alendronate administration. The illustrations
shown are for the (A) control, (B) acidosis, and (C) acidosis+BPP mice.
61
3.1.2.2 Blood and urine chemistries and assessment of acidosis and treatment
Approximately 300 µL of blood was collected from non-anesthetized mice via
submandibular puncture procedures on days 0 and 14 of acidosis induction (N=8
mice/experimental group). After blood collection, gauze was placed on the site of collection to
stop the bleeding before the mouse was placed back in their cage. A Heska Epoc blood gas
analyzer (Loveland, CO, USA), which is calibrated for each test card before a blood sample is
inserted, was used to determine various metrics of the blood, including pH, bicarbonate (HCO3-),
calcium (Ca2+), potassium (K+), and sodium (Na+). Urine pH and mouse weights as well as food
and liquid consumption were measured every 3 days. Urine was collected via manual
expression and its pH was measured using Hydrion pH strips (4.5-7.5) with a resolution of 0.5
pH units. Food consumption was measured by taking a baseline measurement and then
weighing the food after three days. Liquid consumption was measured by taking a baseline
measurement and then measuring the liquid after three days.
3.1.2.3 Mechanical testing of bone
The mice were thawed, and left femurs were dissected on the day of testing. Three-point
bend testing was done to measure the femur macroscale mechanics (N=8 mice/experimental
group) using a Biomomentum (Laval, Canada) Mach-1 Mechanical Tester (v500csst with a 3-axis
motion controller). During testing, the femurs were placed in a bath of 1X phosphate buffered
saline (PBS) at 37°C. The femurs were placed in a three-point bend rig (Biomomentum) with an
8 mm span and the posterior side facing up. They were then loaded at a rate of 0.1 mm/s until
failure. Displacement was recorded using the stage displacement and the load was recorded with
a 25 kg load cell. After mechanical testing, the femurs were wrapped in 1X PBS-soaked gauze
and frozen at -20°C. Load vs. displacement curves were used to determine structural mechanical
properties, such as stiffness, maximum load, and yield load. The area moment of inertia and
62
centroid distance as well as the span length were used to normalize the load and displacement
values to calculate stress vs. strain curves. Custom MATLAB code was used to determine the
material mechanical properties modulus, maximum stress, and toughness.
3.1.2.4 Raman spectroscopy of bone
The mice were thawed, and left femurs were dissected on the day of testing. Three-point
bend testing was done to measure the femur macroscale mechanics (N=8 mice/experimental
group) using a Biomomentum (Laval, Canada) Mach-1 Mechanical Tester (v500csst with a 3-axis
motion controller). During testing, the femurs were placed in a bath of 1X phosphate buffered
saline (PBS) at 37°C. The femurs were placed in a three-point bend rig (Biomomentum) with an
8 mm span and the posterior side facing up. They were then loaded at a rate of 0.1 mm/s until
failure. Displacement was recorded using the stage displacement and the load was recorded with
a 25 kg load cell. After mechanical testing, the femurs were wrapped in 1X PBS-soaked gauze
and frozen at -20°C. Load vs. displacement curves were used to determine structural mechanical
properties, such as stiffness, maximum load, and yield load. The area moment of inertia and
centroid distance as well as the span length were used to normalize the load and displacement
values to calculate stress vs. strain curves. Custom MATLAB code was used to determine the
material mechanical properties modulus, maximum stress, and toughness.
3.1.2.5 Structural analysis of bone
After Raman spectroscopy analysis, all the left femurs used for mechanical testing were
dehydrated in graded ethanol rinses up to 70% ethanol and imaged using a Scanco 50
microcomputed tomography (μCT) system (μCT 50, Scanco Medical, Bruttisellen, Switzerland)
(N=8/experimental group). The scans were run at an energy of 55 kV and current of 145 μA with
a Cu Kα X-ray source. Scans were done over an angular range of 180o with a step size of 0.36o
(500 projections) and an acquisition time of 400 ms to obtain a 16 µm voxel size. The experimental
63
data was used to determine the (1) fracture area parameters, (2) cortical parameters, and (3)
trabecular parameters. 30 μCT sections on each side of the fracture were examined using BoneJ
on ImageJ (U. S. National Institutes of Health, Bethesda, Maryland, USA) [152] to measure
centroid distance and area moment of inertia near the fracture site. The cortical thickness (Ct.Th)
was determined by selecting a region of interest starting at the beginning of the fracture on the
distal side and ending at 100 μCT sections going towards the condyles. Trabecular parameters
were measured both in the distal epiphysis and in the distal diaphysis. For the distal epiphysis,
μCT sections were selected between the start of the trabecular region on the distal end to the
beginning of the growth plate. For the distal diaphysis, a region of interest containing 100 sections
proximal from the end of the growth plate was selected. Using the Bruker CTan software, both
the cortical and trabecular segmentation procedures included global thresholding, despeckling
black spots less than 10 pixels from the images, ROI shrink-wrapping, and morphological
operations. Thresholding values were selected by an experienced user after a preliminary
examination of the data set and set to equal values across all samples. The trabecular parameters
obtained were bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), trabecular
number (Tb.N), and trabecular spacing (Tb.Sp).
3.1.2.6 Statistical analysis
Statistical analysis and graphical visualization were done in GraphPad Prism version
9.4.0. Day 0 is the day of acidosis induction right before giving the acidosis groups ammonium
chloride. Day 14 is two weeks of graded dosing of ammonium chloride in acidosis drinking water,
or for the control mice, two weeks of being maintained in the vivarium. Due to the normal
distribution of data, parametric statistical testing using two-way ANOVAs or mixed-effects analysis
with post-hoc Bonferroni’s or Tukey’s multiple comparisons tests were used for the blood metrics,
food consumption, urine pH, weight, and fluid consumption. For the compositional, mechanical,
64
and structural data, non-parametric statistical testing was used including Kruskal-Wallis tests with
post-hoc Dunn’s multiple comparisons tests.
3.1.3 Results
3.1.3.1 Blood gas, urine, and weight analysis
The blood gas parameters were measured at Days 0 and 14 for all groups. The
acidosis+BPP group had a reduction in blood pH and HCO3- between Days 0 and 14 (Fig. 3.2A-
B). However, the control and acidosis groups did not have any changes in these parameters. The
acidosis and acidosis+BPP groups had an increase in blood Ca2+, chlorine (Cl-), and Na+ at Day
14 compared to Day 0, while the control group stayed consistent over this time period (Fig. 3.2C-
E). Moreover, there was an increase in glucose with the acidosis+BPP group, but no changes in
blood urea nitrogen (BUN), as observed with the control and acidosis groups (Fig. 3.3A-B). The
acidosis group also had a decrease in lactate from Day 0 to Day 14 (Fig. 3.3C). The base excess
in blood (BE(b)) decreased in both the acidosis and acidosis+BPP groups (Fig. 3.3D). There were
no changes in partial pressure of carbon dioxide (pCO2), partial pressure of oxygen (pO2), and
anion gap, K+ (AgapK) within any of the groups over the span of 14 days (Fig. 3.3E-G).
65
Figure 3.2. Blood gas measurements of before acidosis induction (Day 0) and after 14 days of
acidosis induction (Day 14) for the control, acidosis, and acidosis+BPP groups. The measurements
collected were (A) blood pH, (B) HCO3-, (C) Ca2+, (D) Cl-, (E) Na+, and (F) K+. For blood pH, HCO3-, Ca2+,
and Na+, two-way repeated measures ANOVAs and post-hoc Bonferroni’s tests were used. For Cl- and
K+, mixed-effects analysis and post-hoc Bonferroni’s tests were used.
66
Figure 3.3. Additional blood gas measurements of before acidosis induction (Day 0) and after 14
days of acidosis induction (Day 14) for the control, acidosis, and acidosis+BPP groups. The
measurements collected were (A) glucose, (B) BUN, (C) lactate, (D) BE(b), (E) pCO2, (F) pO2, and (G)
AGapK. For glucose and pCO2, two-way repeated measures ANOVAs and post-hoc Bonferroni’s tests
were used. For BUN, lactate, BE(b), pO2, and AGapK, mixed-effects analysis and post-hoc Bonferroni’s
tests were used.
At Day 0, the urine pH was significantly higher in the groups with acidosis compared to
the control; however, this changed once acidosis was induced. From Day 3 to Day 14, the urine
67
pH in the acidosis and acidosis+BPP groups decreased compared to the control group. Moreover,
on Day 6, the acidosis+BPP had a higher urine pH compared to the acidosis group. On the other
hand, the acidosis group had a higher urine pH compared to the acidosis+BPP group on Day 12
(Fig. 3.4A).
68
Figure 3.4. A decrease in urine pH and food and water consumption over a span of 14 days of
acidosis induction. The measurements collected were (a) urine pH, (b) weight, (C) fluid consumption,
and (D) food consumption. For urine pH, fluid consumption, and food consumption, two-way ANOVAs
and post-hoc Tukey’s tests were used. For weight, mixed-effects analysis and post-hoc Tukey’s tests
were used.
The animal weight between the three groups was similar throughout the 14 days. The
acidosis+BPP groups had a trending increased weight compared to the acidosis group on Day 9
(p=0.0832). On Days 12 and 14, the acidosis group had a trending smaller weight than the control
(respectively, p=0.0947 and p=0.0771) (Fig. 3.4B).
The groups undergoing acidosis were shown to drink and eat less. Days 3 through 14
showed a decrease in fluid consumption for the acidosis and acidosis+BPP groups compared to
the control group (Fig. 3.4C). At Day 3, the acidosis mice were already consuming less food than
the control mice. However, this only continued for Day 6. On Days 6 and 9, the acidosis+BPP
mice were eating less than the control mice (Fig. 3.4D).
3.1.3.2 Assessment of bone tissue composition in response to bisphosphonate and acidosis
There were no differences between the three groups when looking at the exterior and
interior composition in terms of the mineral:matrix ratio, the CO3-:PO43- ratio, and the inverse of
FWHM of the 960 cm-1 peak (Fig. 3.5).
69
Figure 3.5. No changes in composition via Raman spectroscopy after 14 days of acidosis induction.
(A) Raman spectra was used to measure composition on the (B) exterior and (C) interior of femur sample
in order to determine the (D) exterior mineral:matrix ratio, the CO3:PO4 ratio, and the inverse of the FWHM
of the 960 cm-1 peak and the (E) interior mineral:matrix ratio, CO3:PO4 ratio, and the inverse of the FWHM
of the 960 cm-1 peak. For all data, Kruskal-Wallis tests with post-hoc Dunn’s tests were used.
3.1.3.3 Bisphosphonate administration and acidosis effects on whole bone femur mechanics
To determine how structural factors affect mechanical properties of the femurs in each group, the
outer diameter and length were measured. The three groups had no differences for outer diameter
(Fig. 3.6A). However, after 14 days of acidosis, the acidosis+BPP group femurs had a trending
increase in length compared to the control bones (p=0.0811) (Fig. 3.6B). The centroid distance
70
and area moment of inertia were also calculated from microCT data for mechanical influence (Fig.
3.6C-D). For the area moment of inertia, the acidosis+BPP group was higher compared to the
control group. For all the material and structural mechanical properties, there were no significant
differences between the three groups (Fig. 3.7).
Figure 3.6. Structural components that affect mechanical properties. Gross measurements were taken
for (A) outer diameter and (B) length of the femurs while microcomputed tomography was used to determine
(C) centroid distance and (D) moment of inertia. For all data, Kruskal-Wallis tests with post-hoc Dunn’s tests
were used.
71
Figure 3.7. No changes in mechanics via 3-point bend testing after 14 days of acidosis induction.
Structural mechanical properties measured were (A) maximum load, (B) yield load, and (C) stiffness.
Material mechanical properties measured were (D) maximum stress, (E) yield stress, and (F) modulus. For
all data, Kruskal-Wallis tests with post-hoc Dunn’s tests were used.
3.1.3.4 Trabecular and cortical structural analysis in acidosis and bisphosphonate exposed bone
For the epiphysis, the trabecular BV/TV was significantly different, where the acidosis+BPP group
had more bone volume than the control (Fig 3.8C). The diaphysis trabecular metrics had more
significant differences between the groups. The acidosis+BPP group had a higher BV/TV and
Tb.N and a lower Tb.Sp than the control (Fig 3.8F). Additionally, the acidosis group had a trending
higher BV/TV (p=0.0549) and significantly higher Tb.N and lower Tb.Sp than the control (Fig
3.8F). There were no changes in the Ct.Th among the three groups (Fig 3.8I).
72
73
Fig 3.8. BPP administration impacted distal diaphysis trabecular parameters after 14 days of
acidosis induction. In order to determine trabecular parameters in the (A) distal condyle, (B) microCT
sections were used to examine (C) BV/TV, Tb.Th, Tb.N, and Tb.Sp. In order to determine trabecular
parameters in the (D) distal shaft, (E) microCT sections were used to examine (F) BV/TV, Tb.Th, Tb.N, and
Tb.Sp. In order to determine cortical parameters in the (G) shaft, (H) microCT sections were used to
examine (I) Ct.Th. For all data, Kruskal-Wallis tests with post-hoc Dunn’s tests were used.
3.1.4 Discussion
The relationship between BPP, acidosis, and bone health remains unexplored. However,
there is a need to understand these interactions, especially with respect to the CKD population
that presents with both bone disease, which could benefit from BPP treatment, and metabolic
acidosis. Here we used an established murine model of diet-induced metabolic acidosis to isolate
the effects of BPP on the pH dysregulation and bone health of the mice.
Much like CKD patients that exhibit acid accumulation but no reduction in blood pH or
HCO3-, the acidosis mice in this study also presented with eubicarbonatemic or preclinical
metabolic acidosis at Day 14 as expected for this model [160,209,213–215]. Despite exhibiting
pH and HCO3- levels on par with Day 0 controls, the urine pH and Base Excess (BE(b)) were
reduced with administration of the NH4Cl, further supporting this hypothesis. However, the
acidosis+BPP group showed a significant decrease in both pH and HCO3- with 14 days of NH4Cl
administration, suggesting that the alendronate treatment exacerbated the severity of acidosis in
the mice. Like the acidosis group, the acidosis+BPP group also exhibited the reduced BE(b) and
urine pH with NH4Cl administration.
There were no significant differences between the acidosis and acidosis+BPP groups in
terms of food and water consumption, so the diet patterns of the mice should not have influenced
the extent of acidosis experienced. The similar increases in blood Cl- also indicated that similar
levels of NH4Cl were consumed by the two groups. This further suggested that alendronate
exacerbated the metabolic acidosis, resulting in lower body pH and HCO3- levels. This could be
74
due to reduced osteoclast activity in the acidosis+BPP group, preventing the release of
bicarbonate and other buffering agents from bone, leading to less buffering of the blood.
Interestingly, both the acidosis and the acidosis+BPP groups had an increase in
circulating Ca2+ and Na+. Such an increase is generally used as an indicator of bone dissolution
or increased remodeling [153,154]. This would suggest that despite the administration of
alendronate in the acidosis+BPP group, there is still a net release of ions from the bone material.
To determine whether this release would have significant effects on the bone function, these
bones were tested for compositional, structural, and mechanical changes.
The acidosis+BPP group exhibited an increase in BV/TV at both the epiphysis and the
diaphysis at 14 days compared to controls. This was accompanied by an increase in trabecular
number and a decrease in trabecular spacing in the diaphysis, suggesting that the alendronate
induced a small increase in bone volume compared to the healthy control. Similar changes were
seen in the acidosis group for the diaphysis; although, the extent of the change was larger for the
acidosis+BPP group. The reason for the increase in bone volume with acidosis is unclear, but the
greater accumulation of bone tissue in the epiphysis and diaphysis of the acidosis+BPP group
suggests that alendronate treatment led to increased bone deposition despite the measured
increase in acidosis. This is in agreement with other studies showing an increase in trabecular
volume with BPP treatment in rats with CKD, although their acidosis status was not reported
[208,216]. Although there were structural changes to the bone with acidosis and acidosis+BPP,
there were no measurable changes in the bone composition or bone mechanics at 14 days. In a
previous study of our acidosis model, we saw changes in toughness between Days 1 and 14 [43].
Since this current study only looks at one time point, we might have only observed a recovery in
the bone mechanics, which might be the reason for a lack in change after 14 days of acidosis
[43].
Additionally, the blood gas results showed an increase in glucose in the acidosis+BPP
groups over the span of 14 days. Although alendronate and other bisphosphonates have an
75
association with decreasing insulin resistance, which in turn would decrease blood glucose
[217,218], acidosis has the opposite effect [82,219]. Even though the acidosis+BPP group
received alendronate, it seems that the effect of acidosis was greater than that of the acidosis
group, potentially increasing glucose levels due to an increase in insulin resistance. We also saw
a decrease in lactate in the acidosis group, which has been seen in previous studies [209].
However, the lactate might not have decreased in the acidosis+BPP group because there was a
balance between the acidosis-mediated lactate lowering and the elevation of lactate levels due to
a potential decrease in insulin resistance from the alendronate [220]. Together, these results
suggest that when administering BPP to individuals with CKD and other co-morbidities, it is also
essential to consider other systemic effects, such as insulin resistance, especially since CKD and
diabetes are so often comorbidities [221].
3.1.5 Conclusion
The administration of BPPs could be a useful tool for the treatment of bone disease in
individuals with CKD; however, the relationship between BPP, acidosis, and bone health
remained unclear. Therefore, in this study, we used a model of diet-induced metabolic acidosis
to isolate the effects of BPP on acid accumulation and bone health. We found that the
administration of alendronate to mice undergoing acid dosing led to a decrease in blood pH and
HCO3- levels compared to acidotic mice, suggesting that BPPs could lead to increased acidosis.
We hypothesize that this is a result of reduced osteoclast activity resulting in decreased bone
resorption and thus decreased release of buffering ions. The administration of alendronate also
had more impactful effects on bone volume, suggesting that alendronate does promote bone
growth; however, this change was minimal compared to the acidosis group and had no effect on
the composition or mechanics of the bones. Our data suggests the prescription of BPPs in
individuals with acidosis should be a case-by-case basis with a balance of concerns about
worsening acidosis and bone disease risks. However, acidosis in these scenarios could
76
potentially be combated with the supplementation of alkali treatment [70]. Therefore, the dual
administration of alkali treatment and BPP administration needs to be further explored.
77
Chapter 3.2 Potassium bicarbonate, not sodium bicarbonate, maintains acidosis-
mediated bone dissolution
This chapter has been reviewed and is currently undergoing revisions for publication.
3.2.1 Introduction
To determine the effect of sodium bicarbonate and potassium bicarbonate on bone health
when exposed to an ongoing acidic challenge, our study used an established, diet-induced mice
model that utilizes graded amounts of ammonium chloride in the drinking water [42–44,209]. After
inducing acidosis in adult mice for 14 days, we continued acidosis for an additional seven days at
a constant concentration of ammonium chloride along with supplementation of either potassium
bicarbonate or sodium bicarbonate. After the seven days of bicarbonate supplementation, the
femurs from the mice were excised to examine any impact on the mechanics, structure,
composition, and cellular properties to the bone. Additionally, acidosis induction was confirmed
via blood gas analysis before and after bicarbonate treatment. This study established the
differences between the effects of potassium bicarbonate and sodium bicarbonate on systemic
acid-base balance and murine bone health.
3.2.2 Materials and Methods
3.2.2.1 Induction of metabolic acidosis and administration of sodium bicarbonate and potassium
bicarbonate
The Institutional Animal Care and Use Committee (IACUC) at UConn Health approved all
animal experimental procedures. These procedures also complied with the National Institutes of
Health guide in terms of care and handling of laboratory animals (NIH Publications No. 8023,
revised 1978). 4-5-month-old, male CD-1 mice from Charles River Laboratories (Worcester, MA)
acclimated to the vivarium environment for 7 days before any experimentation. After these seven
78
days, mice were separated into 5 groups (N=15 mice/group): control, acidosis + potassium
bicarbonate (A+KHCO3), acidosis + sodium bicarbonate (A+NaHCO3), potassium bicarbonate
only (KHCO3-only), and sodium bicarbonate only (NaHCO3-only) (Fig. 3.9). Control mice had no
changes in their diet. For the A+KHCO3 and A+NaHCO3 groups, metabolic acidosis was induced
by changing their drinking water with an aqueous solution of ammonium chloride (NH4Cl) and 5%
sucrose. NH4Cl dosing started at 0.2M and every 3 days was increased by 0.1M for 14 days and
then remained constant at 0.5M for 7 more days [209]. At 14 days, the A+KHCO3 and A+NaHCO3
groups were given food (Envigo 2018) supplemented with either 1.603% of potassium
bicarbonate or 1.345% sodium bicarbonate, respectively. These groups of mice were sacrificed
after 21 days of experimentation via CO2 asphyxiation. The KHCO3-only and NaHCO3-only groups
were given food (Envigo 2018) supplemented with either 1.603% of potassium bicarbonate or
1.345% sodium bicarbonate, respectively, for seven days, and were sacrificed via CO2
asphyxiation on the seventh day of experimentation. The control mice were sacrificed via CO2
asphyxiation at different times compared to the other groups. The femurs were characterized for
mechanical, structural, compositional, and histological properties as described below. The
percentages of the potassium and sodium bicarbonate in the food were calculated based on mice
experiments done by Robey et al. [222–224] that used 200mM sodium bicarbonate in the drinking
water, which is equivalent to 3.2 g/kg/day in mice, along with drinking water consumption data
from a previous study in the lab [43].
79
Fig. 3.9. Methods for bicarbonate supplementation and acidosis induction. These schematics show
how the experiments for the (A) control group, (B) potassium bicarbonate only and sodium bicarbonate only
groups, and (C) acidosis plus either potassium bicarbonate or sodium bicarbonate supplementation groups
were conducted.
3.2.2.2 Blood and urine chemistries and assessment of acidosis and treatment
To determine if the mice were in metabolic acidosis, we measured various blood gas
parameters, such as blood pH and blood bicarbonate (HCO3-) levels (N=15 mice/group/day).
Blood was collected via submandibular bleeds [150]. 200-300 µL of blood was collected per
mouse at each time of collection. The samples were immediately tested using a Heska PoC Epoch
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blood-gas analyzer (Loveland, CO, USA). Baseline measurements were taken either seven days
before the start of the experiment (A+KHCO3 and A+NaHCO3) or the start of
experimentation/seven days before sacrifice (control, KHCO3-only, and NaHCO3-only). For
A+KHCO3 and A+NaHCO3 groups, blood was also collected on day 14 and day 21. For control,
KHCO3-only, and NaHCO3-only groups, blood was again collected on day of sacrifice. Other blood
gas parameters that were analyzed were ions, such as Ca2+, Na+, and Cl-. Urine pH was also
analyzed using pH strips with increments of 0.5. Baseline urine pH was collected at the same time
baseline blood gas was done. It was then collected starting at day 0 and then every 3 days until
sacrifice.
3.2.2.3 Assessment of bone molecular composition using Raman Spectroscopy
Raman spectroscopy was used to determine the compositional properties of the exterior
and interior of the femurs (N=3 femurs/group). A Witec alpha 300 Raman Spectrometer (Witec,
Ulm, Germany) with a laser wavelength of 785 nm, laser power of 65mW or less, an acquisition
time of 2 sec x 30, and an objective of 50X was used to obtain spectra. Before data acquisition,
the femurs for the exterior measurements were left in the carcass at -20°C, and then dissected,
cleaned of soft tissue, and wrapped in gauze soaked in 1X PBS at -20°C. Right before analysis,
the femurs were thawed, and the periosteum was removed using fine grained sandpaper. For the
internal measurement, the femurs were dissected and frozen in 30% sucrose at -80°C before
being embedded into Optimal Cutting Temperature (OCT) medium, frozen at -80°C, and then
cryosectioned along the coronal plane. For the external measurements, five spectra were taken
at the midshaft as well as the proximal and distal ends of the femur on the anterior side for a total
of 15 measurements. For the internal measurements, three spectra were taken at the proximal,
distal, and midshaft regions on the medial and lateral sides of the femur. Before fitting, the spectra
were cropped to a range of about 200cm-1-1800 cm-1 and had the background corrected and any
cosmic rays removed. The samples were then averaged based on location per sample. The
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spectra were then fitted assuming a Lorentzian shape function, as described in our previous
studies [43,44,209].
3.2.2.4 Whole-bone mechanical analysis
Three-point bend mechanical testing was performed to determine the mechanical
properties of the femoral samples (n=10 mice/group) [209]. After sacrifice, the mice were stored
at -20°C. On the day of testing, the femurs were dissected from the thawed mice and cleaned of
any soft tissue. The femurs were then placed in a Biomomentum Mach-1 (v500csst with a 3-axis
motion controller) 3pt bend apparatus in 1X phosphate buffered saline heated up to 37°C using a
small portable immersion heater. Each sample was tested a rate of 0.1 mm/s using a 25kg load
cell. MATLAB custom code was used to convert force vs. displacement curves to stress vs. strain
curves by normalizing to the span length, bone area moment of inertia, and bone centroid
distance. Additionally, this code was able to calculate structural mechanical properties, such as
maximum load, stiffness and energy to fracture, and material mechanical properties, such as
maximum stress, modulus, and toughness.
3.2.2.5 Structural analysis
After mechanical testing, microcomputed tomography (µCT) was performed on the femurs
to determine any structural changes due to concurrent acidosis induction and bicarbonate
treatment (n=10 mice/group). Samples were first dehydrated in increasing concentrations of
ethanol up to 70%. After dehydration, µCT data was collected using a Scanco 40 µCT system
(Scanco Medical, Bruttisellen, Switzerland) at a resolution of 16 µm. From the µCT scans, cortical,
trabecular, and fracture area data was obtained, as done previously [43,44,209]. Bruker CTan
program was used to calculate the cortical and trabecular parameters of the femurs. For cortical
bone analysis, the region of interest was determined by selecting a µCT slice that was about 345
slices away from the distal tip of the femur and then analyzing 100 slices proximal to that slice.
82
For the trabecular bone, both the distal shaft and the distal condyles were analyzed. The region
of interest for the distal shaft trabecular bone was 100 slices proximal to the growth plate. The
region of interest for the condyle trabecular bone was between the tip of the distal end of the
femur and the start of the growth plate. BoneJ, a plug-in ImageJ was used to determine the
moment of inertia and centroid distance around the fracture site [152]. The region of interest for
the fracture site measurements was 30 slices from each side of the fracture with an increment of
10 slices.
3.2.2.6 Bone apatite analysis
Five bones from each condition used in microcomputed tomography was further
dehydrated in a desiccator for seven days and then crushed with a mortar and pestle. Attenuated
Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy was used to determine
the amount of A-type, B-type, and labile carbonate in the femoral samples. For each sample, a
Nicolet Magna-IR 500 spectrometer was used by taking 64 scans with a resolution of 2 cm-1 from
4000-400 cm-1 range. Using a range of 900-750 cm-1, the A-type, B-type, and labile peaks in the
ν2 CO3 peak were fit using a Gaussian fit function with initial peak centers of 880 cm-1, 873 cm-1,
867 cm-1 [225]. Additionally, areas under the phosphate (1200 - 900 cm-1), amide I (1720 - 1585
cm-1), and carbonate (890 - 850 cm-1) peaks were calculated using the peak area tool from OMNIC
Spectra Software [226–228]. Before the areas were measured, the spectra were each baseline
corrected. These areas were then used to calculate mineral:matrix (phosphate:amide I) and
carbonate:phosphate peaks.
3.2.2.7 Ionic composition analysis
Using the samples from ATR-FTIR analysis, five femoral samples from each group were
analyzed using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) to
determine the amount of calcium (Ca), phosphorus (P), sodium (Na), and potassium (K) with in
83
the bone tissue. The samples were tested in a Perkin Elmer Optima 7300 Dual View ICP-OES at
the Center for Environmental Sciences and Engineering laboratory at the University of
Connecticut. Standard quality assurance procedures were utilized, such as initial and continuing
calibration checks and blanks, duplicate samples, preparation blanks, post digestion spiked
samples, and laboratory control samples.
3.2.2.8 Toluidine blue staining
Traditional non-fluorescent toluidine blue histological staining was performed on femurs
(n=3-5 femurs/group). After sacrifice, the femurs were immediately dissected and fixed in 4% PFA
at 4oC for 2 days. The femurs underwent demineralization using 14% EDTA, then dehydration via
graded ethanol washes prior to toluidine blue staining. Osteoblasts and osteoclasts in the
trabecular bone were analyzed using the OsteoMeasure (OsteoMetrics, Atlanta, GA, USA) image
analysis system. Data obtained from femur histology was reported according to the guidelines
published by the ASBMR, specifically quantifying osteoblast (Ob.N) or osteoclast (Oc.N) number
and osteoblast (Ob.S) or osteoclast (Oc.S) surface relative to measurements of bone mineral
[189].
3.2.2.9 Statistical analysis
Statistical analysis and graphical visualization were done in GraphPad Prism. Any
significant p values for comparisons between A+KHCO3 and NaHCO3-only and between
A+NaHCO3 and KHCO3-only were not included on graphs. For all statistical tests, we used a
significance level of 0.05. For all normally distributed data, comparisons between groups and
between day 0 and day 7 were made using one-way and two-way ANOVAs and post-hoc Tukey’s
tests. For data that was not normally distributed, non-parametric statistical tests were used. Data
that used non-parametric tests were blood pO2, amount of calcium and phosphorus determined
by ICP-OES, and mineral:matrix from interior Raman spectroscopy. Since statistical analysis of
84
the blood pO2 data was not possible in Graph Prism, R was used to determine any significance
using a Kruskal-Wallis test followed by a Dunn’s test. All data expressed within the graphs and
the text are the mean ± standard deviation.
3.2.3 Results
3.2.3.1 Confirmation of acidotic induction and bicarbonate administration
Before bicarbonate food was given to the mice (Day 0), the blood pH was significantly
lower in the A+KHCO3 (7.203 ± 0.131, p=0.0321) and A+NaHCO3 (7.169 ± 0.142, p=0.0026)
groups compared to the control (7.324 ± 0.094). The blood pH also decreased from KHCO3-only
to A+KHCO3 and from NaHCO3-only to A+NaHCO3 (Fig. 3.10A). After seven days of bicarbonate
food (Day 7), the blood pH remained lowered between KHCO3-only and A+KHCO3 and between
NaHCO3-only and A+NaHCO3 (Fig. 3.10A). Moreover, at Day 0, there was a significant decrease
in HCO3- for A+NaHCO3 (15.486 ± 5.430 mmol/L) compared to the control group (20.673 ± 4.783
mmol/L, p=0.0265) (Fig. 3.10B). This decrease was not maintained over the seven days; however,
there was a decrease in HCO3- for the A+KHCO3 compared to KHCO3-only (p=0.0052).
For the baseline measurements (Day 0), calcium (Ca2+) was higher in the
acidosis+bicarbonate groups compared to the control (Fig. 2C). On Day 7, there were no more
significant differences in Ca2+ between any of the groups. For sodium (Na+) at Day 0, there were
differences between the control group and the acidosis+bicarbonate groups. Additionally, the
acidosis+bicarbonate groups had higher Na+ than their respective bicarbonate-only groups. After
seven days of bicarbonate administration, the differences between the control and NaHCO3-only
groups with the A+NaHCO3 group were eliminated (Fig. 3.10D); however, Na+ remained elevated
in the A+KHCO3 (154.467 ± 2.588 mmol/L) group compared to the control (151.267 ± 2.120
mmol/L, p=0.0097) and KHCO3-only (150.5 mmol/L, p=0.0013) groups. There were no significant
differences between any of the groups for potassium (K+) at any of the time points (Fig. 3.10E).
85
At the beginning of the experiment (Day 0), the acidosis+bicarbonate groups had more
chlorine (Cl-) than the control and their respective bicarbonate only groups. After seven days of
bicarbonate food and continued acidosis induction, the A+NaHCO3 (124.25 ± 2.864 mmol/L)
group still had elevated Cl- levels compared to the control (120.286 ± 2.644 mmol/L, p=0.046) and
the NaHCO3-only (118.357 ± 3.177 mmol/L, p=0.0008) groups (Fig. 3.11F). For both time points,
there was a decrease in urine pH for the acidosis+bicarbonate groups compared to the control
and their respective bicarbonate-only groups (Fig. 3.11J). At Day 0, the control and bicarbonate-
only groups had higher body weights compared to the acidosis+bicarbonate groups. However,
with Day 7, the weights were all similar for all groups (Fig. 3.11K).
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Figure 3.10. Blood gas was partially rescued with bicarbonate supplementation. Some of the blood
gas parameters analyzed were (A) blood pH, (B) bicarbonate (HCO3-), (C) calcium (Ca2+), (D) sodium
(Na+), (E) potassium (K+), and (F) chlorine (Cl-). (G) A schematic showing the blood gas changes before
and after bicarbonate supplementation.
87
Figure 3.11. Additional blood gas parameters, which were (A) glucose, (B) base excess, (C) anion gap K,
(D) lactate, (E) partial pressure of oxygen, (F) partial pressure of carbon dioxide, (G) blood urea nitrogen,
(H) hematocrit, and (I) hemoglobin. Other parameters measured were (J) urine pH and (K) weight.
Day 0 Day 7
0
100
200
300
Glucose (mg/dL)
0.0317
Day 0 Day 7
0
5
10
15
20
Lactate (mmol/L)
Day 0 Day 7
0
10
20
30
40
BUN (mg/dL)
Day 0 Day 7
-30
-20
-10
0
10
BE(b) (mmol/L)
0.0389
0.0042
0.0140
0.0462
0.0465
0.0009
0.0405
Day 0 Day 7
0
50
100
150
pO
2
(mmHg)
Day 0 Day 7
0
10
20
30
40
50
Hct (%)
Day 0 Day 7
0
10
20
30
AGapK (mmol/L)
0.0044
0.0011
0.0375
Day 0 Day 7
0
20
40
60
pCO
2
(mmHg)
Day 0 Day 7
0
5
10
15
20
cHgb (g/dL)
Day 0 Day 7
5
6
7
8
Urine pH
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
Day 0 Day 7
20
30
40
50
60
Weight (g)
0.0011
<0.0001
0.0022
0.0006
A B C
D E F
G H I
J K
Control KHCO
3
only A + KHCO
3
NaHCO
3
only A + NaHCO
3
88
3.2.3.2 Assessment of internal and external bone tissue composition in response to bicarbonate
exposure
We used Raman spectroscopy to determine how bicarbonate administration impacted the
mineral composition of the femurs. On the exterior of the bone, there were not many changes
(Fig. 3.12) The mineral:matrix ratio was not different among any of the groups. However, the
carbonate:phosphate ratio (CO3:PO4) for the KHCO3-only group (0.258 ± 0.008022) significantly
increased compared to the A+KHCO3 group (0.02419 ± 0.007935, p=0.0251) (Fig. 3.12C).
Internally, there are no changes for any of the groups for mineral:matrix, CO3:PO4, and full width
half maximum (FWHM) of the 960 cm-1 peak (Fig. 3.13).
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Figure 3.12. Minimal changes in exterior compositional parameters of femurs. Raman spectroscopy
was conducted on (A) three locations of the anterior side of the femur. Measurements taken from Raman
was (B) mineral:matrix, (C) carbonate:phosphate, and (D) the full width half maximum of the 960 cm-1
peak. (E) A schematic showing the interactions between the acidosis induction and potassium
bicarbonate supplementation with the blood gas and Raman results.
Figure 3.13. Raman spectroscopy was measured (A) internally at three locations with six points at each
location. Interior Raman spectra were used to calculate (B) mineral:matrx, (C) carbonate:phosphate, and
(D) the full width half maximum of the 960 cm-1 peak.
3.2.3.3 Influence of bicarbonate on whole bone femur mechanics
To examine if bicarbonate alongside acidosis impacted the mechanical properties of bone,
3pt bend testing was performed. For all the materials and structural mechanical properties
analyzed, there were no differences found between any of the groups (Fig. 3.14).
A
B C D
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Fig. 3.14. The femurs were mechanically tested to calculate (A) maximum load, (B) maximum stress, (C)
yield load, (D) yield stress, (E) stiffness, (F) modulus, (G) work, and (H) toughness.
3.2.3.4 Potassium bicarbonate decreased trabecular bone in distal shaft
Control
KHCO3 only
A + KHCO3
NaHCO3 only
A + NaHCO3
0
10
20
30
40
50
Max. Load (N)
Control
KHCO3 only
A + KHCO3
NaHCO3 only
A + NaHCO3
0
10
20
30
40
50
Yield Load (N)
Control
KHCO3 only
A + KHCO3
NaHCO3 only
A + NaHCO3
0
50
100
150
200
Stiffness (N/mm)
Control
KHCO3 only
A + KHCO3
NaHCO3 only
A + NaHCO3
0
5
10
15
Work (N-mm)
Control
KHCO3 only
A + KHCO3
NaHCO3 only
A + NaHCO3
0
50
100
150
200
250
Max. Stress (MPa)
Control
KHCO3 only
A + KHCO3
NaHCO3 only
A + NaHCO3
0
50
100
150
200
250
Yield Stress (MPa)
Control
KHCO3 only
A + KHCO3
NaHCO3 only
A + NaHCO3
0
2
4
6
8
Modulus (GPa)
Control
KHCO3 only
A + KHCO3
NaHCO3 only
A + NaHCO3
0
2
4
6
8
10
Toughness (MPa)
AB
C
E
G H
F
D
91
Structural analysis was conducted on the distal epiphysis, the distal diaphysis, and the
midshaft cortical bone. Within the distal diaphysis (Fig. 3.15A-B), the A+KHCO3 group (8.597 ±
1.426 %) had a lower bone volume (BV/TV) compared to the KHCO3-only group (12.49 ± 2.618
%, p=0.0364) (Fig. 3.15C). The A+KHCO3 group also had an increased trabecular thickness
(Tb.Th.) and trabecular spacing (Tb.Sp.) and decreased trabecular number (Tb.N) compared to
the control (Fig. 3.15D-F). In addition, A+KHCO3 group (3.965 ± 0.4124 1/mm) had a lower Tb.N
compared to KHCO3-only (4.072 ± 0.2385 1/mm, p=0.0332) (Fig. 3.15D). In the epiphysis and
midshaft cortical bone, there were no differences seen between any of the groups (Fig. 3.16).
92
Figure 3.15. Trabecular bone loss observed with acidosis and potassium bicarbonate
suplementation. Trabecular structural measurements were calculated in the (A) distal shaft. (B)
Microcomputed tomography images were used to measure (C) bone volume to total volume (BV/TV), (D)
trabecular thickness (Tb.Th), (E) trabecular number (Tb.N), and (F) trabecular spacing (Tb.Sp.). (G) A
schematic showing the effect of acidosis and potassium bicarbonate treatment (A+KHCO3) on the
trabecular bone in the distal shaft compared to the control group.
93
Figure 3.16. Trabecular structural measurements were calculated in the (A) distal condyle. (B)
Microcomputed tomography images were used to measure (C) bone volume to total volume (BV/TV), (D)
trabecular thickness (Tb.Th), (E) trabecular number (Tb.N), and (F) trabecular spacing (Tb.Sp.). Cortical
structural measurements were calculated in the (G) diaphysis. (H) Microcomputed tomography images
were used to measure (I) cortical thickness.
3.2.3.5 Increase in sodium ions with potassium bicarbonate administration under acidotic
conditions
Crushed murine femurs were used in ICP-OES analysis to determine the levels of ions
under simultaneous acidotic induction and bicarbonate treatment (Fig. 3.17). The amount of
sodium (Na+) in the bones from the A+KHCO3 (4769 ± 181.2 µg/g) group increased compared to
Distal Condyle
Trabecular Bone
Growth plate
Control KHCO3only A + NaHCO3
A + KHCO3NaHCO3only
Cortical Bone
Control KHCO3only A + NaHCO3
A + KHCO3NaHCO3only
A B
CD E F
G
H
I
94
the control (4389 ± 215.9 µg/g, p=0.0274) (Fig. 3.17A). Additionally, the amount of potassium (K+)
in the bones from the A+KHCO3 (452.5 ± 65.77 µg/g) group decreased compared to the control
(562.6 ± 46.91 µg/g, p=0.0329) (Fig. 3.17B). There were no differences seen in calcium and
phosphorus between any of the groups (Fig. 3.17C-D).
Figure 3.17. Minimal changes to ionic composition of femurs. The ionic composition of the femurs
was analyzed using ICP-OES and measured levels of (A) sodium (Na), (B) potassium (K), (C) calcium
(Ca), and (D) phosphorus (P). (E) A schematic showing the interactions between the acidosis induction
and potassium bicarbonate supplementation with the blood gas, Raman, and ICP-OES results.
95
3.2.3.6 FTIR-measured mineral carbonate-type and mineral content were not altered
To examine if the type of carbonate in the bone mineral was being impacted by acidosis
coinciding with bicarbonate treatment, ATR-FTIR analysis was conducted. There were no
changes seen in the type of carbonate seen in the mineral based on wavenumber (Fig. 3.18A) or
normalized area (Fig. 3.18B). Additionally, there were no differences between any of the groups
with CO3:PO4 (Fig. 3.18C) and mineral:matrix (Fig. 3.18D), as measured by FTIR.
Figure 3.18. FTIR spectra were used to calculate (A) wavenumber and (B) normalized area for each
carbonate type (A, B, and labile) as well and (C) carbonate:phosphate and (D) mineral:matrix.
A-type B-typeLabile
855
860
865
870
875
880
885
Carbonate type
Wavenumber (cm-1)
Control
KHCO
3
only
A + KHCO
3
NaHCO
3
only
A + NaHCO
3
A-type B-typeLabile
0.0
0.2
0.4
0.6
Carbonate type
Normalized area
A
B
96
3.7 Cellular influence on bone due to acidosis and bicarbonate treatment
There were no changes in osteoclast perimeter (Oc.Pm/B.PM), osteoclast number
(N.OC/T.Ar. and N.Oc./B.Pm.), eroded surface (E.Pm/B.Pm), osteoblast perimeter
(Ob.Pm/B.Pm), or osteoblast number (N.Ob/T.Ar and N.Ob./B.Pm) when looking at toluidine blue-
stained histological sections for each group (Fig. 4.19).
Figure 3.19. (A) Toluidine blue stained slides of the femurs were used to determine (B) osteoclast
perimeter to bone perimeter, (C) number of osteoclasts to total area, (D) number of osteoclasts to bone
perimeter, (E) eroded perimeter to bone perimeter, (F) osteoblast perimeter to bone perimeter, (G)
number of osteoblasts to total area, and (H) number of osteoblasts to bone perimeter. Red arrows
indicate osteoblast.
3.2.4 Discussion
When patients present with metabolic acidosis (MA), physicians commonly give them
bicarbonate supplements to recover physiological blood pH and bicarbonate levels. Although MA
has been shown to negatively impact bone functionality, few studies have looked at the effect of
bicarbonate supplements on bone health after exposure to an acidic challenge. To help bridge
this gap, we examined the effects sodium bicarbonate (NaHCO3) and potassium bicarbonate
(KHCO3) on the bone of acidotic mice.
3.2.4.1 Acidosis mice underwent acid challenge before bicarbonate treatment
In this study, the mice readily consumed the NH4Cl solution over the course of 14 days
before the Day 0 measurements were made. Although fluid intake was not directly measured, the
mice in both acidosis groups exhibited increased blood Cl- levels compared to controls,
suggesting continued consumption of NH4Cl solutions. According to previous studies using this
NH4Cl graded murine model, it was expected that the mice would exhibit acidosis or acidemia by
Day 14 [44,209]. We found that at Day 0, before administration of any bicarbonates, both acidosis
groups exhibited reduced blood pH as compared to control groups confirming the induction of
97
acidemia, which is a reduction of blood pH. Metabolic acidosis is clinically characterized by a
decrease in both blood pH and HCO3- levels. Only the A+NaHCO3 group exhibited a decrease in
blood HCO3- as compared to control at Day 0. This suggests that acidosis was more strongly
induced in the A+NaHCO3 than the A+KHCO3 group at Day 0 and that the A+KHCO3 group was
in subclinical acidosis [44,160,209,213–215]. Due to variations in blood values, both clinical and
subclinical levels of acidosis have been measured using the NH4Cl graded murine model;
although, acidemia is always induced after 14 days [44,209]. In agreement with previous studies
of acidosis, both acidosis groups have increased levels of Ca2+ and Na+ in the blood compared to
controls, potentially indicating an occurrence of bone dissolution [30,88,115,116,170]. Overall, we
can confidently say that the acidosis groups are undergoing a systemic acid challenge before
administration of bicarbonates.
3.2.4.2 Bicarbonate administration only partially rescues acidosis/acidemia
Administration of NH4Cl has been associated with decreased animal body weight [43]. In
this study, the acidosis groups at Day 0 exhibited lower body weights than the control groups.
However, administration of bicarbonates recovered this body weight to control levels at Day 7,
suggesting that both NaHCO3 and KHCO3 improve general health in acid challenged mice. There
was no increase or decrease in blood pH or HCO3– between Day 0 and Day 7 for any of the
groups, in agreement with a study by Lemann et al. that found that blood pH was not altered due
to KHCO3 and NaHCO3 supplementation in healthy men [24]. This likely indicates that a higher
dose of bicarbonates may be needed to change the blood parameters back to normal levels.
However, administration of both bicarbonate types somewhat rescued the blood pH under acidotic
conditions compared to the control while remaining lower than the bicarbonate-only groups. This
suggests that bicarbonate administration raises the blood pH in the bicarbonate-only groups more
effectively than in the acidosis groups, where there remains a strong acid challenge. This seems
especially true in the A+KHCO3 group, where the blood HCO3- remained low and the Na+
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remained high as compared to the KHCO3 only group. This was not seen in the NaHCO3 groups
where any differences in blood HCO3- and Na+ between the A+NaHCO3 and NaHCO3-only groups
seen at Day 0 were resolved at Day 7. This suggests that NaHCO3 may be more effective at
rescuing acidosis than KHCO3. However, the continued increase in Cl- after seven days of
bicarbonate supplementation in the A+NaHCO3 group but not the A+KHCO3 group may point to
a higher intake of NH4Cl solution in the A+NaHCO3 group. This potential increase in NH4Cl water
consumption for the A+NaHCO3 group could be due to the increase in Na+ intake, leading them
to feel more dehydrated [229,230]. Further evidence that bicarbonate administration is improving
the acidemia/acidosis is seen by the rescue of blood Ca2+ levels in the acidosis groups compared
to controls. Increases in blood Ca2+ levels with acidosis are associated with bone dissolution
[30,88,115,116,170] and reduced calcium resorption in the renal tubule [231]. This recovery in
Ca2+ levels could therefore point to either reduced bone dissolution or enhanced kidney function
with bicarbonate administration.
3.2.4.3 Acidosis and bicarbonate treatments do not affect kidney function
To test for kidney function, blood urea nitrogen (BUN) was measured, and no changes
were observed as a function of treatment or time point. This suggests that neither the acidosis
nor the treatments significantly affected the kidney function. Further, urine pH, another indicator
of kidney function, remained low in the acidosis groups even with bicarbonate supplementation,
indicating that the kidneys were able to continue excreting acid from the NH4Cl administration. On
the other hand, both bicarbonate only groups increased in urine pH due to bicarbonate
supplementation, which is similar to a study that found that women and men had an increase in
urine pH when given KHCO3 [135]. Together these results suggest that the measured changes in
blood ion content are not primarily controlled by changes in kidney function but by changes in
bone dissolution.
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3.2.4.4 KHCO3 is less protective against bone dissolution than NaHCO3 likely due to increase
blood Na+
To analyze the impact of bicarbonate treatment on bone properties, we analyzed the blood
ion levels as well as the mechanical, compositional, structural, and cellular properties of the
femurs. In addition to the decrease in Ca2+ levels with treatment in the acidosis groups, the
administration of KHCO3 and NaHCO3 also reduced blood Na+ levels between days 0 and 7 in
the acidosis groups. Na+ is generally expected to increase with bone dissolution, again suggesting
that bicarbonate administration may play a role in reducing bone dissolution and recovery of bone
structure and function. Examination of bone structure, composition, and mechanical function
showed that there was no change in the NaHCO3 groups while the KHCO3 groups, which
maintained more severe acidosis effects even after treatment, exhibited compromised structural
and compositional parameters, but no mechanical changes. Analysis of the trabecular bone within
the femoral distal shaft shows that at Day 7, there is an increase in trabecular thickness and
trabecular spacing accompanied by a decrease in trabecular number for A+KHCO3 compared to
the control. This points to preferential loss of smaller trabeculae over larger trabeculae. As
described by Krieger et al., this could have been caused by either cellular resorption by
osteoclasts or physiochemical dissolution [27]. Increased blood Na+ as seen in the A+KHCO3
group has also been linked to increased osteoclast activity [63]. However, the lack of change in
histological osteoclast values suggests that, at the doses provided here, there is no cellular
involvement, and that the dissolution is primarily physiochemical. Both the type and level of bone
loss is similar to that seen after 14 days of NH4Cl administration [44,209]; therefore, this bone
loss suggests that the administration of KHCO3 was not sufficient to recover acidosis induced
damage over the course of 1 week. The A+NaHCO3 group was able to return to control levels,
suggesting that the enhanced effects of the bicarbonate treatment on the HCO3- and blood Na+
may have created an environment for bone recrystallization or growth.
100
Compositionally, the A+KHCO3 group exhibited decreased exterior CO3:PO4 ratio as well
as increased Na+ content. These again point to physiochemical interactions between the bone
and the blood/body fluids. Decreased pH as seen in acidemia/acidosis has been shown to
preferentially remove carbonate from bone apatite [26,45,232]. Therefore, the reduction in CO3
levels suggests once again that the A+KHCO3 group is still affected by the reduced pH, while
NaHCO3 is being more protective. This is of great interest as the A+KHCO3 mice exhibit the
highest levels of blood Na+. This correlation between high blood sodium, bone mineral loss, and
preferential removal of CO3 has been shown by us and others [26,233].
Although NaHCO3 was expected to increase blood Na+ levels, it failed to do so and instead
administration of KHCO3 lead to higher levels of blood Na+, resulting in increased bone loss and
CO3 dissolution. This reinforces the importance of understanding how the body regulates K+ and
Na+ levels concurrently. Finally, the A+KHCO3 group, with the high blood Na+, also exhibited
increased Na+ content in the bone mineral itself. We have shown that bone apatite readily uptakes
Na+ substitutions, leading to increased dissolutions rates [233,234]. Overall, these results suggest
that administration of KHCO3 was less successful in regulating acidemia/acidosis, resulting in
increased bone loss and modifications, as compared to NaHCO3.
Clinically, previous studies have suggested that high potassium diets are associated with
increased bone mineral density and improved bone structure, especially in women [62,235].
However, it is unclear in these studies if the benefits arise from increased K+ or the improved
quality of K+ containing foods and diets (leafy greens, fruits, etc.). Others have shown that
supplementation of potassium bicarbonate reduces biochemical markers of bone turnover and
reduced dietary calcium loss contrary to what was seen here [135,236]. This discrepancy may
arise from the presence of acidosis in the mice examined in this study compared to others, where
KHCO3 was provided to individuals without acid challenge. In acidosis, K+ levels become
dysregulated due to changes in ionic secretion and renal uptake [237], thus the effects of
potassium supplementation may not follow those seen in studies on healthy individuals. For
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example, administration of KHCO3 in this study failed to significantly change blood K+ levels while
elevating Na+ levels. Instead, it seems like blood K+ levels may decrease with acidosis. It is well
established that K+, Na+, and Cl- have a complex relationship in the body. Under the specific
conditions of this experiment, it seems that the concomitant administration of K+ and Cl- did not
result in elevated blood K+ but instead promoted the release of Na+. This reminds us of the
complex effects of ionic balance in pathological conditions like acidosis.
3.2.5 Limitations and future studies
This study was able to determine the impact of bicarbonate treatment on acidotic mice as
well as illustrate a difference between sodium bicarbonate and potassium bicarbonate. However,
some additional experiments and studies in the future will help bring further insight into this topic.
It would have been useful to have done a group with only acidosis for 21 days to compare this
elongated acidosis induction to the bicarbonate and acidosis groups. Moreover, it would have
been impactful to determine the urine levels of K+, Ca2+, and Na+ as well as serum bone
remodeling markers, such as procollagen type 1 N-terminal propeptide (P1NP) and crosslinked
C-telopeptide of type I collagen (CTX). Additionally, future studies could look at the combination
of bisphosphonate and bicarbonate in acidotic mice to see if using both treatments would help
reduce bone loss synergistically [44]. Varying the dosage of the bicarbonate in the food could also
be interesting to study to determine the optimal amount of bicarbonate needed to prevent bone
loss under acidotic conditions. Only a constant dose of NaHCO3 and KHCO3 was used in this
study because that was typically used in previous studies. However, clinically, there are multiple
ways of administration (tablets, injections, and infusions) for bicarbonate administration and
various doses can be used on one patient depending on the severity of their symptoms of
metabolic acidosis throughout their life [124]. So, using a single dose of bicarbonate is not entirely
reflective of the clinical setting. Additionally, this study exclusively used male mice, it would be
important to study the impact of bicarbonate treatment on acidotic female since acidosis affects
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male mice differently than female mice [238–241]. Lastly, there is currently no mechanism
describing the results obtained from this study. Further studies need to be conducted to determine
the mechanisms behind the relationship of Na/K in bicarbonate treatment for acidotic patients.
3.2.6 Conclusions
As expected, an acidic challenge was generated in both acidosis groups before
bicarbonate treatment using a previously established model. After bicarbonate treatment was
administered, the acidic challenge seen in the acidosis groups were partially rescued. However,
the A+KHCO3 group still seemed to be undergoing acidemia, as made evident by the unexpected,
continued increase in sodium content after seven days of KHCO3 administration. While the
A+NaHCO3 group had control level bone properties, the A+KHCO3 group had some changes
within the bone structure and composition there were indicative of bone dissolution. Even though
bone loss due to cellular changes might have been expected, there were no osteoclast or
osteoblast changes seen for any of the groups, leading us to believe the structural and
compositional alterations seen in the A+KHCO3 group were physiochemical. Moreover, since
exposure to sodium can lead to bone dissolution and integration into the bone, the imbalance of
sodium ions within the A+KHCO3 group might be culprit for bone loss seen in this group.
Conclusively, under an acid challenge, the bones from mice given NaHCO3 seemed to have
experienced a protective effect while bone from mice given KHCO3 experienced bone loss due to
an increase in sodium content in the blood. The differences seen between these two bicarbonate
treatments illustrates the importance of studying the effects of acidosis treatments on bone
function and health.
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Chapter 4: Sexual differences in potential protective mechanisms against acid-
mediated bone dissolution
4.1 Introduction
Like many studies within the medical field, most studies about metabolic acidosis are
conducted with a focus on men and male animals [242,243]. This mostly likely happens to reduce
any hormonal effects that might impair studies’ findings [244]. However, studies have shown that
female mice do not increase variability in data [239,242]. Despite these results, exclusively male
animals are typically used in studies and findings from male subjects are usually implemented on
women when it comes to diagnosis and treatment, leading to poor outcomes for women [140–
143]. Because of this, the NIH has mandated the use of female subjects and women in preclinical
studies [139].
Very few studies have researched sexual dimorphism in those with acidosis. One known
study that used a model of graded loading of ammonium chloride in the drinking water conducted
a transcriptomic analysis of cardiac tissue from male and female mice. In the female mice with
acidosis, they saw a lowered cardiac muscle contractility and estrogen response, while in the
male mice with acidosis, there was an increase in dilated cardiomyopathy [42]. Another study,
using hydrochloric acid in the food to induce acidosis, found that male and female mice have
different mechanisms of renal ammonia response to acid loading [138].
It is well established that sexual differences exist in healthy and osteoporotic patients in
terms of bone. These differences in bone start around ages sixteen to seventeen, where the bone
mineral content in men begins to be greater than in women. Additionally, bones from men were
shown to be stronger not because they have a higher bone mass, but because of how their bone
mass was distributed [245]. Osteoporosis, a metabolic bone disease that causes low bone mineral
density, also reveals the presence of sexual dimorphism of bone tissue. This disease tends to
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mainly affect postmenopausal women due to their longer lifespans and a sudden decrease in
estrogen levels compared to men [246]. Because bone health is sex-dependent, it is important to
establish how acidosis affects bone tissue in women.
In this study, acidosis was induced in male and female mice using an established model
of graded-dosing of ammonium chloride solution in the drinking water [42–45,209]. We found that
acidosis was induced in male mice, but much more difficult to induce in female mice. Additionally,
there were no phenotypical changes in bone using Raman spectroscopy, microcomputed
tomography, and flexural mechanics. However, using NanoString analysis, there was shown to
be sex- and time-dependent differences between male and female mice due to acidosis.
Additionally, these differences in genetic expression might have revealed a protective mechanism
against osteoclastic bone dissolution due to acid loading.
4.2 Methods and Materials
4.2.1 Diet-induced Metabolic Acidosis in Male and Female Mice
3–4-month-old, wild-type, CD-1 retired male breeders and 3–4-month-old, wild-type, CD-
1 virgin female mice from Charles River Laboratory were used in this study. This study was
approved by the Institutional Animal Care and Use Committee (IACUC) at the University of
Connecticut Health Center (UConn Health) and complied with the regulations set by the National
Institutes of Health (NIH). The male and female mice were split into six separate groups (9
mice/group/sex): Day 3 control, Day 3 acidosis, Day 7 control, Day 7 acidosis, Day 14 control,
and Day 14 acidosis. Each of the six groups were further split into three subgroups of A, B, and
C where 3 mice were in each subgroup. Acidosis induction for each subgroup started at the same
time. Each subgroup on their corresponding day (Day 3, 7 or 14) were sacrificed at the same
time. For example, on Day 7 of experiments/acidosis induction, twelve mice were sacrificed total
(three Day 7 control male mice, three Day 7 acidosis male mice, three Day 7 control female mice,
and three Day 7 acidosis female mice). Acidosis induction was done using the graded-dosing of
105
ammonium chloride (NH4Cl) model established in our lab [42–45,209]. The beginning dose was
0.2 M NH4Cl + 5% sucrose. Every three days, the dose of NH4Cl increased by 0.1 M ending at a
dose of 0.6 M NH4Cl + 5% sucrose by Day 12 and remaining constant until Day 14. The control
mice were given water provided by the UConn Health Center for Comparative Medicine. The
water of the control mice was also replaced every three days. Days 3, 7, and 14 control and
acidosis mice were sacrificed via carbon dioxide asphyxiation followed by cervical dislocation
after, respectively, three, seven, and fourteen days of acidosis induction. Mice were either then
frozen immediately at -20°C or their femurs were dissected and processed after euthanasia.
4.2.2 Blood Gas and Urine pH Data Collection
About 300 µL of blood was collected from non-anesthetized male and female mice (7-9
mice/group) on the day of sacrifice via submandibular bleeds using 5 mm lancets and the
technique developed by Golde et al [150]. Right after blood collection from an individual mouse,
a Heska Element Point of Care (Element POC) blood gas and electrolyte analyzer (Loveland, CO,
USA) was used to analyze the blood. An individual Heska Element POC card was used for each
mouse and calibrated before inserting the blood into the card. To stop the bleeding, a piece of
gauze was used on the site of collection. Urine was collected while holding the mouse. Sometimes
abdominal pressure was added for further collection. Urine pH was measured using Hydrion pH
strips (4.5-7.5) with increments of 0.5 pH units.
4.2.3 Weight, Food Consumption, and Water Consumption
Baseline mouse weights, food, and water were measured on the first day of acidosis
induction (Day 0) for each subgroup. Mouse weight and food were measured using a scale and
water was measured using a graduated cylinder. These measurements were taken every three
days and on the day of sacrifice. At the baseline and every three days afterwards, the acidosis
106
mice were given 200 mL of ammonium chloride + sucrose solution while the control mice were
given 200 mL of water in their fluid dispenser.
4.2.4 Three Point Bend Mechanical Testing of Femurs
Mice were thawed and femurs were dissected on the day of testing (8 femurs/group). Ten
to twenty femurs were dissected per testing day. A Biomomentum (Laval, Canada) Mach-1
Mechanical Tester (v500cst with a 3-axis motion controller) with a three-point bend set-up was
used to analyze the macroscale mechanics of the femoral samples. During the testing process,
the samples were placed in a 1X PBS bath and heated to 37°C using an immersion heater and
temperature controller. The femurs were placed on a three-point bend rig with an 8 mm span and
were tested with the posterior side facing upwards at a loading rate of 0.1 mm/s until failure. A 25
kg load cell was used to measure force, and displacement was measured from the stage
displacement. After testing, femurs were individually wrapped in gauze soaked with 1X PBS and
frozen at -20°C. Load vs. displacement curves were used to measure structural mechanical
properties. Stress vs. strain curves were calculated using the area moment of inertia, span length,
and centroid distance using custom MATLAB code. The area moment of inertia and centroid
distance were determined via microcomputed tomography, which is described below.
4.2.5 External and Internal Raman Spectroscopy of Femurs
After mechanical testing, half of the femur samples (4 femurs/group) were thawed at room
temperature and compositional analysis was done using a Witec Alpha 300 Raman Spectrometer
(Ulm, Germany). A 785 nm laser with a grating of 300 g/mm and a power of 65 mW or less was
used for data collection. Each spectrum was collected with a 50X objective, center wavelength of
about 887 nm, and an acquisition time of 2 seconds x 30 acquisitions. For external analysis, five
point measurements were taken on the anterior side of the femur samples at the distal, midshaft,
and proximal locations. The measurements were taken laterally to medially across the surface of
107
the bone. For internal analysis, six point measurements were taken on the distal fracture surface
as done in our previous study [44]. Using the Witec Program 5.3 software, the external and
internal spectra were cropped to 200 – 1800 cm-1, background corrected, and cleared of cosmic
rays. The five single point, external measurements for each sample were averaged together to
create a single spectrum for each location. The six single point, internal measurements for each
sample were also averaged together to create a single spectrum. Within the Witec Program 5.3
software, a Lorentzian shape function was used to fit each of the spectra to measure the peak
areas and full width half maximums (FWHM) of these three peaks: the 960 Δcm-1 ν1 phosphate
(PO4) peak, the 1003 Δcm-1 phenylalanine collagen peak, and the 1070 Δcm-1 carbonate peak
[155,247]. The mineral:matrix ratio was calculated from the ratio of the 960 Δcm-1 and 1003 Δcm-
1 peak areas. The carbonate:phosphate (CO3-:PO43-) ratio was calculated from the ratio of the
1070 Δcm-1 and 960 Δcm-1 peak areas.
4.2.6 Microcomputed Tomography of Femurs
After completing Raman spectroscopy data collection, all the femurs used for mechanical
testing (8 femurs/group) were then dehydrated in graded ethanol rinses (30%, 50%, 70%). These
samples were then placed in a holder using dental wax and imaged using a Scanco 50
microcomputed tomography (μCT) system (μCT 50, Scanco Medical, Bruttisellen, Switzerland) at
UConn Health’s MicroCT Imaging Facility. The scans were done at a 16 µm voxel size. The scans
were used to calculate the (1) fracture area parameters, (2) cortical parameters, and (3) trabecular
parameters. To measure centroid distance and area moment of inertia near the fracture site, four
μCT sections with an increment of 10 μCT sections on each side of the fracture were examined
using the BoneJ plug-in on ImageJ (U. S. National Institutes of Health, Bethesda, Maryland, USA)
[152]. The cortical thickness (Ct.Th) was measured by selecting a region of interest starting at the
beginning of the fracture on the distal side and ending at 100 μCT sections going towards the
condyles. Trabecular parameters were measured both in the distal epiphysis and in the distal
108
diaphysis. For the distal diaphysis, a region of interest containing 100 μCT sections proximal from
the end of the growth plate were analyzed. For the distal epiphysis, μCT sections were analyzed
between the start of the trabecular region on the distal end to the beginning of the growth plate.
The trabecular parameters obtained were bone volume/total volume (BV/TV), trabecular
thickness (Tb.Th), trabecular number (Tb.N), and trabecular spacing (Tb.Sp). Using the Bruker
CTAn software, both the cortical and trabecular segmentation procedures were used including
global thresholding, despeckling black spots less than 10 pixels from the images, ROI shrink-
wrapping, and morphological operations. Thresholding values were equal across all samples.
4.2.7 NanoString RNA Analysis of Femurs
On the day of each sacrifice, femurs used for NanoString analysis were immediately
dissected (5 samples/group). The ends were of the femurs were cut off and the shaft was cleared
of bone marrow using 1X PBS. Afterwards, the femurs were snap frozen in liquid nitrogen and
then frozen at -80°C. RNA was extracted by homogenizing the tissue in Trizol and phasing
separating from the organic phase using chloroform. The RNA was then purified using a Qiagen
RNeasy Mini Kit. A NanoDrop OneC (Thermo Fisher Scientific) was used to determine RNA
quantity and quality.
All RNA samples (all had an A260/A280 higher than 1.7) were analyzed using a
NanoString nCounter Analysis System at the Genomics Facility at Brown University. Before
analysis, all the samples were diluted to 10 ng/uL using nuclease-free water. The RNA was
quantified using selection of 97 genes (NanoString Osteoporosis Panel with the addition of
DMP1).
4.2.8 Statistical Analysis
Using Prism 10, two-way ANOVAS with post-hoc Tukey’s and Sidak’s multiple comparison
tests were used to compare the control and acidosis groups for both the male and female mice
109
for each day (Days 3,7, and 14). Normality of the data was checked using QQ plots. A p-value of
less than 0.05 was used to determine significance. All of graphs are mean ± standard deviation.
4.3 Results
4.3.1 Sexual Differences in Blood Gas Analysis
To monitor the health of the mice as well as determine whether the acidosis induction was
successful, blood gas data was collected on the day of sacrifice for each of the mice. The
differences in blood gas parameters between the male and female mice fluctuated throughout the
fourteen day of acidosis induction. For blood pH, there was a difference between the male
acidosis and female acidosis groups at Day 3 that disappeared at Day 7 and reappeared by Day
14 (Figs. 4.1A-C). There was also a difference between these two groups at Day 14 with the blood
bicarbonate levels (cHCO3-) (Fig. 4.1F). There was a significant decrease in blood pH and
bicarbonate levels between the male control and male acidosis groups at Day 14 (Fig 4.1C), but
there was only a significant decrease in blood bicarbonate levels for the female acidosis group
compared to its respective control at Day 14 (Fig. 4.1F). There was not an increase in calcium
levels until Day 7 for both males and females with acidosis, which continued into Day 14 (Figs
4.1H-I). Moreover, sodium levels started to increase in male mice with acidosis at Day 7 (Fig.
4.1K), but female mice with acidosis did not see an increase in sodium until Day 14 (Fig. 4.1L).
There were also sexual differences in other blood gas parameters over the span of
fourteen days. At Day 14, the female acidosis group had a higher total carbon dioxide (TCO2)
than the male acidosis group. At this same day, there was significant decrease in TCO2 in the
male acidosis group compared its respective control (Fig. 4.2F). Chlorine (Cl-) significantly
increased with the male acidosis and female acidosis groups starting at Day 7 continuing into Day
14, with a difference between the two acidosis groups on Day 14 (Fig. 4.2I). For Days 3 and 14,
there was a decrease in lactate between the both the males and females for the control groups
110
and acidosis groups (Figs 4.2J and 4.2L). This difference in lactate amount between the male and
female mice was also present on Day 7 in the control groups (Fig. 4.2K).
111
Figure 4.1. Acidosis was induced in male mice, but not female mice, after 14 days. Various blood gas
parameters that were measured include (A-C) blood pH, (D-F) bicarbonate (cHCO3-), (G-I) calcium (Ca2+),
and (J-L) sodium (Na+). The left column is all from Day 3, the middle column is Day 7, and the right column
is Day 14.
Control Acidosis
6.0
6.5
7.0
7.5
8.0
Blood pH
Day 3
0.0019
0.0281
Control Acidosis
6.0
6.5
7.0
7.5
8.0
Blood pH
Day 7
0.0003
Control Acidosis
6.0
6.5
7.0
7.5
8.0
Blood pH
Day 14
Male
Female
0.0002
<0.0001
0.0040
Control Acidosis
0
10
20
30
cHCO3- (mmol/L)
0.0181
Control Female
0
10
20
30
cHCO3- (mmol/L)
0.0011
Control Acidosis
0
10
20
30
cHCO3- (mmol/L)
0.0002
<0.0001
0.0284
0.0176
Control Acidosis
0.0
0.5
1.0
1.5
2.0
Ca2+ (mmol/L)
Control Acidosis
0.0
0.5
1.0
1.5
Ca2+ (mmol/L)
<0.0001
0.0007
<0.0001
0.0172
Control Acidosis
0.0
0.5
1.0
1.5
Ca2+ (mmol/L)
<0.0001
<0.0001
<0.0001
<0.0001
Control Acidosis
120
140
160
180
Na+ (mmol/L)
Control Acidosis
120
140
160
180
Na+ (mmol/L)
0.0452
0.0031
Control Acidosis
120
140
160
180
Na+ (mmol/L)
0.0004
<0.0001
0.0013
0.0182
AB C
D FE
G IH
KJ L
112
Figure 4.2. Sexual dimorphism in other blood gas parameters. These other blood gas parameters
include (A-C) partial pressure of oxygen (pO2), (D-F) total carbon dioxide (TCO2), (G-I) chlorine (Cl-), and
(J-L) lactate. The left column is all from Day 3, the middle column is Day 7, and the right column is Day 14.
113
In order to determine the functionality of the kidneys during acidosis induction, blood urea
nitrogen (BUN), base excess (BE), and anion gap, K (AGapK) were calculated. For BUN, there
were not many differences seen between male and female mice (Fig. 4.3A-C). However, base
excess of the blood (BE(b)) and base excess of the extracellular fluid (BE(ecf)) had some
significant differences. For both BE(b) and BE(ecf), the female acidosis was higher than the male
acidosis group at Day 3 and Day 14 (Figs 4.3G and 4.3I). Additionally, for both base excess
parameters, the male acidosis group was lower than the male control groups for Days 7 and 14
(Figs 4.3E and 4.3H). On the other hand, acidosis in the female mice only resulted in an decrease
in BE(ecf), but not BE(b), at Day 14 (Fig. 4.3F and 4.3I). There were no changes in AGapK due
to acidosis, but the AGapK in the male acidosis group was higher than the female acidosis group
at Day 14 (Fig. 4.3L). All the blood gas analysis results can be found in Table 4.1.
114
Figure 4.3. Male acidosis mice had a base excess than female acidosis mice. Other blood gas
parameters measured include (A-C) blood urea nitrogen (BUN), (D-F) base excess of the blood (BE(b)) (G-
I) base excess of extracelluar fluid (BE(ecf)) and (J-L) anion gap, K (AGapK). The left column is all from
Day 3, the middle column is Day 7, and the right column is Day 14.
115
Table 4.1. Table detailing of the blood gas data for each day, condtion, and sex.
116
4.3.2 Sexual Dimorphism in Weight, Urine pH, and Food and Water Consumption
Weight data and urine pH data was gathered to ensure the mice were still healthy while
undergoing acidosis. Female mice weighed less than male mice for both the control and acidosis
groups for all the days (Fig. 4.4). Additionally, the female acidosis group weighed less than the
female control group on Day 14 (Fig. 4.4G). For urine pH, female acidosis mice had more acidic
urine than male acidosis mice at Day 0 (Fig. 4.5A). However, this difference disappeared after
Day 3. There was a difference in urine pH between the male control and female control groups
on Day 3, with the female control group having a lower urine pH (Fig. 4.5B). For all the days,
except Day 0, the urine pH for the male acidosis was lower than in their respective controls (Figs
4.5B-G). This is almost true for the female acidosis mice with an exception on Day 12 (Fig. 4.5F).
Figure 4.4. Female weigh less than male mice. Mice were weighed every three days during
experimentations and on the day of sacrifice. These days include (A) Day 0, (B) Day 3, (C) Day 6, (D) Day
7, (E) Day 9, (F) Day 12, and (G) Day 14.
ControlAcidosis
0
20
40
60
Day 0
Weight (g)
<0.0001
<0.0001
<0.0001
<0.0001
ControlAcidosis
0
20
40
60
Day 3
Weight (g)
<0.0001
<0.0001
<0.0001
<0.0001
ControlAcidosis
0
20
40
60
Day 6
Weight (g)
<0.0001
<0.0001
<0.0001
<0.0001
ControlAcidosis
0
20
40
60
Day 7
Weight (g)
Male
Female
<0.0001
<0.0001
<0.0001
<0.0001
ControlAcidosis
0
20
40
60
Day 9
Weight (g)
<0.0001
<0.0001
<0.0001
<0.0001
ControlAcidosis
0
20
40
60
Day 12
Weight (g)
<0.0001
<0.0001
<0.0001
<0.0001
ControlAcidosis
0
20
40
60
Day 14
Weight (g)
<0.0001
<0.0001
<0.0001
0.0114
<0.0001
AB C D
E GF
117
Figure 4.5. Urine pH decreased with acidosis in both male and female mice. Urine pH was measured
every three days during experimentations and on the day of sacrifice. These days include (A) Day 0, (B)
Day 3, (C) Day 6, (D) Day 7, (E) Day 9, (F) Day 12, and (G) Day 14.
Male mice did not have any changes in their eating or drinking habits due to acidosis
induction over the span of fourteen days. However, female mice did drink and eat less due to
acidosis induction. For the Day 7 timepoint, the female mice ate less between Day 3 and Day 6
and Day 6 and Day 7 (Fig 4.6D). They also drank less of the ammonium chloride liquid compared
to regular water between Day 3 and Day 6 (Fig. 4.7D). For the Day 14 timepoint, there was similar
trend as Day 7 where the female acidosis mice consumed less food between Day 3 and Day 14
(Fig. 4.6F) and drank less at all days compared the female control mice (Fig. 4.7F).
Control Acidosis
5
6
7
8
Urine pH
Day 0
0.0468
0.0049
Control Acidosis
5
6
7
8
Urine pH
Day 3
0.0022
<0.0001
<0.0001
<0.0001
<0.0001
Control Acidosis
5
6
7
8
Urine pH
Day 6
<0.0001
<0.0001
<0.0001
<0.0001
Control Acidosis
5
6
7
8
Urine pH
Day 7
Male
Female
0.0030
0.0048
0.0013
0.0022
Control Acidosis
5
6
7
8
Urine pH
Day 9
0.0058
<0.0001
0.0015
Control Acidosis
5
6
7
8
Urine pH
Day 12
0.0069
0.0078
Control Acidosis
5
6
7
8
Urine pH
Day 14
0.0004
0.0016
0.0003
0.0013
AB C D
E F G
118
Figure 4.6. Female mice ate less food when under acidotic conditions. Food consumption data for (A-
B) mice sacrificed on Day 3, (C-D) mice sacrificed on Day 7, and (E-F) mice sacrificed on Day 14.
119
Figure 4.7. Female mice drank less of the NH4CL solution than water. Fluid consumption data for (A-
B) mice sacrificed on Day 3, (C-D) mice sacrificed on Day 7, and (E-F) mice sacrificed on Day 14.
Control
Acidosis
0
5
10
15
Fluid Consumed (mL/mouse/day)
Male
Day 3 - Day 0
Day 6 - Day 3
Day 9 - Day 6
Day 12 - Day 9
Day 14 - Day 12
0
5
10
15
Fluid Consumed (mL/mouse/day)
Male
0.0014
Day 3 - Day 0
Day 6 - Day 3
Day 7 - Day 6
0
5
10
15
20
25
Fluid Consumed (mL/mouse/day)
Male
Control
Acidosis
0
5
10
15
Fluid Consumed (mL/mouse/day)
Female
Day 3 - Day 0
Day 6 - Day 3
Day 7 - Day 6
0
5
10
15
20
25
Fluid Consumed (mL/mouse/day)
Female
0.0007
Control
Acidosis
Day 3 - Day 0
Day 6 - Day 3
Day 9 - Day 6
Day 12 - Day 9
Day 14 - Day 12
0
5
10
15
Fluid Consumed (mL/mouse/day)
Female
<0.0001 0.0380 0.0362 0.0059 0.0077
Day 3
Day 7
Day 14
A B
DC
FE
120
Figure 4.8. Sexual dimorphism in drinking and eating habits under acidosis conditions. This data is
from the Day 14 group of male and female mice, showing (A) fluid consumption under control conditions,
(B) fluid consumption under acidosis conditions, (C) food consumption under control conditions, and (D)
food consumption under acidosis conditions.
Between Days 3 and 6, the male control mice consumed more fluid than the female control
mice for the Day 14 groups (Fig. 4.8A). However, at all the days, the male acidosis mice
consumed more fluid than the female acidosis mice (Fig. 4.8B). This trend could also be seen in
the food consumption data. Between Days 3 and 6, and Days 6 and 9, the male control mice ate
more food than the female control mice for the Day 14 groups (Fig. 4.8C). However, at all the
days, the male acidosis mice ate more food than the female acidosis mice (Fig. 4.8D).
Day 3 - Day 0
Day 6 - Day 3
Day 9 - Day 6
Day 12 - Day 9
Day 14 - Day 12
0
2
4
6
8
10
Fluid Consumed (mL/mouse/day)
Control (Day 14)
0.0024
Day 3 - Day 0
Day 6 - Day 3
Day 9 - Day 6
Day 12 - Day 9
Day 14 - Day 12
0
5
10
15
Fluid Consumed (mL/mouse/day)
Acidosis (Day 14)
Male
Female
<0.0001 <0.0001 <0.0001 0.0033 0.0002
Day 3 - Day 0
Day 6 - Day 3
Day 9 - Day 6
Day 12 - Day 9
Day 14 - Day 12
0
5
10
15
Food Consumed (g/mouse/day)
Control (Day 14)
0.0236 0.0360
Day 3 - Day 0
Day 6 - Day 3
Day 9 - Day 6
Day 12 - Day 9
Day 14 - Day 12
0
5
10
15
Food Consumed (g/mouse/day)
Acidosis (Day 14)
<0.0001 <0.0001 <0.0001 0.0026 0.0027
AB
C D
121
4.3.3 No sexual differences in mineral composition via Raman spectroscopy
There was no difference seen in the mineral:matrix ratio, carbonate:phosphate ratio, and
the FWHM of the 960 cm-1 peak between the sexes and conditions for internal and external
measurements (Figs. 4.9 and 4.10).
Figure 4.9. Lack of changes in the internal composition of male and female femurs exposed to acid.
Raman spectroscopy was used to measure the (A-C) mineral to matrix ratio, (D-F) carbonate to phosphate
ratio (CO3:PO4), and (G-I) full width half maximum (FWHM) of the 960 cm-1 peak. The left column is all from
Day 3, the middle column is Day 7, and the right column is Day 14.
Control Acidosis
0
20
40
60
80
Mineral:matrix
Day 3
Control Acidosis
0.0
0.1
0.2
0.3
0.4
CO3:PO4
Control Acidosis
0
5
10
15
20
25
960 peak FWHM (Dcm-1)
Control Acidosis
0
20
40
60
80
Day 7
Mineral:matrix
Control Acidosis
0.0
0.1
0.2
0.3
0.4
CO3:PO4
Control Acidosis
0
5
10
15
20
25
960 peak FWHM (Dcm-1)
Control Acidosis
0
20
40
60
80
Day 14
Mineral:matrix
Control Acidosis
0.0
0.1
0.2
0.3
0.4
CO3:PO4
Male
Female
Contrrol Acidosis
0
5
10
15
20
25
960 peak FWHM (Dcm-1)
ACB
D FE
G IH
122
Figure. 4.10. Lack of changes in the external composition of male and female femurs exposed to
acid. Raman spectroscopy was used to measure the (A-C) mineral to matrix ratio, (D-F) carbonate to
phosphate ratio (CO3:PO4), and (G-I) full width half maximum (FWHM) of the 960 cm-1 peak. The left column
is all from Day 3, the middle column is Day 7, and the right column is Day 14.
4.3.4 Sexual differences in trabecular bone, but not cortical bone
C
o
n
t
rol
KH C
O
3
only
A
+
KH CO3
NaHCO
3
only
A
+
N
a
H
CO3
-0.5
0.0
0.5
1.0
1.5
2.0
Oc.Pm/B.Pm (%)
C
on
t
ro
l
KH C
O
3
only
A
+
KH CO3
NaHCO
3
only
A
+
N
a
H
C
O3
0.0
0.5
1.0
1.5
2.0
N.Oc/B.Pm (µm-1)
C
o
n
t
rol
KH C
O3 only
A
+ KH C
O
3
NaHCO
3
only
A
+ N
aHC
O
3
0
1
2
3
4
Ob.Pm/B.Pm (%)
C
o
n
t
rol
KH CO3
only
A
+
KH C
O
3
NaHCO
3
only
A +
Na
H
C
O
3
0
2
4
6
8
10
N.Ob/B.Pm
C
o
nt
rol
KH CO
3
only
A
+
KH C
O3
NaHCO 3 only
A +
N
a
H
C
O
3
-2
0
2
4
6
8
N.Oc/T.Ar (mm-2)
Cont
ro
l
KH
C
O
3
only
A
+
KH
C
O
3
NaHCO
3 only
A
+ N
a
H
C
O3
0.0
0.5
1.0
1.5
E.Pm/B.Pm (%)
C
o
n
tro
l
KH C
O
3
only
A
+ KH CO3
NaHCO
3
only
A
+
N
aHC
O
3
0
10
20
30
40
N.Ob/T.Ar
Control KHCO3only A + KHCO3
NaHCO3only A + NaHCO3
A
CB D E
F G H
Control Acidosis
0
20
40
60
Day 3
Mineral:matrix
Control Acidosis
0.0
0.1
0.2
0.3
CO3:PO4
Control Acidosis
0
5
10
15
20
960 peak FWHM (Dcm-1)
Control Acidosis
0
10
20
30
40
50
Mineral:matrix
Day 7
Control Acidosis
0
5
10
15
20
960 peak FWHM (Dcm-1)
Control Acidosis
0.0
0.1
0.2
0.3
CO3:PO4
Control Acidosis
0
10
20
30
40
50
Day 14
Mineral:matrix
Control Acidosis
0.0
0.1
0.2
0.3
CO3:PO4
Male
Female
Control Acidosis
0
5
10
15
20
960 peak FWHM (Dcm-1)
A B C
ED F
IHG
123
For Days 3 and 7, the male mice had more trabecular bone volume in the condyles than
the female mice, but by Day 14, this difference is no longer significant (Figs. 4.11A-C). There
were minimal differences in trabecular thickness within the condyles, except at Day 14 between
the control groups (Figs. 4.11D-F). At all days, the female mice had a lower number of trabeculae
in the condyles than the male mice for both the control and acidosis groups (Figs. 4.11G-I). Lastly,
the spacing between the trabeculae was bigger in the female mice compared to the male mice
for Days 3 and 7, but not at Day 14 (Figs. 4.11J-L).
Within the distal shaft, similar trends were seen in the trabecular bone between the male
and female mice as seen in the condyles. The female mice had a lower trabecular bone volume
compared to the male mice in only the control group at Day 3 (Fig. 4.12A). This significant
difference in trabecular BV/TV continued at Day 7 for both the control and acidosis groups, but
then the BV/TV leveled out between the male and female mice for both conditions (Figs. 4.12B-
C). The trabeculae in the acidosis group were thicker in the female mice than the male mice at
Days 3 and 14 but were thicker in the control group in the female mice for Day 7 (Figs. 4.12D-F).
The male control and acidosis mice had more trabeculae compared to the female control and
acidosis mice for Days 3 and 7, but this was only evident in the control group at Day 14 (Figs.
4.12G-I). Lastly, within the distal shaft, there was more spacing between the trabeculae in the
female control mice compared the male mice at Day 3 (Fig. 4.12J). This significant difference
continued onto Day 7 including a difference between the male and female mice in the acidosis
group. However, these differences became insignificant by Day 14 (Figs. 4.12K-L).
There were no changes in the cortical thickness due to sexual differences and acidosis
(Figs. 4.13A-C).
124
Control Acidosis
0
20
40
60
80
Day 3
BV/TV (%)
0.0074
0.0015
0.0139
Control Acidosis
0
20
40
60
80
BV/TV (%)
Day 7
0.0002
<0.0001
0.0018
0.0006
Control Acidosis
0
20
40
60
80
Day 14
BV/TV (%)
Male
Female
Control Acidosis
0.00
0.05
0.10
0.15
0.20
Tb.Th (mm)
Control Acidosis
0.00
0.05
0.10
0.15
0.20
Tb.Th (mm)
Control Acidosis
0.00
0.05
0.10
0.15
0.20
Tb.Th (mm)
0.0420
Control Acidosis
0
2
4
6
Tb.N (1/mm)
0.0119
0.0005
0.0047
Control Acidosis
0
2
4
6
Tb.N (1/mm)
<0.0001
<0.0001
<0.0001
<0.0001
Control Acidosis
0
2
4
6
Tb.N (1/mm)
0.0241
0.0062
0.0443
Control Acidosis
0.0
0.1
0.2
0.3
Tb.Sp (mm)
0.0067
0.0026
0.0030
0.0012
Control Acidosis
0.0
0.1
0.2
0.3
Tb.Sp (mm)
0.0064
0.0037
0.0055
0.0031
Control Acidosis
0.0
0.1
0.2
0.3
Tb.Sp (mm)
0.0392
ACB
FD E
IHG
J K L
125
Figure 4.11. Female mice have less trabecular bone than male mice within their condyles. The
trabecular parameters measured were (A-C) bone volume to total volume (BV/TV), (D-F) trabecular
thickness (Tb.Th), (G-I) trabecular number (Tb.N), and (J-L) trabecular spacing (Tb.Sp). The left column is
all from Day 3, the middle column is Day 7, and the right column is Day 14.
126
Figure 4.12. Female mice have less trabecular bone than male mice within the distal shaft. The
trabecular parameters measured were (A-C) bone volume to total volume (BV/TV), (D-F) trabecular
thickness (Tb.Th), (G-I) trabecular number (Tb.N), and (J-L) trabecular spacing (Tb.Sp). The left column is
all from Day 3, the middle column is Day 7, and the right column is Day 14.
Control Acidosis
0
10
20
30
40
Day 3
BV/TV (%)
0.0061
0.0117
Control Acidosis
0
10
20
30
40
Day 7
BV/TV (%)
<0.0001
<0.0001
0.0049
<0.0001
Control Acidosis
0
10
20
30
40
BV/TV (%)
Day 14
Male
Female
0.0115
Control Acidosis
0.00
0.05
0.10
0.15
Tb.Th (mm)
0.0106
0.0377
0.0027
Control Acidosis
0.00
0.05
0.10
0.15
Tb.Th (mm)
0.0132
0.0047
Control Acidosis
0.00
0.05
0.10
0.15
Tb.Th (mm)
0.0085
0.0032
0.0102
0.0039
Control Acidosis
0
1
2
3
4
5
Tb.N (1/mm)
0.0006
0.0135
0.0007
0.0151
Control Acidoiss
0
1
2
3
4
5
Tb.N (1/mm)
<0.0001
<0.0001
<0.0001
<0.0001
Control Acidosis
0
1
2
3
4
5
Tb.N (1/mm)
0.0063
0.0009
Control Acidosis
0.0
0.2
0.4
0.6
0.8
1.0
Tb.Sp (mm)
0.0076
0.0011
Control Acidosis
0.0
0.2
0.4
0.6
0.8
1.0
Tb.Sp (mm)
0.0005
<0.0001
0.0007
<0.0001
Control Acidosis
0.0
0.2
0.4
0.6
0.8
1.0
Tb.Sp (mm)
AB C
FED
H IG
J K L
127
Figure 4.13. No differences in cortical thickness between male and female mice. Cortical thickness
(Ct.Th.) was calculated for (A) Day 3, (B) Day 7, and (C) Day 14 of acidosis induction.
4.3.5 Differences in mechanical properties of femurs between male and female mice
In order to determine if acidosis affects both the mechanical properties of bone in male
and female mice, three-point bend mechanical testing was conducted on their femurs. There are
structural properties that impact the mechanical properties. Female mice have a lower centroid
distance, moment of inertia, and outer diameter than male mice for both the control and acidosis
groups (Figs. 4.14A-I). However, male and female mice have similar lengths with or without
acidosis (Figs. 4.14J-L).
When it comes to mechanical properties dependent on structure, there were time-
dependent differences between the male and female mice. For Days 3 and 7, femurs from the
female mice had a lower maximum and yield loads for both the control and acidosis groups (Figs.
4.15A-B and Figs. 4.15D-E). However, this disappeared by Day 14, and there was only a
difference between male acidosis and female acidosis groups for maximum and yield load (Fig.
4.15C and 4.15F). The female acidosis mice had lower work than male acidosis mice for only Day
3 (Fig. 4.15G). For the control and acidosis groups, work continued to be lower in the female mice
compared to the male mice at Day 7 (Fig. 4.15H). These differences in work diminished by Day
14 (Fig 4.15I). There were no differences seen with femur stiffness between condition and sex
(Figs. 4.15J-L).
Control Acidosis
0.0
0.1
0.2
0.3
0.4
0.5
Day 3
Ct.Th (mm)
Control Acidosis
0.0
0.1
0.2
0.3
0.4
0.5
Day 7
Ct.Th (mm)
Control Acidosis
0.0
0.1
0.2
0.3
0.4
0.5
Ct.Th (mm)
Day 14
Male
Female
ACB
128
Figure 4.14. Structural parameters of femurs for male and female mice. The structural parameters
measured were (A-C) centroid distance, (D-F) moment of inertia, (G-I) outer diameter, and (J-l) length. The
left column is all from Day 3, the middle column is Day 7, and the right column is Day 14.
Control Acidosis
0.0
0.2
0.4
0.6
0.8
1.0
Centroid Distance (mm)
Day 3
0.0012
0.0010
0.0115
0.0095
Control Acidosis
0.0
0.2
0.4
0.6
0.8
1.0
Day 7
Centroid Distance (mm)
0.0003
<0.0001
0.0154
0.0004
Control Acidosis
0.0
0.2
0.4
0.6
0.8
1.0
Day 14
Centroid Distance (mm)
Male
Female
0.0004
0.0001
<0.0001
Control Acidosis
0.0
0.1
0.2
0.3
0.4
0.5
Moment of Inertia (mm4)
0.0002
0.0003
0.0005
0.0007
Control Acidosis
0.0
0.1
0.2
0.3
0.4
0.5
Moment of Inertia (mm4)
<0.0001
<0.0001
0.0006
<0.0001
Control Acidosis
0.0
0.1
0.2
0.3
0.4
0.5
Moment of Inertia (mm4)
0.0065
0.0002
<0.0001
<0.0001
Control Acidosis
0.0
0.5
1.0
1.5
2.0
2.5
Outer Diameter (mm)
0.0002
0.0001
<0.0001
<0.0001
Control Acidosis
0.0
0.5
1.0
1.5
2.0
2.5
Outer Diameter (mm)
<0.0001
<0.0001
<0.0001
<0.0001
Control Acidosis
0.0
0.5
1.0
1.5
2.0
2.5
Outer Diameter (mm)
0.0150
<0.0001
<0.0001
<0.0001
Control Acidosis
0
5
10
15
20
Length (mm)
Control Acidosis
0
5
10
15
20
Length (mm)
Control Acidosis
0
5
10
15
20
Length (mm)
A CB
E FD
G IH
K LJ
129
Figure 4.15. Structural mechanical properties of femurs for male and female mice. The structural
parameters measured were (A-C) maximum load, (D-F) yield load, (G-I) work, and (J-l) stiffness. The left
column is all from Day 3, the middle column is Day 7, and the right column is Day 14.
ControlAcidosis
0
10
20
30
40
50
Day 3
Max. Load (N)
<0.0001
<0.0001
<0.0001
<0.0001
ControlAcidosis
0
10
20
30
40
50
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Max. Load (N)
<0.0001
<0.0001
0.0002
<0.0001
ControlAcidosis
0
10
20
30
40
50
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Max. Load (N)
Male
Female
0.0193
0.0019
0.0004
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0
10
20
30
40
50
Yield Load (N)
0.0012
0.0018
0.0001
0.0002
ControlAcidosis
0
10
20
30
40
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Yield Load (N)
0.0001
<0.0001
0.0030
<0.0001
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0
10
20
30
40
50
Yield Load (N)
0.0257
0.0384
ControlAcidosis
0
2
4
6
8
10
Work (N-mm)
0.0048
0.0051
ControlAcidosis
0
2
4
6
8
10
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0.0281
0.0023
0.0261
ControlAcidosis
0
2
4
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8
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100
150
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ACB
D FE
G IH
J K L
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Figure 4.16. Material mechanical properties of femurs for male and female mice. The structural
parameters measured were (A-C) maximum stress, (D-F) yield stress, (G-I) toughness, and (J-l) modulus.
The left column is all from Day 3, the middle column is Day 7, and the right column is Day 14.
Control Acidosis
0
50
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0.0054
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0.0150
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0
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0.0316
0.0205
0.0030
0.0019
Control Acidosis
0
5
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0.0095
0.0070
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0.0336
0.0305
A
D
B C
FE
K
G H I
L
J
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Unlike the structural mechanical properties, the maximum stress and yield stress were not
different between the sexes and conditions (Figs. 4.16A-F). At Day 3, the male control mice
femurs were tougher than the female control mice (Fig. 4.16G). This difference switched at Day
7 where the male acidosis femurs were tougher than the female acidosis mice (Fig. 4.16H). This
difference disappeared by Day 14 (Fig. 4.16I). The modulus was higher in the female acidosis
mice compared to the male acidosis at all three days (Fig. 4.16J-L). Additionally, the female
control mice had a higher modulus than the male control mice at Day 3 (Fig. 4.16J).
4.3.6 Alterations in RNA expression of bone due to acidosis
To see how acidosis affects the genetic expression of bone in male and female mice,
NanoString analysis on RNA samples was conducted. When considering the influence of acidosis
on bone regardless of sex and time, there was an upregulation of Esr2, Tnfrsf11B, Twist1, Calca,
Nos3, Fos, and Ldha (Fig. 4.17).
Figure 4.17. Acid exposure upregulates expression of specific genes independent of sex and day.
A volcano plot of the influence of acidosis on the genetic expression of murine femurs. A p-value of 0.05
was used to determine significance.
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When separating out the genetic expression data by sex, some differences between the
female and male mice appeared. For the female mice, acidosis resulted in an upregulation of
Esr2, Jun, Cyp19a1, Vegfa, Calca, and Ldha (Fig. 54.18A). For the male mice, acidosis results in
an upregulation in Twist1, Tnfrsf11b, Nos3, Esr2, and Comt, while there was a downregulation in
Prl due to acidosis (Fig. 54.18B).
Figure. 4.18. Sexual difference in genetic expression due to acidosis. Volcano plots separated by (A)
female and (B) male mice independent of day. A p-value of 0.1 was used to determine significance.
To see the influence of time on the genetic expression of acid-exposed bone, Days 3, 7,
and 14 were separated out along with sex. For the female mice, acidosis resulted in an
upregulation in Esr2 and a downregulation in Nog and Bglap at Day 3, an upregulation in Nos
and Pth at Day 7, and a upregulation in Jun and Sost and a downregulation in Sfrp1 and Calcr
at Day 14 (Figs. 4.19A-C). For the male mice, acidosis resulted in an upregulation in Gusb, Fos,
Nos3, Tshr, and Tnfrsf11b at Day 3, an upregulation in Esr2 and Dbp and a downregulation in
Tnfrsf11a at Day 7, and an upregulation in Enpp1 at Day 14 (Figs. 4.19D-F).
log(fold change)
-log10(p-value)
Is the p-value less than 0.1?
A
B
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Figure 4.19. Genetic expression difference due to sex and time in acid-exposed femurs. Volcano
plots separated by sex and time. Female mice data can be seen for (A) Day 3, (B) Day 7, and (C) Day 14.
Male mice data can be seen for (D) Day 3, (E) Day 7, and (F) Day 14.
4.4 Discussion
To determine the influence of sex on acidosis induction in wild-type, CD-1 mice, metabolic
acidosis was induced in both male and female mice using our lab’s established graded dosing of
ammonium chloride in the drinking water. As expected, the male acidosis mice had a reduction in
their blood pH and bicarbonate levels after 14 days of acidosis induction. This trend was seen in
our other studies using the same strain, sex, and model system [42–45,209]. However, the female
acidosis mice only had a reduction in bicarbonate compared to the female control group at Day
14. Even though there are little to no studies looking at the sexual differences in blood gas
measurements of acidotic mice [42], there could be a few reasons for this difference in acidosis
Is the p-value less than 0.1?
log(fold change)
-log10(p-value)
A
B
C
D
E
F
134
induction between male and female mice. One reason is that the female mice have a higher blood
pH and amount of blood bicarbonate than the male mice. This could be due to female mice having
less acidic by-products during their normal activities, which is made evident by lower lactate levels
in the female mice compared to the male mice. A study found that even when men and women
exercise the same amount, women tend to have lower amounts of lactate in their plasma [248].
Moreover, the base excess (BE) was much higher in the female acidosis mice than the male
acidosis mice, providing evidence that the blood pH and bicarbonate levels are higher in the
female acidosis mice [249]. Additionally, the male and female mice had very few differences in
blood urea nitrogen (BUN) levels, illustrating similar kidney function. It was expected that since
the female mice weighed less than the male mice, it would take less ammonium chloride water to
induce acidosis in the female mice. However, because of this slight difference in blood pH and
bicarbonate, a higher dosing of ammonium chloride might be needed in the female mice to induce
acidosis as seen in the male mice. Another factor could be that the female acidosis mice drank
less of the ammonium chloride fluid compared to the male acidosis mice over the span of the
fourteen days of experimentation. The female acidosis mice also ate less food compared to the
female control mice and male acidosis mice. There were very few differences between male
control and female control mice in terms of food and fluid consumption; even though, a study
found that body weight can influence eating and drinking habits [229]. However, it seems like
ammonium chloride strongly impacts these habits in the female acidosis mice. Combining these
two factors together, it might be necessary to use a gavage to administer ammonium chloride to
female mice to induce metabolic acidosis.
Despite little to no changes in blood pH and bicarbonate levels in the female acidosis mice,
these mice were still excreting acid in their urine, as made evident by a decrease in urine pH over
14 days due to the addition of ammonium chloride in their drinking water. Because of this
presence of acid within the body, there could be some bone dissolution occurring in both the male
and female mice due to acidosis induction. By Day 7, both the female and male mice undergoing
135
acidosis induction had elevated blood calcium levels that continued into Day 14. This elevation in
calcium could be evidence of bone dissolution [28,40,116,192,194,250,251]. Nonetheless, the
potential bone dissolution seen in both male and female mice was not enough to alter the
composition, structure, and flexural mechanics of their femurs. Although there were
compositional, structural, and mechanical differences between the male and female mice; within
the sex, there were no substantial changes to the bone due to acidosis. The differences seen
between sexes also fluctuated throughout the fourteen days of experimentation, potentially
illustrating a high bone turnover rate. Younger mice (3–4-month-olds) were used in this study
compared to the typical age used in past studies (4–6-month-olds) that utilized this particular
model of diet-induced metabolic acidosis. Although three-month-old mice are considered
skeletally mature, their ability to remodel bone is most likely higher than in six-month-old mice. A
study found that 6-month-old wild-type, C57BL/6 mice decreased in bone formation rate (BFR/BS)
of the endocortical bone compared to their 3 month old counterparts. Additionally, in the same
study, osteocalcin and CTX both seemed to decrease from 3 to 6 month old mice, further
illustrating a decrease in bone turnover [252]. Although we used CD-1 mice in this study rather
than C57BL/6 mice, it can be assumed that CD-1 mice follow a similar pattern in their bone tissue.
It is hypothesized that there is an age-related dependency, despite assuming skeletal maturity,
with our graded-dose NH4Cl model of metabolic acidosis.
Despite not observing a phenotypical change within the bone, there were some sex-
dependent gene expression differences between control and acidosis mice. When only looking at
the changes in both male and female mice combined with acidosis, there was an upregulation in
genes such as Esr2, Tnfrsf11b, and Calca. Esr2, which is an estrogen receptor, is found in both
osteoblasts and osteoclasts [253–255], and it may play a role in bone homeostasis, especially
since estrogen influences bone health in both men and women [256]. Estrogen deficiency can
cause osteocyte apoptosis [257,258], osteoclastogenesis [259,260], and imbalanced bone
turnover [256]. In our mice with acidosis, there seems to be an increase in genetic expression of
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estrogen receptors, possibly leading to an increase in estrogen activation. Tnfrsf11b encodes for
a protein called osteoprotegerin (OPG), which is released by osteoblasts and binds to RANKL to
inhibit the activation of osteoclasts precursors via RANK [261–263]. Our model seems to illustrate
a need to prevent osteoclastogenesis via OPG. Other studies have shown that osteoclast activity
increased in bone with acid exposure [38,264,265]. In our previous studies using the same model,
we found a decrease or no change in osteoclast number or activity [43,149]. Potentially, there is
a balance between activation of osteoclasts due to acid exposure and protective mechanisms of
bone dissolution occurring in the body, resulting in a lack of cellular changes. Further protective
mechanisms were demonstrated by an increased expression of Calca. Calca encodes for
calcitonin, which is a hormone that regulates calcium balance by reducing calcium in the blood
[266,267]. This demand to rebalance the calcium levels could be due to the increase in blood
calcium levels seen in both male acidosis and female acidosis mice at Days 7 and 14. Also,
calcitonin has shown to rapidly reduce the ruffled boundary of osteoclasts, diminishing their ability
to resorb bone [268,269]. The combined upregulation in Tnfrsf11b and Calca demonstrate the
body’s ability to possibly counteract the activation of osteoclasts to prevent bone dissolution due
to acidosis, which could be another reason why no changes were seen phenotypically in the bone
tissue of these mice.
When the sexes are separated out from the gene expression data from femurs, sexual
differences begin to appear, which was also seen in heart tissue taken from CD-1, male and
female, mice undergoing acidosis using the same model of induction [42]. Both sexes have an
upregulation in Esr2, but Calca is only upregulated in female acidosis mice and Tnfrsf11b is only
upregulated in male acidosis mice compared to their respective controls, illustrating that the
proposed protective mechanisms against bone dissolution under acidic conditions might be sex
dependent.
When looking at the sex and time dependency of the RNA expression data, there were
evident variations in gene expression between the days and sexes. Upregulation in Esr2
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continued to be present in male and female mice, but only for male mice at Day 3 and only for
female mice at Day 7. For the female and male mice, the genes that had modified expression due
acidosis over the fourteen days varied. At the early timepoint (Day 3), osteoblastic differentiation
and bone morphogenetic proteins (BMPs) signaling were seemingly altered due to the
downregulation of Bglap [270,271] and Nog [272]. At the intermediate timepoint (Day 7), an
upregulation in PTH correlates to other studies who have seen heightened levels of PTH and PTH
receptors due to the presence of acidosis [110,111]. By Day 14, osteocyte expression of SOST
was upregulated, suggesting an increase in osteoclast resorption by antagonizing Wnt activity
[273–276]. Sfrp1 was underexpressed at this day as well, which could increase osteoblastic
differentiation [277]. Even though the incidence of sclerostin might have increased by Day 14 in
the female acidosis mice due to a heightened expression of SOST, it seems like the body was
trying to compensate for this potential osteoclastic resorption by increasing osteoblastic
differentiation through the reduction of the Wnt antagonist Srfp1. Male mice also saw a variation
in genetic expression over time, but for different genes. Male mice with acidosis saw an increase
in Tshr, a receptor for the protein called thyroid-stimulating hormone (TSH), which has been found
to inhibit the formation of osteoclasts [278–281]. TSH secretion with acidosis is complicated, with
some studies showing an increase in TSH with acidosis [282] while others have shown no change
in TSH levels with acidosis [283]. By Day 7, there was an reduction in the expression of Tnfrsf11a,
which makes a protein on the surface of osteoclast called RANK [284]. At Day 14, Calca and
Enpp1 were upregulated, suggesting a potential reduction in osteoclast activity and an increase
in osteoblastic differentiation [285]. These fluctuations might be due to bone turnover, which can
occur over a span of fourteen days in mice, but the lack of changes in osteoclastic differentiation
and activity due to acidosis might demonstrate that both female and male mice with acidosis have
inherent protective mechanisms to prevent further bone dissolution due to acid exposure.
Although there are currently no previous studies that have shown this result, most acidosis studies
look at short timeframes (hours to a few days) or long timeframes (weeks to months). Our study
138
is one of the few that has looked at an intermediate timepoint (14 days), which might be why other
studies are not capturing a recovery mechanism within bone. Moreover, the increase in blood
calcium might suggest that there is physiochemical dissolution occurring, but at such a small rate
that it has not impacted the structure, composition, and mechanics of the bone yet. Longer studies
of acidosis induction in male and female mice may be needed to observe phenotypical changes
in bone.
Although we were able to mimic clinical metabolic acidosis in male mice, the ability to do
this in female mice was challenging. To overcome this, older female mice with less effective
kidneys could be used in acidosis studies. Additionally, a gavage could be used to ensure that
the mice consumed equal amounts of placebo and ammonium chloride solution. Another limitation
of this study is that both the male and female mice did not show any phenotypical changes in
femoral tissue using the analytical techniques used in this study. As mentioned, one of the
reasons for this is that younger mice (3 months old) were used compared to the mice used in
previous studies utilizing this model (4-6 months old). To determine if there is an age-dependency
of skeletally mature mice using this model, it would be useful to use the model on various ages
(3, 4, 5, and 6 months) for both male and female mice. To determine if there are different levels
of bone turnover in these different ages, static and dynamic histological analysis could provide
insight into the behavior of bone cells. Additionally, CTX, a bone resorption marker, and P1NP, a
bone formation marker, could also be measured in the serum to observe changes in bone
turnover. Also, this study did not utilize histological analysis to look at osteoclast (TRAP staining),
osteoblast (toluidine blue staining), and osteocyte (silver nitrate staining) number and behavior.
In future studies, it would be necessary to do this type of analysis to validate the RNA genetic
expression data.
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4.5 Conclusion
To determine the presence of sexual dimorphism in bone exposed to acid, metabolic
acidosis was induced in male and female CD-1 mice using an established model of graded-dosing
of ammonium chloride. The composition, structure, mechanics, and genetics of their femurs were
studied. Using blood gas analysis, the male acidosis mice showed signs of metabolic acidosis by
Day 14, with a reduction in blood pH and bicarbonate. On the other hand, blood bicarbonate in
the female acidosis mice decreased at Day 14, suggesting the female mice may have been in
subclinical metabolic acidosis. Although there were sexual differences of bone, acidosis induction
did not seem to change any further parameters in the femurs. This could be due to the fact that
3-month-old mice were used in this study rather than 4 -6 month old mice. Lastly, sex- and time-
dependency was seen in the genetic expression of bone exposed to acid compared to their
controls. There also might be a protective mechanism occurring in the both the male and female
mice, causing a lack of bone dissolution under acidotic conditions. Conclusively, metabolic
acidosis presents itself differently in male and female CD-1 mice. These differences are
supplemented by the potential, sex-dependent pathways in which bone protects itself from
dissolution under acidotic conditions.
140
Chapter 5: Conclusions and Future Directions
5.1 Conclusions
Overall, our diet-based graded-model of ammonium chloride (NH4Cl) for metabolic
acidosis induction was able to mimic what is seen clinically in patients with this disease, such as
a reduction in blood pH and bicarbonate levels. The mice given this diet had increases in blood
calcium and sodium levels, potentially indicating bone dissolution, along with increases in blood
chlorine levels, suggesting ingestion of the ammonium chloride solution. Various studies using
this model were conducted to not only determine its repeatability, but also observe how metabolic
acidosis-mediated bone loss can be treated and how it differs between males and females.
Temporal differences between our new model and the traditional flat-dose model of NH4Cl
were measured by analyzing blood gas data along with femoral composition, structure, and
mechanics. The flat-dose model was found to not be as effective at mimicking metabolic acidosis
via blood gas parameters compared to the graded dose model, which showed a reduction in blood
pH and bicarbonate levels for fourteen days. Additionally, there were no changes in bone with the
flat dose model. The graded dose model showed bone modifications in the early timepoints but
saw a recovery in bone parameters by Day 14, which illustrated the importance of studying the
temporal effects of acidosis on bone. Additionally, this particular study found that physiochemical
processes might be at play during the early changes, but that cellular behavior was not impacted
at any of the timepoints, which could indicate the presence of protective mechanisms preventing
further bone loss. The existence of protective mechanisms was further supported by studies done
in this dissertation. CTX levels were not altered at Day 3 and Day 14 of acidosis induction in mice
compared to their respective controls. Additionally, sex-dependent genetic data in acidotic mice
temporally illustrated a prevention of osteoclastic resorption and a facilitation of osteoblastic
differentiation. Combining these results demonstrate that (1) even if osteoclast resorption is
increased with acid exposure, the body is potentially changing the expression of various genes
141
within bone tissue to combat this and (2) physiochemical dissolution may be governing the bone
changes seen in the early timepoints of this model due to the inhibition of osteoclast resorption.
Although bisphosphonates are not commonly used in patients with low renal function, its
ability to reduce osteoclast activity could be beneficial to patients with metabolic acidosis.
However, our study found that bisphosphonates worsen acidosis, potentially due to the lack of
buffer release under acidotic conditions. This treatment might be useful if combined with
bicarbonates. Bicarbonate treatment for metabolic acidosis have been widely studied and are
currently clinically used, but the effect of bicarbonates on bone has not been as well researched.
We found that differences in acidosis recovery and bone composition differed between sodium
bicarbonate and potassium bicarbonate administration with acidotic mice. Sodium bicarbonate
was able to return the composition, structure, and mechanics of bone to control levels, while
potassium bicarbonate saw a reduction in trabeculae and an integration of sodium into the bone.
These differences between the two types of bicarbonate treatment were most likely attributed to
an increase in blood sodium in the blood of the mice treated with potassium bicarbonate. Although
the reason for this increase in blood sodium with potassium bicarbonate is not yet well understood,
it is important to decipher how these two treatments impact bone health.
5.2 Future Directions
Although there are inconsistencies in bone modifications with the graded-dosing model
across different studies, this could have been due to different ages of skeletally mature mice
utilized within these studies. As mentioned in Chapter 4, there could be an age dependency
occurring within the model, with younger mice having higher bone remodeling rate, leading to
lessened effects of acidosis on bone. To determine if there is a potential age-dependency, a study
on mice of ages starting at 12 weeks and ending at 12 months using the grade-dosing model
could be useful. This would not only give insight into how age plays a role in metabolic acidosis
but help us determine the reliability and repeatability of our model.
142
If the repeatability of the graded-dosing model continues to be a problem, it may be
necessary to use a different technique to administer ammonium chloride. One solution is creating
a longer study that lasts a few months rather just two weeks. The graded dosing could be either
elongated so that the changing the dosing is every week rather than every three days and/or the
increments of increased loading could be smaller. For example, instead of having increments 0.1
M of NH4Cl, it might be better to have increments 0.05 M every three days. One concern for this
modification is that at higher doses, the mice might stop consuming the ammonium chloride water
all together, leading to dehydration, illness, and detrimental weight loss. If ammonium chloride
induction in liquid is not sufficient, then combining ammonium chloride in the water and the food
could be a potential solution. No current studies have looked at this combination yet, only at food
and water separately [41], but having lower doses of ammonium chloride in both the food and
water might be a better alternative than having high doses of ammonium chloride in only the
water. One problem that might occur with this solution is the occurrence of discrepancies in food
and water consumption. If this occurs, then administration of ammonium chloride using a daily
gavage could be utilized to ensure that the mice are ingesting ammonium chloride at similar rates.
Since acidosis has a huge impact of urinary calcium excretion [286,287], it would be useful
to be able to detect in changes in this metric in our model of acidosis. Additionally other ions and
molecules could be measured in the urine, such as sodium, chlorine, and phosphate levels.
Colorimetric assays could be utilized to analyze these urinary parameters. In order to obtain large
enough amounts of urine from the mice, a method using a disposable plastic tray has been
established [288].
Even though work in this dissertation was able to look at sodium bicarbonate and
potassium bicarbonate along with bisphosphonates as a form of treatment for acidosis and acid-
exposed bone, it only studied one concentration of this treatment. Conducting a study on the
dose-dependency on the recovery of acid-exposed bone could provide insight into the exact dose
needed to improve bone health under acidotic conditions. Wong et al. found that sodium and
143
potassium incorporate into the carbonate locations within biomimetic apatite using different
mechanisms [234]. Additionally, in biomimetic apatite, potassium had a protective effective over
dissolution [233] rather than a negative impact, unlike what was seen in this dissertation. Further
research into bicarbonate treatment for acidotic mice could also illustrate how various doses affect
the bone dissolution and remineralization behaviors of in vivo murine bone.
Other treatments that could be tested to observe its effects on the health and functionality
of acid-exposed bone are sodium citrate [289] and potassium citrate [290]. Sodium citrate was
found to be as effective as sodium bicarbonate at correcting metabolic acidosis. Additionally,
sodium bicarbonate discontinuation was higher than that of sodium citrate most likely due to lower
gastrointestinal tolerance [291]. Potassium citrate treatment in renal transplant patients was also
found to be effective at correcting metabolic acidosis with a potential to improve bone health [290].
A study of not only looking at the effects of citrate on the recovery of acidosis-mediated bone loss,
but also a comparative study between citrate and bicarbonate treatments would be impactful. It
would help determine if current treatments (bicarbonate and citrate) for correcting metabolic
acidosis are also effective at returning bone to their healthy standards.
144
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Appendices
8.1 Weight, Urine pH, and Blood Gas Collection Protocol
Created by Mikayla Moody (August 2021)
Supplies
• Gauze
• 5mm Goldenrod animal lancets
• 1mL syringes
• PST microtubes with LH
• Element POC (EPOC) HESKA cards
• 70% ethanol
• pH paper (usually in the range of 4.5-7.5)
• Plastic beaker
Equipment
• Scale
• Heska EPOC blood gas analyzer
• Light box
• Sharps container
Steps
1. Make sure you have all the necessary supplies on the cart or already in the room where
the mice are located in the animal tower (most likely room B7010).
2. Go to LB005 in the basement (code is 0135) in the animal tower to pick up the blood gas
analyzer. Just unplug it from the charging cable.
3. Go back up to the room and get the urine and weight collection set-up.
Weight and urine collection
4. Use the plastic beaker to put the mice in to weigh them.
5. Since the mice will most likely move around a lot in the
beaker, the weight on the scale will fluctuate. Look at the
numbers and record the weight that shows up the most.
6. Next, restrain the mouse and hold them in a way that their
back is facing the ground and their stomach is facing
upward. Put the pH strip onto their pee, put the mouse back
in the cage, and then image the pH strip under the light box
next to the pH scale using your phone’s camera. Then
record the urine pH.
7. Record urine every time you give the mice solution (either starting an experiment or
changing their solution) and when you do a baseline measurement.
Example of urine sample on pH
strip in light box
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Blood gas sample and data collection
8. After doing urine and weights, set up the bench for blood collection. You will need gauze,
lancets, microtubes, 70% ethanol, Heska cards, the Heska machine, sharps container,
and syringes.
Note: If you are doing this by yourself, it is recommended that you uncap
the tube and put the tube in something that will hold it up right. For
example, you can use tape as shown in the picture.
Note: Only use 70% ethanol to clean up any messes that are created.
Do not use the Clidox spray because it has chlorine dioxide, which will
influence the chloride measurements in the blood gas data. Only clean
with this after being done with this part.
9. Log into the Heska machine. You can type into anything for User ID and the Password but
can’t use the same thing for both. For example, you can use for User ID: # and Password:
* but not User ID: # and Password: #
10. While the machine is configuring, type the sample name and experiment details in the
comment section (you can ask this by clicking on the second tab on the top left).
11. Once you have the information done, you can return to the original tab. At this point, you
can either put in a EPOC card and then collect the blood or you can collect the blood first
and then put the EPOC card in.
Note: It is only recommended to put the EPOC card in first if you have done many
submandibular bleeds before and believe that you can get enough blood quickly. When
you enter the card into the machine, it will take about 170 seconds to calibrate before you
Example of the set-up for blood collection
Set-up of microtube
when working by
yourself
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can put the blood sample. Please wait until after the calibration is done before putting the
blood sample in.
12. To do the submandibular bleeds, make sure that you have microtube uncapped and ready
to go as well as has gauze nearby to stanch the wound. Scruff the mouse and make sure
you have the animal tightly enough that its head doesn’t move around much, but not too
tightly that it is suffocating. You can tell when a mouse is suffocating when it is having a
hard time breathing and its mouth is opening and closing quickly.
13. Next, puncture the cheek using a lancet, let go of the lancet, and quickly grab the
microtube. If someone is help you, have them hold the microtube close to the mouse’s
face before puncturing and then having them move in quickly. Make sure to get about 200-
300 microliters of blood. Try to not get more than that because then the mouse could be
losing too much blood.
Note: I would have three lancets removed from the paper package and ready to be used
before doing the bleed just in case you need to do multiple punctures and the lancet gets
dull.
YouTube video showing use of lancets:
https://www.youtube.com/watch?v=niTVnEAHOko&ab_channel=VideoProtocolsCom
14. Once you have gotten enough blood/getting close to having enough blood, you can start
pushing the microtube against the mouse’s face to start stopping the bleeding. After doing
that for a few seconds, gently press the gauze on their face. Make sure the bleeding has
completely stopped before placing the mouse back in their cage.
Note: One thing that can help the mouse recover faster is putting food and new white
cage paper into the cage. This just helps them feel comfortable or safer (in Mikayla’s
opinion).
15. Put the used Heska cards, syringes, and lancets in the biohazard sharps container
Getting blood gas data from computer
16. Once done with blood collection, go back to room where you go the Heska machine. Plug
the Heska machine back in to charge.
These are images of (a) the ideal location of the puncture, (b) holding the lancets perpendicular to the mouse’s face, and
(c) how to collect the blood after puncture [1].
(a)
(b)
(c)
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17. Log into computer using your UCHC credentials
18. Open up EDM lite application on the desktop.
19. Click the X on the top right of Heska machine when it says to retry synchronization (you
may have to log back in). Let the reader configure and then click the X on top right
again.
20. The blood gas data should then automatically load onto the EDM lite program. Once the
data is done uploading to the computer, there should be summary on the Heska
machine.
21. Click the X on Heska machine and turn off by clicking the power button on the top
portion and the bottom portions of the machines.
22. In the EDM program, choose the current day at the top (so the current day is both for
from and to). To get the data, select all of the data, click the CSV button, and then save
the file on the desktop. You can also make PDFs for each of the blood collection
samples. You can’t select all samples at once, so you must click on each sample
separately and press the PDF button each time. Then, email the data to yourself (use
Internet Explorer).
Images of (a) the machine showing that it is sending the data to the
computer and (b) the machine summarizing the data it sent.
(a)
(b)
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23. Then log off the computer and make it go to sleep.
Reconnecting Heska machine back to Bluetooth
Sometimes the Heska machine might disconnect from the Bluetooth, which allows it to send the
data over to the computer. In order to fix
this, click the settings the top right in the
EDM lite program. Then click “Connect
epoc Hosts”. Under that, click “Allow
epoc Hosts to connect using Bluetooth”.
Hopefully that makes it work!
Updating Heska machine software
Images of the (a) EDM Lite program with the new data uploaded on it and (b) the location of the CSV
and PDF buttons on the EDM lite program.
(a)
(b)
CSV button
PDF button
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Sometimes the Heska machine software will need
updating. You will know this because it will be stated below
on the login screen of the machine.
First, you need to go to settings and then go to “Update
epoc hosts”. Click the “Download upgrade file” and then
upload that file in the “Select epoc Host upgrade file”.
Next, log into the Heska machine using the administrator
login. You do this by taking the top part of the machine off
and then scanning the administrator barcode by using the
yellow button.
Then click the “Tools” button on the bottom left, then click
“Perform Update”, and then “From EDM”. Then follow the
rest of the steps and it should upgrade [2]!
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8.2 Histology and Histomorphometry Femur Preparation Protocol
Created by: Mikayla Moody
Date: May 28, 2021
Updated: May 30, 2021
Supplies:
• Ethanol (30%, 50%, & 70%)
• Tissue cassettes
• 10X PBS
• 4% PFA in 1X PBS
• 14% EDTA
• 15 mL Falcon tubes
• 30% sucrose in 1X PBS
• 1.5 mL microvials
• OCT
• Disposable base molds
• Aluminum foil
• Japanese tape
• Glass jars
• Slide holder
Steps:
1. After euthanatizing the mice, dissect the femurs (use dissection protocol). After dissecting,
place the femurs in separate 15mL Falcon tubes with 4% PFA (in 1X PBS).
2. Leave in 4% PFA (in 1X PBS) for three days on the rocker at 4°C. Make sure that the rocker
is gently rotating (every time the rocker it used, go about halfway with the rocking).
3. After three days, rinse the femurs in 1X PBS, 3 times for five minutes, each at 4°C. Put about
10mL of PBS in each tube for each rinse. You can use the same tubes that they are in and
remember to replace the 1X PBS for each rinse. Put them on the rocker during the five minutes.
You can put the 4% PFA into the general non-hazardous waste container.
For Histology Samples
4. After the three rinses, put at least 12-14mL of 14% EDTA solution in each tube to decalcify
the bones. Put the samples back on the rocker at 4°C.
5. Change the 14% EDTA solution every other day. Change on days 3, 5, 7, 9, and 11. Day 1 is
the first day it was put in EDTA. You can put the old EDTA into the general non-hazardous
waste container.
6. On the 12th day in EDTA, rinse the samples in 1X PBS, 3 times for five minutes, each at 4°C.
7. Next, put the femurs in individual cassettes. Use a pencil to write the sample names on the
cassettes. If you are only doing a few samples (for example: five bones), you can do the 30%
and 50% dehydration in their individual tubes and wait to put them in cassettes and the jar for
the 70% step.
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8.Then put the samples in a glass jar to dehydrate them and add 30% ethanol to the jar for 15
minutes at 4°C. Shake the jar every couple of minutes to make sure that all parts of the bones
are being dehydrated. If you are doing this step in individual tubes, you can put about 10mL in
each tube and put it on the rocker for 15 minutes. Please put all ethanol waste in the solvent
hazardous waste container.
9. Then, remove the 30% ethanol and add 50% ethanol for 15 minutes at 4°C. Shake the jar
every couple of minutes. If you are doing this step in individual tubes, you can put about 10mL
in each tube and put it on the rocker for 15 minutes.
10. Lastly, remove the 50% ethanol and add in 70% ethanol. If you haven’t done so already, put
the samples in cassettes before putting in the jar with 70% ethanol. The samples can then be
placed in the fridge (4°C) until they are submitted to the Histology Core. Make sure to the bring
the samples along with slide holders and the Histology Core form filled out (email Zhifang Hao
to see when you can bring them to her).
11. Once stained, see Microscopy protocol to image the samples.
For Histomorphometry Samples
4. After the three 1X PBS rinses, add at least 10mL of 30% sucrose (in 1X PBS) to the tubes.
5. Put the tubes on the rocker at 4°C for at least 12-24 hours.
6. After 12-24 hours in 30% sucrose at 4°C, place the samples upward in the -80°C freezer.
Once the samples have frozen, you can put them in a sample box and place them back in the -
80°C freezer. Keep them in the freezer until all samples are ready to be embedded and
sectioned.
7. Once all samples are ready, defrost the bones overnight by placing them in the 4°C fridge.
8. When defrosted, take the bones out of the Falcon tubes and put them in 1.5mL microvials in
OCT for 30 minutes in order to let the OCT infiltration into the bones. Put the 30% sucrose
solution in non-hazardous waste.
9. Take the bones out of the microvials and place them in base molds with the condyles facing
up and femoral head facing downward. Additionally, make sure that the bone is centered.
Additionally, make sure to label the mold with the sample name.
10. Before adding more OCT, check to see there aren’t any bubbles at the bottom of the mold.
You can pop the bubbles by moving the bones around a bit in the OCT that was left over from
infiltration and/or getting a needle and popping them.
11. Add more OCT to the mold until the enter bone is covered. You want to add the OCT
directly over the bone.
12. Make sure there are little to no bubbles on or near the bone. It is okay if there are tiny
bubbles on the sides the mold. Bubbles can also be moved around or popped with a needle.
13. Once it is all good, make space in the -80°C freezer, so that the bottom of it is exposed and
the mold can be placed directly on the metal. This allows it is be frozen from the bottom up and
helps get rid of bubbles.
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14. Once frozen, wrap each mold in aluminum foil and put them in a sample box.
15. Give to the Histology Core to section them (email Zhifang Hao to see when you can bring
them to her). Bring the sample box at once along with Japanese tape. You don’t want them to
be glued down, just sectioned onto the tape.
16. Once sectioned, see Microscopy protocol for imaging.
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8.3 Mouse Femur Three Point Bending Protocol
VERSIONS
V1: Paige Woods (June 22nd, 2018)
V1.2: Edited by Brian Wingender (June 28th, 2018)
V1.3: Edited by Paige Woods (June 29th, 2018)
V1.4: Edited by Mikayla Moody (June 17th, 2024)
1. PURPOSE
This procedure is used to determine the mechanical properties of a mouse femur that has been
previously preserved via freezing in -80°C. This is done by testing the isolated femur under
three point loading bend test using the Mach-1 mechanical testing machine.
2. SAFETY
This protocol requires the handling of animal cadaver tissue and delicate equipment. Proper
training in the use of the Mach-1 and in handling biohazardous materials is required, and the
latter can be learned in ‘Biological Safety in Animal Research’ offered by the division of
Environmental Health and Safety at UCONN to all students and employees.
3. EQUIPMENT
Mach-1 Mechanical Tester Model v500csst (Biomomentum – MA009)
Chamber for Sample Holder (76.2mm) (Biomomentum – MA626)
Single Axis Load Cell (25kg; S/N 1626027) (Biomomentum – MA299)
Calibration Weight (500g; MA3371217A-7) (Biomomentum – MA337)
Three Point Bending Kit (Biomomentum – MA097)
4. MATERIALS
Biological Material:
Mouse femur sample(s)
Solutions:
250 mL 1X PBS solution (made from Gibco DPBS (10X) ref: 14200-075)
1X PBS solution in squirt bottle (Bottle: Fisherbrand Polyethylene 250mL Wash Bottle 03-409-
23T)
Deionized H2O
Supplies:
Clean gauze (1 1inx3in strip per femur sample) (Fisherbrand 3x3 inch Non-Woven Gauze Cat.
No. 22-028-560)
Benchtop paper
Flat edge tweezers
1.5mL micro centrifuge (MCT) snap top tubes (1 per femur sample) (Fisherbrand Cat No. 05-
408-137)
Surgical pad
Heater
Temperature Controller
Stand for heater
5. PROCEDURE
5.1 Chamber Assembly
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Place the black O ring on the outer surface around the clear chamber wall. Run finger under O
ring, between the rubber and the chamber wall, to reduce the chance of the material twisting.
Push the O ring down to the edge of the wall.
Figure 1: Chamber Wall and O Ring
On a bench top, place the chamber wall on the base plate such that the O ring is in contact with
the edge of the circular base plate area. Then place the circular metal plate on top and secure
with four screws. Tighten the screws across from each other sequentially, not in a circular
pattern, and tighten each screw a few times keeping the pattern across the plate, if the screws
are tightened all the way down at once the seal may be faulty. The screws should be tightened
until the top plate is flush against the bottom.
Fill the chamber half way with deionized water and place it on a dry paper towel. Check for any
leaking or bubbles and allow to sit for approximately 15 minutes while the rest of the software
and equipment is set up.
5.2 Software Set Up
Power on the computer. Power on the Newport Motion Controller box (Model ESP301), located
underneath the desktop monitor, using the switch on the left hand side in the back.
Open the “Mach-1 Motion” software using the desktop shortcut. For the first pop up window
select the option for MA0561123-CSS which allows for testing compression and tension shear
along two axes, whereas the MA0561123-CST option is used for torsion. In the next pop up
window click the “Start” button and system will automatically run through an initialization
sequence, which enables communication between all of the system components. Allow the
system to warm up before performing load cell calibration and/or running a test sequence, it
requires about fifteen minutes for the transducers to reach a steady state and for the load
readings to stabilize. Click “Manual Controls”, select Axis = Position (z) and Velocity = Medium,
and click and hold “Up” until the Z stage reaches its upper limit.
5.3 Load Cell Calibration
The 25kg load cell should be installed on the Z stage of the Mach-1. If it is not, install it by gently
placing it on the attachment screw on the Z stage and turning it secure. The 150g load cell is
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EXTREMELY delicate and should only be handled by those who have been trained. DO NOT
REMOVE if it is already attached.
Gently screw the load cell calibration weight attachment into the load cell, turning it until stable
but not tightened all the way.
Click the “Load Cell” button at the center bottom area of the Mach-1 Motion window. Make sure
that the 25 kg load cell (SN 1626027) is highlighted has an active status. Click on the “Calibrate”
button at the bottom of the window.
In the next window, select the appropriate calibration weight from the drop down menu. Since
this experiment uses the 25kg load cell, the calibration weight is 500g (MA3371217A-7).
A window will pop up that says “Install the calibration weight,” do this and click “OK”. Another
window will pop up that says “Remove the calibration weight,” do this and click “OK”. Then
gently remove the calibration weight attachment from the load cell and record the calibration
factor that is displayed on the screen in the calibration log (located in the top drawer under the
computer. If the calibration factor is more than ±5% off of 1.000 then the calibration process
should be repeated, and if it is still off then the experiment should be put on hold and the
machine should be examined by a technician.
5.4 Apparatus Set Up
Empty the deionized water from the sample chamber and dry it. Place the pieces from the three
point bending kit in the chamber such that the track is aligned vertically with the screws. Cover
the XY stage of the Mach-1 with lab benchtop paper to protect from any possible leaks, with the
shiny, plastic-y side facing down and the absorbent side facing up. Place the chamber on the
stage of the Mach-1 and secure with provided screws. Attach the loading piece, also called the
blade, to the underside of the load cell on the Z stage. Adjust the stage positions by clicking the
“Manual Controls” button at the bottom of the window and selecting the desired stage from the
drop down menu. Adjust until the loading piece can fit between the two supports while the
supports are 8mm apart. Ensure that the rubber bands on the supports are out towards the end
of the metal rod, and that the rubber bands on the loading piece are pushed in towards the
center. This is shown in the image below.
Figure 2: Three Point Bending Configuration for Mouse Femur
A heater can be used to mimic the temperature of the human body in the 1X PBS bath. Set up
the heater in the bath using the stand to hold it up over the bath, as seen in the picture on right.
Track
Blade
Supports
Rubber
Bands
176
Don’t plug it into the temperature controller until you have set-up the heater. Make sure that the
heater is not touching the plastic chamber wall or the metal 3pt bend apparatus or anything else
but 1X PBS. Once you have set-up the heate, plug in the heater into the heating plug of the
temperature controller. Plug in the thermocouple into the 1X PBS before pluggin in the
temperature controller. It should automatically turn on when plugged in and it should be set to
37C already.
Temperature Controller
Spread the surgical pad out on the benchtop adjacent to the machine so that there is an area to
thaw femur samples, remove them from their containers, and rewrap them and package them
after they are broken. Keep the tweezers on the surgical pad.
Then add to the chamber approximately 250mL of 1X PBS solution. This is used so that the
sample tissue doesn’t dry out.
Remove the femur from its tube and gauze wrapping and place the femur across the two
supports with the orientation that keeps the condyles facing up and the head of the femur facing
down. This orientation is shown in the image below.
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Figure 3: Femur Orientation [1]
In the Mach-1 Motion software click the “Show Camera” button at the bottom of the window.
Move the Mach-1 camera so that is perpendicular to the three point bending track and
perpendicular to the femur. This set up is shown in the image below. The camera’s macro lens
works best when it is positioned as far as possible from the sample, then it should be zoomed in
and focused until a clear image of the femur is provided on the screen.
Figure 4: Mach-1 Set Up With Camera
5.5 Three Point Bending Test
For the three point bending test, click the “Load Sequence” button on the left side of the window
and select the “Mouse_femur_3pt_bend”. If this sequence is not already created, it can be made
by combining four different functions in the following order: Find Contact, Zero Position, Zero
Load, Move Relative. The parameters for the Find Contact and Move Relative functions are
shown in the images below (the other functions do not require specifications).
178
Figure 5: Find Contact Parameters Figure 6: Move Relative Parameters
Note that these settings are optimized for testing in submerged liquid, they could be altered for
other conditions.
Once the sequence is loaded, ensure that the Move Relative data will be saved by selecting a
file location via the “Browse Data Files” button and naming a new text file. Similarly, to record
camera images during the move relative function the check box under the camera controls must
be checked. The data will not be automatically saved unless these are done.
Click “Manual Controls” and use the drop down menu to select the Z stage motion. Set the
velocity to low. Move the Z stage down until it is approximately 2mm away from the surface of
the femur. Close the manual controls window and click “Execute” on the left side of the screen.
The loading piece will move down 3 mm after coming into contact with the femur and the bone
will break. It may not break completely in half within the time period of the test. An image of this
is provided below.
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Figure 7: Post Test Broken Femur
Use the manual controls to raise the blade to the maximum position, allowing room for sample
retrieval. Use the tweezers to remove the broken femur from the test chamber. If the sample
needs to be preserved then wrap the bone in a strip of gauze, place in a MCT tube and apply
PBS solution with the squirt bottle until the gauze is soaked. This can then be stored in -80°C
freezer.
If more than 1 sample is tested, place the next sample in the testing chamber, move the z stage
down to 2mm away from the femur, create a new file location via “Browse Data Files” and click
“Execute” again. Make sure to change the file name for saving between tests.
5.6 Disassembly
Using the manual controls, move the Z stage to its highest position. Remove the loading piece
from the load cell. Remove the testing chamber from the XY stage and empty the PBS. Remove
all the screws and wash all components with soap. Once everything is removed from the Mach-
1 (except the load cell can be left in place), move the Z stage down to its lowest position. Exit
the motion software and power off the motion controller box with the switch in the back on the
left side. Disinfect any tools or surfaces that came into contact with the bone tissue and dispose
of the surgical pad, any contaminated gauze and gloves in biological waste.
6. DATA ANALYSIS
Open the Mach-1 Analysis software. Clicking on the yellow file folder in the top left section of the
window, select the folder that contains the data files from the three point bending test. Click on
one test sample file, then click on “Move Relative” which appear in tab under that test sample. A
curve should appear on the right side of the window. Using the drop down menus at the top of
the window, ensure that the y axis of the plot is force in gf (gram force) and that the X axis is
position of the Z stage in millimeters.
For this data analysis, five data values are collected from the Mach-1 Analysis software for each
sample that undergoes testing. These values are the overall maximum load, the yield load,
stiffness, the post-yield deformation (PYD), and the energy to fracture (ETF). The table
below shows how each of these values is represented on the Mach-1 Analysis curve.
180
Value
Description
Units
Max Load
The highest y value on the entire curve
gf
Yield
Load
The y value of the point where the curve deviates from linearity
gf
Stiffness
The slope of the linear portion of the curve
gf/mm
PYD
The ∆x between yield point and fracture point
mm
ETF
The area under the curve
gf*mm
To find the maximum load, set the Analysis drop down menu in the lower right to “Min & Max”
and move the cursors so that they are on either side of the entire graph. Record the max value.
This is shown in the image below.
Figure 8: Maximum Load
181
To find the yield load, leave the software on “Min & Max” and locate the point at which the left
side of the curve is no longer linear. Move the right hand cursor to this point and record the max
value. This is somewhat subjective and may vary depending on the curve.
Figure 9: Yield Load
182
To find the stiffness of the bone material, find the slope of the linear region of the graph. Leaving
one cursor on the yield point, move the left hand cursor up the curve a little to disregard the foot
region in the beginning of the curve. Change the Analysis drop down menu to “Slope” and
record the value.
Figure 10: Stiffness
Foot region
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To find the PYD, find the change in the position between the yield and the point of fracture. For
this change the Analysis drop down menu to “Cursors” and move the left hand cursor to the
point at which the curve drops off (the fracture point), while leaving the other cursor in place on
the yield point. Record the delta x value.
Figure 11: Post-Yield Displacement
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To find the ETF, find the area under the curve. Change the Analysis drop down menu to
“Integral” and place the cursors on either side of the curve. Record the integral value.
Figure 12: Energy to Fracture
These data values can be analyzed as they are or used to calculate mechanical properties of
the mouse femur, such as yield stress, ultimate stress, and Young’s modulus.
7. REFERENCES
[1]M. Silva, "Bone Mechanical Testing by Three-Point Bending", Washington University
Musculoskeletal Structure and Strength Core, 2016.
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8.4 Moment of Inertia MicroCT Analysis with Bone Protocol
VERSIONS
V1: Created by Katya Morozov (June 28th, 2018)
V1.2: Edited by Paige Woods (July 25th, 2018)
V.1.3: Edited by Erica Doyle (July 7th, 2022)
V.1.4: Edited by Nikhil Menon (January 10th, 2024)
1. EQUIPMENT AND SOFTWARE
External hard drive large enough to hold microCT data
Computer capable of processing large TIFF files
FIJI/ImageJ Software – December 2015 version (https://imagej.net/Fiji/Downloads)
BoneJ Plug-In for ImageJ (http://bonej.org/legacy)
Zip file with correct versions is uploaded to lab drive and on the external hard drive – “fiji-
java6-20170530”
Microsoft Excel - “MicroCT ImageJ Output Data Template” File
2. NOTES
Throughout the procedure, keep only one image window open in ImageJ, otherwise you may
accidentally compute calculations on the wrong image.
Before starting the procedure, open ImageJ ensure that the BoneJ plug-in is available. If not,
you should check that you are using the older version and restart the program. The BoneJ plug-
in is not compatible with the most recent version of ImageJ.
3. PREPARATION
Download all the TIFF image sequence files from the external hard drive to your computer’s
hard drive. Keep them separated in folders by sample number.
Before you start analysis, it is helpful to set up an excel spreadsheet to copy the data into.
Shown below is the template is used for this data analysis. One of these rectangular sections
should be made for each sample.
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Figure 1: Empty Data Sheet Template
The pink cell at the bottom of column L averages the 8 measurements for R1 to give a single
value for the distance from the centroid to the outer surface of the bone.
The yellow cell at the bottom of column N is set to average the 8 values for Imin, which are the
minimum values of the second moment of area around the major axis. Once all of the data has
been entered, this cell will show the final output for the moment of inertia (in mm^4) that can be
used in further calculations.
4.PROCEDURE – Without Code
1. File → import in Fiji → image sequence
a. Created new folder titled “Aligned hm1” based on the sample name
b. Double click on first image in tiff stack – keep everything the same in sequence
box
c. Select “ok” in the sequence options window
2. Image → Properties
a. Change the “Unit of length” to mm
b. Set “Pixel width”, “Pixel height”, and “Voxel depth” to match scan resolution
i. For Renata’s standard scan, all of these are 0.008
c. Confirm dimensions be going to Analyze → Set Scale
i. For Renata’s standard scan, scale should be 125 pixels/mm
3. Plug-ins → BoneJ → Optimize threshold
a. Check boxes for “threshold only” and “Apply Threshold”, keep all other settings
the same
b. Hit ok
4. Click process → noise → de-speckle
a. Click yes to warning box (you may lose some trabecular bone detail)
5. Crop bone in order to get rid of outside speckles
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a. Click box selection tool, and enclose the sample roughly an inch from bone
perimeter
i. Note: Make sure this crop is larger, if it's too small it will make cropping in
the future more difficult
b. Image → Crop
6. Edit → Invert
a. Do this to make bone white on a black background, which is necessary for
moments of inertia to run properly
b. "Yes" to warning
7. Click Plug ins → BoneJ → Moment of inertia
a. Leave bone min and max as is
b. Uncheck “Show axes (3D)”
c. Click "ok"
d. This is for re-aligning the bones along the z-axis and figuring out volume, it’s not
for calculating actual moment of inertia
8. Copy results from results pop-up into excel sheet and delete results from the ImageJ
table
9. New stack will appear that’s aligned (will be slightly off center)
10. Invert image again (make sure bone is white)
11. Crop out all of white space bordering sample
a. Use circle of square to surround sample and then click image → crop
b. Crop again if any outside white border is visible
c. Add black fills to leftover white gaps
12. Scroll through and figure out the last proximal slide where bone is intact (start at fracture
area and move proximally to first slide where bone is completely intact)
a. Subtract 30 slides from the "last proximal slice"
Ex) 89 is proximal last slice and 59 is proximal first slice
Ex) 247 is distal first slice and 277 is distal last slice
See Figure 2 for diagram of slices – should have a total of 4 slices per side, each
slice 10 slides apart
b. Record the slices on excel sheet
Figure 2: Bone Slices Diagram
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i. Ideally, the slices that the calculations are performed on should resemble
an ellipse as much as possible
ii. If trochanter is visible, this will distort centroid and MOI values, so only
compute “slice geometry” on distal side
iii. Similarly, if a significant amount trabecular bone is included on distal side
that is unable to be cropped out, only compute “slice geometry” on
proximal side
13. Re-save aligned stack
a. File → Save as → Image Sequence
b. Make sure you save as TIFF
c. Save in new "Aligned Folder" in same sample file
d. Add space after naming file then click ok
14. Do slice-by-slice artifact removal only on the 8 slices of interest if large artifacts or
trabecular bone detail needs to be removed from ROI
a. File → Open... to open individual image where ROI starts
i. In the folder, select and edit the images that are one slice less than the
actual slice (for some reason slice numbering differs by 1 in folder vs. in
ImageJ stack)
b. Use shape selection tool to enclose the artifacts
c. Press ctrl+F to fill in selection with black
d. Press ctrl+shift+o to open next slice
e. Pop up will ask if you want to save changes, click enter to confirm "yes"
f. Pop up will ask if you would like to replace image, press enter to confirm "yes"
g. Go to other end of bone and repeat process
h. Close aligned stack and initial stack that were used earlier
i. Will ask if you would like to save changes to the older aligned stack (pre
artifact removal), say "no"
15. Re-open aligned stack with removed artifacts
a. File → Import → Image Sequence…→ double click first image in new aligned
stack
b. Double check to make sure artifacts are indeed removed on correct slices
16. Go to correct slice you wish to calculate information on
a. Plug-ins → BoneJ → Slice geometry
b. Keep everything the same except change bone in drop-down menu to femur
c. Hit ok Note: This is to obtain PMI (Polar moment of Inertia in mm^4)
Example) slice 66
d. Results (including Imin and Imax) will pop up in new window along with
annotated image
i. Close image but keep Results window open to collect results
17. Add 10 slices to previous slice and repeat steps 16
Example) slice 76
18. Add another 10 slices and repeat steps 16
Example) slice 86
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19. Add another 10 slices and repeat steps 16
a. Example) slice 96
20. Record data from results window on excel sheet (row 9 on the template image, Figure 1)
i. Paste the data 1 row to the left of the table so the values align with the
proper columns
21. Repeat steps 16-20 starting at distal first slice
a. Example) Slice Geometry on slices 202, 212, 222, and 232
22. Calculate average Imin and Imax
23. Link R1 (centroid) and Imin (moment of inertia) values from microCT sheet to “Material
Properties from 3 Point Bend” sheet to determine material properties
5. PROCEDURE – With Code
1. File → import in Fiji → image sequence
a. Created new folder titled “Aligned hm1” based on the sample name
b. Double click on first image in tiff stack – keep everything the same in sequence
box
c. Select “ok” in the sequence options window
2. Plugins → Macros → Edit
a. Navigate to “macros” folder in fiji file
b. Open “Calculate-MOI.ijm” and “Calculate-slice-geometry.ijm”
3. Run Calculate-MOI.ijm
a. Click Run in editing window
b. To check if code worked properly:
i. There should be a subfolder called “aligned” with all the thresholded
aligned pictures. Check that the number of pictures in folder is same as in
program
ii. Image should be white bone on black background
4. Select final image stack and perform artefact removal on slices of interest as described
above in step 14
5. Run Calculate-slice-geometry.ijm on slices of interest
a. On left side of fracture, start macro on slice furthest away from fracture site
i. E.g. slice 66 if set is 66,76,86,96
b. On right side of fracture, start macro on slice closest to fracture site
i. E.g. slice 200 if set is 200, 210, 220, 230
c. Copy values to Excel spreadsheet
6. RESULTS
After this procedure the data sheet should look something like this:
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Figure 3: Completed Data Sheet
The values for the centroid distance (in pink) and the moment of inertia (in yellow) can be
inserted or linked to another data sheet for the purpose of calculating mechanical properties of
the bone sample.
7. TROUBLESHOOTING
● The imageJ online forum is useful in terms of finding solutions to commonly experienced
problems.
● For information on correctly setting the scale for the specific scanning resolution, this
website is a helpful resource.
● When dealing with large files, the program often requires more memory than the
computer initially allots. You can check your computer’s RAM and allocate up to 75% of
the ram by selecting Edit → Options → Memory & Threads and adding more to the
maximum memory.
● To speed up ImageJ, click on the imageJ status bar to collect all of the “garbage” and
increase the processing speed.
● If you are unsure about the measured values, the uCT lab also calculates MOI values,
which can be found on uCT data sheet for each sample, and these can be referenced to
ensure ImageJ is in the right range.
