Effects of Vitamin D, K1, K2 and Calcium on Bone Formation by Osteoblasts in Vitro
by
Charlene Elaine Lancaster
A thesis submitted in conformity with the requirements for the degree of Master of Science
Graduate Department of Cell and Systems Biology University of Toronto
© Copyright by Charlene Elaine Lancaster 2015
ii
Effects of Vitamin D, K1, K2 and Calcium on Bone Formation by
Osteoblasts in Vitro
Charlene Elaine Lancaster
Master of Science
Graduate Department of Cell and Systems Biology
University of Toronto
2015
Abstract
Bone loss is a major health problem that most people will face and thus research focusing on
enhancing bone formation is of great importance. Although there have been many cell biology
articles focusing on the effects of vitamin D, K1 or K2 on bone formation in vitro, there has yet to
be a consensus amongst the literature. Through the use of several meta-analyses of past vitamin
studies, we found that supplementation of vitamin D, K1 and K2, along with the combination of
K2 + 1,25D, increased mineralization, while not consistently changing all of the other parameters
associated with bone formation. In addition we quantified the area of von Kossa stained bone
nodules in calcium, vitamin K1, vitamin K2, 25D and 1,25D treated human and mouse osteoblast
cultures and found that mineralization levels varied depending on the presence of ascorbic acid
or the organism from which the cell lines were derived.
iii
Acknowledgments
I would first like to thank my supervisor, Dr. Rene Harrison, for her guidance, assistance writing
my thesis and sharing with me her substantial knowledge of cell biology. I learned not only
about cell biology, but also about myself during my Master’s degree in your lab. I would also
like to express my gratitude towards my committee, Dr. Bebhinn Treanor and Dr. Blake
Richards, for their helpful suggestions and guidance. In addition, I would like to thank Dr.
Mauricio Terebiznik, Dr. He song Sun, Dr. Aarthi Ashok and Dr. Shelley Brunt for their
feedback on the many projects that I juggled throughout my graduate studies. Furthermore, I
would like to express my gratitude to Dr. Marc Cadotte for teaching me the basics of statistics all
the way up to advanced statistics (at least for cell biologists!).
Many thanks go out to the students within Dr. Terebiznik’s and Dr. Treanor’s laboratories, who
supported me throughout my graduate studies. I am very fortunate to have the opportunity to
work with a brilliant group of students within Dr. Harrison’s laboratory. In particular, I would
like to thank Alex Sin, Kewei Xu, Sadek Shorbagi and Mathieu Poirier for teaching me many
new laboratory techniques, supporting me and filling my life with so much more laughter. I
would also like to thank Cara Fiorino and Urja Naik for providing me with me so much support,
for patiently teaching me techniques from day one onward and for always making time for me to
talk out all of my problems. You are two strong, intelligent female scientists, who I will forever
look up to and I know will accomplish so much in whatever field/career you chose to pursue in
the future.
Finally, I would like to express my extreme gratitude to my brother and my parents, who loved
me unconditionally, supported me throughout everything I have pursued, proofread every
assignment I have ever written (including this thesis!) and sacrificed so much of their own lives
for me to pursue my dreams. I could not have done any of this without you!
iv
Table of Contents
Acknowledgments.......................................................................................................................... iii
Table of Contents ........................................................................................................................... iv
List of Tables ................................................................................................................................. ix
List of Figures ..................................................................................................................................x
List of Appendices ....................................................................................................................... xiii
List of Abbreviations ................................................................................................................... xiv
1 INTRODUCTION ......................................................................................................................1
1.1 Osteoblast Mineralization and Collagen Production within Bone .......................................1
1.1.1 Bone Cells and Remodelling ...................................................................................1
1.1.2 Osteoblasts and Mineralization ................................................................................1
1.1.3 Collagen ...................................................................................................................3
1.2 Osteoporosis .........................................................................................................................3
1.3 Space ....................................................................................................................................4
1.4 Vitamins and Calcium..........................................................................................................4
1.4.1 Vitamin C or Ascorbic Acid ....................................................................................4
1.4.2 Vitamin D.................................................................................................................5
1.4.3 Vitamin K.................................................................................................................6
1.4.4 Calcium ....................................................................................................................7
1.5 Overview of Relevant Vitamin Literature: Cell Biology, Clinical and Animal Studies .....7
1.5.1 Cell Biology .............................................................................................................7
1.5.2 Animal Studies .........................................................................................................9
1.5.3 Clinical .....................................................................................................................9
v
1.6 Meta-analysis .....................................................................................................................10
1.7 Rationale and Hypotheses ..................................................................................................10
1.8 Relevance ...........................................................................................................................11
2 METHODS ...............................................................................................................................12
2.1 Meta-analysis and its Statistical Analysis ..........................................................................12
2.2 Reagents and Supplement Solution Preparation ................................................................13
2.3 Cell Culture ........................................................................................................................13
2.4 Treatment with AA and von Kossa (VK) Staining ............................................................14
2.4.1 AA-Primed Treatment ...........................................................................................14
2.4.2 Continual-AA Treatment .......................................................................................14
2.4.3 von Kossa Staining with Silver Nitrate Solution ...................................................14
2.4.4 Quantification ........................................................................................................15
2.5 Collagen Production...........................................................................................................15
2.6 Statistical Analysis of Experiments ...................................................................................16
2.6.1 Individual Vitamin/Calcium VK Statistics ............................................................16
2.6.2 Combination Vitamin/Calcium VK Statistics ........................................................16
2.6.3 Collagen Statistics ..................................................................................................17
3 RESULTS .................................................................................................................................18
3.1 Meta-analysis .....................................................................................................................18
3.1.1 Discussion of the homogeneity within the meta-analyses that were
subanalyzed by the type of experiment. .................................................................20
3.1.2 For the vitamin K1, K2, D and K2 + 1,25D meta-analyses, most of the overall
grand mean effect sizes and the experiment type grand mean effect sizes were
significantly greater than zero. ...............................................................................23
3.1.3 Discussion of the homogeneity within the meta-analyses that were
subanalyzed by cell type. .......................................................................................31
vi
3.1.4 The overall grand mean effect size and most of the cell type grand mean effect
sizes for the vitamin K2 and D meta-analyses were significantly greater than
zero. ........................................................................................................................33
3.2 Effects of vitamin/calcium supplementation on in vitro bone formation using a mouse
and human osteoblast cell line. ..........................................................................................38
3.2.1 Increasing concentrations of calcium resulted in increased bone mineralization
in continual-AA MC3T3 cultures, while bone nodule formation decreased
with increasing vitamin K2 and 1,25D levels. In contrast, mineralization did
not change in mouse osteoblast cultures upon vitamin K1 and 25D
supplementation. ....................................................................................................40
3.2.2 Total mineralized area increased with increasing levels of calcium in
continual-AA Saos-2 cultures, but did not change with the addition of
increasing concentrations of vitamin K1, vitamin K2, 25D and 1,25D. .................48
3.2.3 Bone nodule formation increased with increasing concentrations of calcium,
vitamin K1 and vitamin K2 in AA-primed MC3T3 cultures, while increasing
1,25D levels lead to decreasing bone mineralization. Conversely, the addition
of increasing concentrations of 25D had no effect on bone mineralization. .........55
3.2.4 The addition of 25D + K2 lead to decreased bone mineralization of continual-
AA treated MC3T3 cells as compared to both vitamin K2 and 25D alone. The
other combinations, under AA-primed and continual-AA conditions, resulted
in no change to the level of mineralization obtained from all the singular
vitamin or calcium controls. ..................................................................................63
3.2.5 Bone mineralization levels of Saos-2 cultures supplemented with
combinations of vitamins and calcium under AA-primed and continual-AA
conditions were unchanged as compared to all of the singular vitamin or
calcium controls. ....................................................................................................67
3.2.6 Increasing concentrations of calcium or vitamin did not lead to increased
collagen concentrations within MC3T3 or Saos-2 cultures. ..................................70
4 DISCUSSION ...........................................................................................................................73
4.1 Inferences that can be made from the results of the meta-analyses. ..................................73
4.2 Uncertainty in the use of proliferation measurements within the K2 meta-analysis. .........74
4.3 Considerations for the interpretation of the meta-analyses. ...............................................75
4.3.1 Homogeneity issues within the experiment and cell type subgroups. ...................75
4.3.2 Conflict between the results of the non-directional test and the confidence
interval test. ............................................................................................................75
vii
4.4 Mineralization in calcium or vitamin supplemented mouse, MC3T3, cell cultures
compared to human, Saos-2, cell cultures under continual-AA conditions. ......................76
4.4.1 Adverse effects of 1,25D supplementation on continual-AA treated mouse
osteoblasts as compared to human osteoblasts. .....................................................76
4.4.2 Vitamin K2 addition has different effects on continual-AA treated mouse
osteoblasts in comparison to human osteoblasts. ...................................................77
4.4.3 Human and mouse osteoblast mineralization increases with calcium
supplementation under continual-AA treatment. ...................................................77
4.4.4 The addition of vitamin K1 or 25D has no effect on the amount of
mineralization within the continual-AA treated MC3T3 and Saos-2 cultures. .....78
4.5 Implications of collagen production on mineralization within human and mouse cell
culture. ...............................................................................................................................79
4.6 Vitamin and calcium-induced bone nodule formation under AA-primed conditions as
compared to continual-AA conditions in murine MC3T3 cultures. ..................................80
4.6.1 Mineralization levels resulting from the addition of vitamin K1 or K2 varies
depending on the amount of AA within the MC3T3 culture. ................................80
4.6.2 Calcium supplementation leads to increased mineralization in AA-primed and
continual-AA treated MC3T3 cultures. .................................................................80
4.6.3 The addition of 25D or 1,25D has the same effect on mineralization in AA-
primed and continual-AA treated MC3T3 cultures. ..............................................81
4.7 Most combinations of vitamins and calcium did not have an effect on mineralization
in cultures of the mouse cell line, MC3T3, and the human cell line, Saos-2. ...................81
4.8 Limitations within the mineralization and collagen experiments. .....................................82
4.9 Comparison of our mineralization and collagen experiments with our meta-analyses. ....82
4.10 Advantages and disadvantages of our meta-analyses and bone formation experiments. ..84
4.11 Future Directions ...............................................................................................................84
4.11.1 Additional mineralization experiments and the utilization of other
measurements of mineralization. ...........................................................................84
4.11.2 Effect of vitamin and calcium on other bone cell parameters. ..............................85
4.12 Conclusion .........................................................................................................................85
References ......................................................................................................................................86
viii
Appendices ...................................................................................................................................102
ix
List of Tables
Page
Table 1 The homogeneity test results for the fixed-effects (Qf) and mixed-
effects (Qm) models of each vitamin meta-analysis, where the
meta-analyses are subanalyzed by type of experiment 22
Table 2 Summary of the results of the vitamin K1, K2, D and K2 + 1,25D
meta-analyses that were subanalyzed by type of experiment 30
Table 3 The fixed-effects (Qf) and mixed-effects (Qm) models’ homogeneity
test results of each vitamin meta-analysis, where the meta-analyses
are subanalyzed by cell type 32
Table 4 Summary of the results of the vitamin K2 and D meta-analyses that
were subanalyzed by cell type 37
Table 5 Summary of the results of the calcium or vitamin supplemented
bone formation experiments run on the continual-AA treated MC3T3
cultures 47
Table 6 Summary of the results of the calcium or vitamin supplemented
bone formation experiments run on the continual-AA treated Saos-2
cultures 54
Table 7 Summary of the results of the calcium or vitamin supplemented
bone formation experiments run on the AA-primed MC3T3
cultures 62
x
List of Figures
Page
Figure 1 Overview of meta-analyses performed 19
Figure 2 The overall grand mean effect size and the experiment type grand
mean effect sizes for the vitamin K1 meta-analysis were significantly
greater than zero 26
Figure 3 The majority of the experiment type grand means and the overall
grand mean effect size were significantly positive for the vitamin K2
meta-analysis 27
Figure 4 The overall grand mean effect size and most of the experiment type
grand mean effect sizes were significantly greater than zero for the
vitamin D meta-analysis 28
Figure 5 The overall grand mean effect sizes for the meta-analyses comparing
the results of the combination of vitamin K2 + 1,25D against
vitamin K2 and 1,25D alone were significantly positive 29
Figure 6 The overall grand mean effect size and most of the cell type grand
mean effect sizes were significantly greater than zero for the
vitamin K2 meta-analysis 35
Figure 7 The majority of the cell type grand mean effect sizes and the overall
grand mean effect were significantly positive for the vitamin D
meta-analysis 36
Figure 8 Overview of experiments executed 39
Figure 9 Increasing the concentration of calcium lead to increased bone
mineralization in continual-AA MC3T3 cultures 42
xi
Figure 10 Bone mineralization did not change with increasing levels of
vitamin K1 in continual-AA MC3T3 cultures 43
Figure 11 Increasing vitamin K2 concentration in continual-AA MC3T3
Cultures resulted in decreased bone nodule formation 44
Figure 12 Bone mineralization of continual-AA treated MC3T3 cultures did
not change with increasing 25D concentration 45
Figure 13 Total mineralized area of continual-AA MC3T3 cultures
decreased with increasing levels of 1,25D 46
Figure 14 Increasing levels of bone mineralization were associated with
increasing concentrations of calcium in continual-AA Saso-2
cultures 49
Figure 15 Increasing concentrations of vitamin K1 in continual-AA Saos-2
cultures did not lead to increased bone nodule formation 50
Figure 16 Total mineralized area did not change with increasing levels of
vitamin K2 in continual-AA Saos-2 cultures 51
Figure 17 Increasing levels of 25D in continual-AA Saos-2 cultures resulted
in no change in bone mineralization 52
Figure 18 Bone nodule formation did not change with increasing
concentrations of 1,25D in continual-AA Saos-2 cultures 53
Figure 19 Bone mineralization increased with increasing concentrations of
calcium, when MC3T3 cells were primed with AA 57
Figure 20 Increasing levels of vitamin K1 resulted in greater bone nodule
formation in AA-primed MC3T3 cultures 58
Figure 21 Total mineralized area of AA-primed MC3T3 cultures increased
with increasing concentrations of vitamin K2 59
xii
Figure 22 Increasing concentrations of 25D did not lead to increased bone
mineralization of AA-primed MC3T3 cells 60
Figure 23 Bone mineralization of AA-primed MC3T3 cultures decreased
with increasing levels of 1,25D 61
Figure 24 The supplementation of 25D + K2 resulted in significantly
decreased bone mineralization of continual-AA treated MC3T3
cells as compared to both vitamin K2 and 25D alone. The other
combinations caused no change to the level of mineralization
obtained from all the singular vitamin or calcium controls 65
Figure 25 Amount of MC3T3 mineralization was unchanged for
combinations of vitamins and calcium under AA-primed
conditions as compared to all the appropriate singular vitamin
or calcium controls 66
Figure 26 Combinations of vitamins and calcium caused no change to the
level of mineralization obtained from all the singular vitamin/
calcium supplemented, continual-AA treated, Saos-2 cultures 68
Figure 27 Mineralization levels of Saos-2 cells treated with combinations of
vitamins and calcium under AA-primed conditions were unchanged
as compared to all of the singular vitamin or calcium controls 69
Figure 28 Increasing concentrations of calcium or vitamin did not lead to
increased collagen concentrations within MC3T3 cultures 71
Figure 29 Collagen levels within Saos-2 cultures did not change with
increasing concentrations of calcium or vitamin 72
xiii
List of Appendices
Page
Supplemental Table 1 Articles used in the vitamin K1, K2, D and K2 + 1,25D
meta-analyses 102
Supplemental Methods Meta-analysis Equations 103
Supplemental Figure 1 Overview of the quantification method used to
determine the total mineralized area for each image 107
Supplemental Figure 2 The supplementation of 25D + K2 resulted in
decreased bone mineralization of continual-AA
treated MC3T3 cells as compared to both vitamin K2
and 25D alone. The other combinations caused no
change to the level of mineralization obtained from
all the singular vitamin or calcium controls 108
Supplemental Figure 3 Amount of MC3T3 mineralization was unchanged for
combinations of vitamins and calcium under
AA-primed conditions as compared to all the
appropriate singular vitamin or calcium controls 110
Supplemental Figure 4 Combinations of vitamins and calcium caused no
change to the level of mineralization obtained from
all the singular vitamin/calcium supplemented,
continual-AA treated, Saos-2 cultures 112
Supplemental Figure 5 Mineralization levels of Saos-2 cells treated with
combinations of vitamins and calcium under
AA-primed conditions were unchanged as compared
to all of the singular vitamin or calcium controls 114
xiv
List of Abbreviations
1,25D 1,25-dihydroxyvitamin D
25D 25-hydroxyvitamin D
AA L-ascorbic acid
AIC Akaike’s information criterion
ALP Alkaline phosphatase
BMD Bone mineral density
CI Confidence interval
df Degrees of freedom
DMEM Dulbecco’s modified Eagle’s media
DNA Deoxyribonucleic acid
Grand mean effect size of ALP activity experiments
Grand mean effect size of collagen levels experiments
Grand mean effect size of DNA levels experiments
Grand mean effect size of human cell line experiments
Grand mean effect size of human primary cells experiments
Overall grand mean effect size
Grand mean effect size of murine cell line experiments
Grand mean effect size of mineralization experiments
Grand mean effect size of murine primary cells experiments
Grand mean effect size of osteocalcin levels experiments
Grand mean effect size of osteopontin levels experiments
xv
Grand mean effect size of other experiments
Grand mean effect size of proliferation experiments
ER Endoplasmic reticulum
FBS Fetal bovine serum
FOXO Forkhead box O
Gla Gamma-carboxyglutamic acid
HCl Hydrochloric acid
MEM α Minimum essential media alpha
MGP Matrix Gla-protein
MSC Mesenchymal stem cells
NaOH Sodium hydroxide
NS No significance
PBS Phosphate-buffered saline
PFA Paraformaldehyde
ROS Reactive oxygen species
RXR Retinoic acid X receptor
SEM Standard error of the mean
VDR Vitamin D receptor
VDRE Vitamin D response elements
VK von Kossa
1
1 INTRODUCTION
1.1 Osteoblast Mineralization and Collagen Production within Bone
1.1.1 Bone Cells and Remodelling
Bone is a strong, light-weight, highly dynamic connective tissue, which is composed of three cell
types: osteoblasts, osteocytes and osteoclasts. Osteoblasts are the cells that are responsible for
the production of the calcified extracellular matrix. Osteoblasts make and secrete large quantities
of collagen that organize to form a fibrillar network, as well as other extracellular proteins (i.e.
osteocalcin; Long, 2012). The osteoblasts that become entombed in the extracellular matrix are
considered osteocytes. Osteocytes, the most common cell found within bone, communicate with
each other through canaliculi and play a role in mechanotransduction, detection of damage and
repair of bone (Han, Cowin, Schaffler, & Weinbaum, 2004). Osteoclasts are large,
multinucleated cells that are involved in the resorption of the extracellular matrix. The secretion
of protons results in the acidification of the Howship’s lacuna, which leads to the decalcification
of the extracellular matrix (Nakamura, 2007). Additionally, the secretion of lysosomal enzymes
degrades the organic portion of the extracellular matrix (Nakamura, 2007).
The amount of bone mass in a human body is maintained through an equal balance of bone
formation by osteoblasts and bone resorption by osteoclasts. It is only when the balance between
the two processes is shifted that a disease results. Adult bone turnover in cortical bone, dense
bone that surrounds the marrow space, occurs at a rate of 2-3%/year, while the rate of turnover in
trabecular bone, bone in a lattice pattern within the bone marrow space, is higher due to the
greater involvement of trabecular bone in mineral metabolism (Clarke, 2008).
1.1.2 Osteoblasts and Mineralization
Osteoblasts are derived from pluripotent mesenchymal stem cells (MSCs), which are located on
the abluminal side of blood vessels (Pontikoglou, Deschaseaux, Sensebé, & Papadaki, 2011).
The maturation of osteoblasts depends on two processes: proliferation and differentiation.
Proliferation plays a large role in the early stages of osteoblast maturation (from the MSC stage
to the committed osteoprogenitor stage) and begins to slow down through the later maturation
2
stages (Neve, Corrado, & Cantatore, 2011). Proliferation is an important component of the
maturation of osteoblasts, since osteoblast differentiation and mineralization starts to occur only
after the cells reach confluence (Whitson et al., 1984; Whitson, Whitson, Bowers, & Falk, 1992).
It is believed that cell-cell contacts are the reason for the induction of osteoblast differentiation in
high confluence, AA-treated cultures (Pustylnik et al., 2013). Differentiation begins in the
committed osteoprogenitor stage and continues through to the osteocyte stage, which is
considered to be the terminal differentiation stage of osteoblast maturation (Nakamura, 2007).
Preosteoblasts begin to differentiate based on the presence of several compounds, including
ascorbic acid (AA), dexamethasone, β-glycerophosphate and ipriflavone (Benvenuti et al., 1991;
Czekanska, Stoddart, Richards, & Hayes, 2012). Different stages of osteoblast differentiation can
be characterized by the expression of certain genes (Beck, 2003). Early differentiation is defined
by the expression of high levels of the enzyme alkaline phosphatase (ALP), while late
differentiation is characterized by the expression of osteopontin and osteocalcin (Beck, 2003).
Besides their involvement in the formation of the fibrillar network, osteoblasts also play a role in
the deposition of minerals, in the form of hydroxyapaptite. Hydroxyapaptite crystals are
composed of calcium hydroxyphosphate and there has been much debate about how the crystals
propagate onto the fibrillar extracellular matrix. One theory suggests that matrix vesicles bud
from the plasma membrane and accumulate inorganic calcium and phosphate ions extracellularly
(Anderson, 1995). The hydroxyapaptite crystals form within the vesicles and are deposited onto
the fibrillar network, after the vesicles rupture (Anderson, 1995). A more recent theory suggests
that calcium phosphate stored within the mitochondria is moved to the extracellular matrix using
vesicles that originate from within the cell (Boonrungsiman et al., 2012). The vesicles then
rupture and release calcium phosphate onto the organic network, where it forms the
hydroxyapaptite crystals (Boonrungsiman et al., 2012).
The balance between inorganic phosphate and pyrophosphate levels within bone is critical for
mineralization to occur (Sapir-Koren & Livshits, 2011). Pyrophosphate, which is a by-product of
many metabolic reactions, is a known inhibitor of bone formation (Sapir-Koren & Livshits,
2011). In contrast, inorganic phosphate is needed to produce the hydroxyapaptite crystals. Any
shift within the balance between pyrophosphate and inorganic phosphate within the human body
can lead to diseases that are characterized by insufficient bone mineralization (Sapir-Koren &
3
Livshits, 2011). The increased pool of inorganic phosphate seen within bone is thought to be the
result of the catalytic activity of the enzyme ALP (Beck, 2003).
1.1.3 Collagen
Collagen is a structural biopolymer composed of three procollagen polypeptide strands arranged
to form a right hand triple helical structure (Sherman, Yang, & Meyers, 2015). It is because of
this rope-like helical structure that collagen has the strength it needs to provide structural support
to various parts of the body, including bone, cartilage, dentin and tendons (Sherman et al., 2015).
Collagen is a very abundant protein within the body, where it comprises up to 30% of the mass
of vertebrates (Sherman et al., 2015). Within bone, type I collagen is produced by osteoblasts
and constitutes up to 90% of the organic portion of the extracellular matrix (Sherman et al.,
2015). The synthesis of collagen requires many post-translational modifications (Kivirikko &
Myllylä, 1985). Two of the most critical modifications are the hydroxylation of proline and
lysine residues, given that these modifications help to form the stable triple helices and the
formation of the intra- and inter-molecular cross-links that help to stabilize the collagen fibrils,
respectively (Kivirikko & Myllylä, 1985).
1.2 Osteoporosis
Osteoporosis is a disorder caused by the imbalance of bone formation by the osteoblasts and
bone resorption by the osteoclasts, which ultimately results in changes to the microarchitecture
of the bone (Lau & Guo, 2011). The disorder was originally classified into two categories:
primary osteoporosis, which is bone loss associated with age or hormonal changes (ex.
Postmenopausal Osteoporosis), or secondary osteoporosis, which is bone loss resulting from a
chronic illness (ex. diabetes or immobilization due to an illness) (Lau & Guo, 2011). Disuse
osteoporosis is the result of decreased mechanical loading on the skeleton and can be caused by
immobilization or lack of gravitational forces (Lau & Guo, 2011). It was estimated in 2004 that
35% of postmenopausal Caucasian women have osteoporosis of their hip, spine or distal forearm
(Office of the Surgeon General (US)., 2004), while the prevalence of disuse osteoporosis is
unknown.
Mechanical forces on the bone are sensed by osteocytes, which signal to osteoblasts to build
bone, but in the absence of mechanical loads, osteocytes will signal osteoclasts to resorb bone
4
(Klein-Nulend, Bacabac, & Bakker, 2012). Osteocytes are surrounded by an extracellular space
called lacuna and nearby lacuna are connected by canals called canaliculi. Mechanical loads
cause interstitial fluid within the lacuna to flow through the lacuna-canalicular system, resulting
in conformational changes in osteocyte structures, including stretch-activated ion channels,
integrins and cell-cell adhesions (Klein-Nulend et al., 2012). This allows the influx or efflux of
ions into the osteocyte processes (cell protrusions allowing for cell-cell contact) or the activation
of signalling cascades, both leading to changes in cell morphology (Klein-Nulend et al., 2012).
1.3 Space
Microgravity can affect many physiological processes within the human body, including bone
remodelling (Tamma et al., 2009). During a flight, astronauts can lose 1 to 2% of their bone mass
per month, where most of the bone is lost from the load-bearing regions of the legs and lumbar
spine (Tamma et al., 2009). This loss of bone mass is the result of increased resorption by
osteoclasts and decreased bone formation by osteoblasts (Nabavi, Khandani, Camirand, &
Harrison, 2011). Osteoblasts in microgravity have short, wavy microtubules leading to decreased
focal adhesion sites and decreased functionality of the osteoblasts (Nabavi et al., 2011).
However, there are more resorption pits caused by osteoclasts in microgravity as compared to
ground controls, indicating increased functionality of osteoclasts in microgravity (Nabavi et al.,
2011).
1.4 Vitamins and Calcium
1.4.1 Vitamin C or Ascorbic Acid
Vitamin C or ascorbic acid (AA) is a water-soluble vitamin involved in the synthesis of collagen
and functions also as an antioxidant (Du, Cullen, & Buettner, 2012; Padayatty et al., 2003). In
both functions the anion of ascorbic acid, called ascorbate, acts as a reducing agent. In collagen
synthesis, ascorbate is required to maintain the full activity of the proline and lysine
hydroxylation enzymes (Du et al., 2012; Kivirikko & Myllylä, 1985). As an antioxidant,
ascorbate donates an electron to reactive oxygen species (ROS), including hydroxyl radicals and
peroxyl radicals, thus preventing further oxidative damage to the cells, proteins or lipids (Du et
al., 2012). The addition of ascorbic acid to osteoblasts in cell culture increased collagen synthesis
and accumulation within the culture (Franceschi & Iyer, 1992). The increase in collagen levels
5
resulted in increased gene expression of two osteoblast markers, ALP and osteocalcin
(Franceschi & Iyer, 1992). Therefore AA also plays an indirect role in the differentiation of
osteoblasts (Franceschi & Iyer, 1992).
Most mammals have the ability to produce ascorbic acid from glucose within their liver
(Padayatty et al., 2003). However, the ability to synthesize ascorbic acid was lost in humans,
other primates and guinea pigs, due to a mutation in the enzyme gulonolactone oxidase
(Padayatty et al., 2003). Thus vitamin C is an essential nutrient for humans, meaning that it must
be ingested in order to survive.
Scurvy is the oldest acknowledged nutritional deficiency disease and is the result of insufficient
ascorbic acid levels within the body (Agarwal, Shaharyar, Kumar, Bhat, & Mishra, 2015). The
earliest manifestations of scurvy include low grade fever and irritability, but will eventually
escalate to serious symptoms such as bleeding gums, bone loss and poor wound healing
(Agarwal et al., 2015). If these symptoms are ignored, scurvy will eventually lead to death
(Agarwal et al., 2015).
1.4.2 Vitamin D
Vitamin D is found in two major forms: D2, which is obtained from the ingestion of plants and
fungi, and D3, which is both synthesized in the skin and obtained through the consumption of
other animals (Stephensen et al., 2012). Although these two forms differ in their side chain
structure, both function as a prohormone (Jones, Strugnell, & DeLuca, 1998), eventually leading
to elevated calcium and phosphate serum levels within the body (Jones et al., 1998). Within the
skin a precursor of cholesterol, 7-dehydrocholesterol, is converted into vitamin D3 as a result of
exposure to ultraviolet radiation from sunlight (Jones et al., 1998). Both vitamin D2 and D3 are
first hydroxylated in the liver to form 25-hydroxyvitamin D (25D), which is the major circulating
form of vitamin D (Jones et al., 1998). The second hydroxylation event occurs in the kidneys
resulting in the formation of 1,25-dihydroxyvitamin D (1,25D), which is the active form of
vitamin D (Jones et al., 1998). Although only 1,25D is active, 1,25D, 25D and vitamin D (the
prohormone) can diffuse freely through the plasma membrane of cells (Jensen, 2014).
6
1,25D enters the nucleus and binds the vitamin D receptor (VDR), which is a nuclear
transcription factor (Shearer, 1997). This binding promotes its association with retinoic acid X
receptor (RXR) and the VDR-RXR complex subsequently binds to DNA sequences, known as
vitamin D response elements (VDRE), which modulate the transcription of certain genes
(Kimmel-Jehan, Jehan, & DeLuca, 1997). Increased levels of 1,25D in the serum results in
increased intestinal absorption of calcium, increased reabsorption of calcium filtered by the
kidneys and mobilization of calcium from bone if serum calcium levels cannot be met (Jones et
al., 1998).
1.4.3 Vitamin K
Vitamin K functions as a cofactor for the enzyme gamma-carboxylase, which carboxylates
glutamic acid and results in its conversion to gamma-carboxyglutamic acid (Gla) (Hamidi, Gajic-
Veljanoski, & Cheung, 2013). Gla residues activate the proteins that contain them (Hamidi et al.,
2013). There are three vitamin K-dependent proteins found in bone: osteocalcin, matrix Gla
protein (MGP) and protein S (Hamidi et al., 2013). Osteocalcin, which is a protein essential for
the formation of hydroxyapaptite crystals, needs the gamma-carboxylation of three glutamic acid
residues in order to bind mineral (Hamidi et al., 2013). MGP prevents the calcification of soft
tissue and cartilage and plays a role in normal bone growth (Shearer, 1997), while protein S is an
anticoagulant and its deficiency results in osteonecrosis, where bone tissue dies due to lack of
blood supply (Pierre-Jacques, Glueck, Mont, & Hungerford, 1997) .
There are two forms of vitamin K: vitamin K1 and vitamin K2. Vitamin K1 is synthesized by
plants and thus humans obtain it through consumption of green leafy vegetables, fruits, herbs,
teas, vegetables in the Brassica genus and plant oils (Hamidi et al., 2013). Vitamin K1 is the
major form of vitamin K in the human diet (Hamidi et al., 2013). Vitamin K2 includes a range of
forms, where members are known as menaquinones-n and n is the number of repeating 5-carbon
units (Hamidi et al., 2013). Most menaquinones are produced by bacteria and obtained by
consuming fermented foods (Hamidi et al., 2013). Although menaquinone-4 has a low
bioavailability from food, it is the main form of vitamin K within the human body (Hamidi et al.,
7
2013). It is thus hypothesized that menaquinone-4 can be produced through the conversion of
vitamin K1, menaquinone-7, 8 and 9 (Beulens et al., 2013).
1.4.4 Calcium
Over 99% of the calcium in the body is found within bones and teeth as hydroxyapaptite
(Peacock, 2010). Calcium is involved in many processes within the body including intracellular
signalling, muscle function and nerve transmission (Peacock, 2010). Serum calcium levels are
tightly maintained between 2.2 mM and 2.6 mM (Peacock, 2010). When serum calcium levels
are below 2.2 mM, it leads to a disorder called hypocalcemia, while if levels are above 2.6 mM,
it results in a disorder called hypercalcemia (Peacock, 2010). Both disorders can lead to severe
symptoms, such as seizures, for hypocalcemia (Alhefdhi, Mazeh, & Chen, 2013), or coma, for
hypercalcemia (Ziegler, 2001).
1.5 Overview of Relevant Vitamin Literature: Cell Biology, Clinical and Animal Studies
1.5.1 Cell Biology
The effect of vitamin K1, K2 and D addition to osteoblast maturation parameters that are
indicative of bone formation in cell culture was determined for four cell types: murine cell lines
(where the term murine includes mice and rats), human cell lines, murine primary cells and
human primary cells. The addition of vitamin K2 to murine cell lines increased ALP activity,
osteocalcin levels, calcium levels and protein content and decreased proliferation in some studies
(Akedo et al., 1992; M Yamaguchi, Sugimoto, & Hachiya, 2001), but had no effect on
osteocalcin levels and ALP activity in other articles (Ichiro Iwamoto, Kosha, Fujino, & Nagata,
2002; Ozeki, Aoki, & Fukui, 2008). Vitamin D supplementation also had variable effects on
some parameters in murine cell lines, where in some articles vitamin D addition increased ALP
activity, calcium levels, collagen levels and mineralization staining (Matsumoto et al., 1991;
Ozeki et al., 2008; F. Sato et al., 1991; Widaa, Brennan, O’Gorman, & O’Brien, 2014), but in
other studies decreased mineralization (Masayoshi Yamaguchi & Weitzmann, 2012) or had no
effect on ALP activity (Widaa et al., 2014).
8
The addition of vitamin K2 to human cell lines increased collagen levels and ALP activity, but
decreased apoptotic cell death (Akedo et al., 1992; T. Ichikawa, Horie-Inoue, Ikeda, Blumberg,
& Inoue, 2006; Urayama et al., 2000). However, vitamin K2 supplementation had conflicting
effects on proliferation, where an article reported decreased proliferation (Akedo et al., 1992)
and another article found that vitamin K2 had no effect on proliferation (Urayama et al., 2000).
The addition of vitamin D to human cell lines increased ALP activity, osteocalcin levels,
collagen levels and calcium levels (Franceschi, Romano, & Park, 1988; R Narayanan, Smith, &
Weigel, 2002; van Driel et al., 2006; Woeckel et al., 2010), while it was also reported that
vitamin D had no effect on DNA synthesis or calcium levels (Adluri, Zhan, Bagchi, Maulik, &
Maulik, 2010).
There was only one article looking at the effect of vitamin K1 supplementation on osteoblast
maturation parameters in murine primary cell cultures and they found that vitamin K1 addition
had no effect on ALP activity (Notoya, Yoshida, Shirakawa, Taketomi, & Tsuda, 1995). The
addition of vitamin K2 to murine primary cells increased calcium and total DNA levels (M
Yamaguchi et al., 2001), but had variable effects on ALP activity leading to either an increase in
ALP activity with vitamin K2 supplementation (M Yamaguchi et al., 2001) or had no effect
(Notoya et al., 1995). There was only one article that tested the result of vitamin D
supplementation on bone formation in murine primary cell cultures and they found that vitamin
D addition decreased mineralization staining compared to an untreated control (Masayoshi
Yamaguchi & Weitzmann, 2012).
The addition of vitamin K1 to human primary cells increased calcium and phosphate levels, had
no effect on ALP activity and decreased cell viability (Atkins, Welldon, Wijenayaka, Bonewald,
& Findlay, 2009; Gigante et al., 2008; Koshihara, Hoshi, Ishibashi, & Shiraki, 1996). Vitamin K2
supplementation to human primary cell cultures increased calcium levels, phosphate levels,
mineralization staining and osteocalcin levels, decreased apoptotic cell death and had no effect
on ALP activity (Atkins et al., 2009; Koshihara et al., 1996; Sugimoto, Hirakawa, Ishino,
Takeno, & Yajin, 2007; Urayama et al., 2000). Increased calcium levels, decreased proliferation,
and no change to the phosphate levels in human primary cell cultures were the result of vitamin
9
D supplementation (Atkins et al., 2007; Koshihara et al., 1996). However, the effect of vitamin D
addition to osteocalcin levels, mineralization staining and ALP activity in human primary cells
was variable, where supplementation either increased mineralization staining, osteocalcin levels
and ALP activity (Koshihara et al., 1996; Koshihara & Hoshi, 1997; Zhou et al., 2012) or had no
effect on those parameters (Koshihara et al., 1996; Sugimoto et al., 2007).
1.5.2 Animal Studies
The effect of vitamin K1, K2 and D addition was tested on bone mineral density (BMD) of many
different mice or rat models that were induced to lose bone mass, such that they modelled bone
loss due to hormonal changes (ovariectomy), loss of bone loading (sciatic neurectomy, hind-limb
unloading) or drug-induced bone loss (phenytoin). There was only one animal study using
vitamin K1 supplementation and they found that BMD levels did not change within rats whose
diet was supplemented with vitamin K1 (Binkley, Krueger, Engelke, Crenshaw, & Suttie, 2002).
The addition of dietary vitamin K2 to rodents had variable effects and either increased BMD
(Akiyama, Hara, Kobayashi, Tomiuga, & Nakamura, 1999; Asawa et al., 2004; Ichiro Iwamoto
et al., 2002; Iwasaki, Yamato, Murayama, Sato, et al., 2002; Iwasaki, Yamato, Murayama,
Takahashi, et al., 2002; Iwasaki-Ishizuka et al., 2005; Onodera, Takahashi, Wakabayashi, Kamei,
& Sakurada, 2003) or had no effect compared to an untreated control (Binkley et al., 2002;
Sasaki et al., 2010). There was also only one animal study looking at vitamin D supplementation
and they found that vitamin D addition to diets increased BMD in rats (Ramesh Narayanan et al.,
2004).
1.5.3 Clinical
Vitamin K1, K2 and D supplementation’s effect on bone mineral density (BMD) was assessed on
several groups of human patients, including osteoporotic patients, postmenopausal women and
healthy individuals. The addition of vitamin K1 had no effect on the BMD of patients (Bolton-
Smith et al., 2007; Braam, Knapen, Geusens, Brouns, & Vermeer, 2003). However, the result of
vitamin K2 supplementation had variable effects on patients, where in some studies BMD was
increased with vitamin K2 addition (Iketani et al., 2003; Orimo et al., 1998; Somekawa,
Chigughi, Harada, & Ishibashi, 1999; Ushiroyama, Ikeda, & Ueki, 2002; Yonemura, Fukasawa,
Fujigaki, & Hishida, 2004) and in others BMD did not change (Emaus et al., 2010; I Iwamoto et
10
al., 1999; Knapen, Schurgers, & Vermeer, 2007). Similarly, the addition of vitamin D had
conflicting effects on patients, where vitamin D supplementation was reported to increase BMD
(Ushiroyama et al., 2002; Yonemura et al., 2004) or have no effect on BMD (Somekawa et al.,
1999).
1.6 Meta-analysis
Meta-analysis is an quantitative analysis procedure that is used to mathematically combine
results from previous research articles to make conclusions regarding that field of study (Garg,
Hackam, & Tonelli, 2008; Haidich, 2010). Meta-analyses are commonly used in medical
research in order to make decisions regarding treatment when results of the previous literature
are diverse and conflicting (Haidich, 2010). In addition, the field of ecology readily uses meta-
analyses, as they help readers to explore heterogeneity, identify patterns and allow researchers to
make decisions using the pooled data, all of which would not be possible using individual studies
(Stewart, 2010).
1.7 Rationale and Hypotheses
Just like in the medical and ecological fields, meta-analyses could be used in the cell biology
field to determine the overall effect of a treatment on certain cells, when the previous literature is
diverse and conflicting. In particular, the effect of vitamin K1, D and/or K2 supplementation on
osteoblast maturation parameters in vitro, such as mineralization and ALP activity, is unknown
because the results of past articles fail to consistently agree with one another. Thus, we decided
to undertake several meta-analyses to conclusively determine the effect of vitamin D, K1, K2 and
the combination of K2 + D on several bone formation parameters in different types of osteoblasts
(i.e. cell line vs. primary cells).
Many studies have looked at the effect of vitamin D, K1, K2 and calcium on bone formation in
cell culture, but very few studied all of these treatments within the same paper using the same
measure of osteoblast maturation. In addition, both human and mouse osteoblast cells were used
interchangeably within the literature without the consideration of interspecies differences in the
requirement of the vitamins and calcium. We addressed these holes in the literature by
determining the effect of different concentrations of vitamin D, K1, K2 and calcium on bone
11
mineralization and collagen production within a mouse preosteoblast cell line, MC3T3-E1, and
human osteosarcoma cell line, Saos-2. The concentrations that were chosen for each vitamin and
calcium have either been used in other cell biology studies or are physiologically relevant
concentrations within human serum. The effect of combinations of vitamin D, K1, K2 and
calcium on bone nodule formation in vitro was also tested to see if the combinations resulted in
increased mineralization beyond that of the singular vitamin/calcium effects. We hypothesized
that each vitamin or calcium alone would increase mineralization and collagen production. In
addition, we hypothesized that some combinations of the vitamins and calcium would further
enhance bone mineralization beyond that of the mineralization obtained from a singular vitamin
or calcium control.
1.8 Relevance
Given the prevalence of osteoporosis on earth, studies looking at ways to enhance bone
formation and/or diminish bone resorption are of high importance. In addition, humans are now
planning longer missions into space leading to greater bone loss within astronauts. Learning
about which supplements enhance bone formation in vitro could not only help osteoporosis
patients on earth, but also help astronauts in a microgravity environment.
12
2 METHODS
2.1 Meta-analysis and its Statistical Analysis
An extensive literature search was used to find cell biology studies that looked at the effect of
vitamin D, K1 and/or K2 on osteoblast maturation parameters, including amount of
mineralization, collagen and osteocalcin. For the studies where the data was not available
numerically, the program Plot Digitizer (version 2.6.6, http://plotdigitizer.sourceforge.net/) was
used to extract the information from the figures. All of the papers included some measure of
variation (i.e. standard deviation) and the number of replicates used to produce the mean
measurement. The studies used for this analysis are listed in Supplemental Table 1.
The statistical approach and the equations used are adapted from Cadotte (2006; see
Supplemental Methods for equations utilized). Meta-analysis allows one to determine the overall
effect size of a phenomenon by compiling the results of the related independent studies. In this
case the phenomenon that was characterized was the effect of vitamin D, K1 or K2 on osteoblast
maturation. For each experiment, k, a Hedges’ d value was calculated to determine the effect size
in terms of an unbiased standardized mean difference between a vitamin treated and untreated
group. In most cases, with the exception of measures of cytotoxicity and apoptosis (where a
decrease in value indicates greater osteoblast survival), the mean control values were subtracted
from the mean treated values. A positive effect size thus indicated that vitamin addition
increased a parameter of osteoblast maturation (i.e. amount of mineralization). Individual
experiment effects were then combined into a grand mean effect. Subanalysis was used to further
break the data into smaller groups to assess the effect of the vitamins on a specific osteoblast
maturation parameter or cell type.
Homogeneity of the experiments analyzed in the meta-analysis increases the confidence that the
overall grand mean represents any study looking at the same phenomenon. In order to assess the
homogeneity of the treatment responses, Cochran’s Q test was utilized, where p < 0.05 was
considered significant. If the effects were considered homogeneous (Cochran’s Q was not
significant), a fixed-effects model was used to calculate the grand mean effect. However, if the
effects were considered heterogeneous, a mixed-effects model was utilized to calculate the grand
mean effect. A mixed-effects model differs from that of a fixed-effects model in that it
13
incorporates an estimate of between experiment variance. If the mixed-effects model was used, a
final Cochran’s Q test was employed to determine the homogeneity of this model.
For each grand mean effect and Hedges’d a 95% confidence interval was constructed and
observed as to whether it intersected zero, as this would indicate that the effect size is not
significantly different than zero. As another assessment of the effects’ difference from zero, a
non-directional test, similar to a χ2 test was employed as an independent estimate of the p-values
(where p < 0.05 indicated significance). In this test, either the variances or adjusted variances
were used depending on if a fixed or mixed model was utilized to calculate the grand mean.
2.2 Reagents and Supplement Solution Preparation
Fetal Bovine Serum (FBS) and Dulbecco’s Modified Eagle’s Media (DMEM) were obtained
from Wisent Inc. (St-Bruno, Quebec). Minimum Essential Media alpha (MEM α) without AA
and Phosphate-buffered saline (PBS) was from Gibco (Burlington, Ontario). The following
reagents were purchased from Sigma-Aldrich Inc. (St. Louis, MO): hydrochloric acid (HCl),
sodium hydroxide (NaOH), 2.5% silver nitrate solution, Bouin’s fluid, 1.3% picric acid solution
and Sirius red dye. Paraformaldehyde (PFA) solution was gathered from Canemco Inc.
(Lakefield, Quebec).
Phylloquinone (K1), menaquinone-4 (K2), calcium chloride (CaCl2), 25-hydroxyvitamin D3
(25D), L-ascorbic acid (AA) and β-glycerophosphate disodium salt hydrate (β-glycerophosphate)
were purchased from Sigma-Aldrich Inc., while 1,25-dihydroxyvitamin D3 (1,25D) was acquired
from Enzo Life Sciences (Plymouth, PA). Vitamins K1, K2, 25D and 1,25D were dissolved in
absolute ethanol, while CaCl2, AA and β-glycerophosphate were solubilized in distilled water.
All of the supplement solutions were filtered to avoid contamination. Supplement solutions with
the exception of 1,25D solution, which was stored at -80ºC, were stored at -20ºC.
2.3 Cell Culture
The MC3T3-E1 (subclone 4) murine preosteoblast cell line and Saos-2 human osteosarcoma cell
line were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and
grown in MEM α without AA or DMEM, respectively, at 37ºC with 5% CO2. Both types of
media were supplemented with 10% heat-inactivated FBS. For each experiment, 100,000 cells
14
were plated in each well of a 6-well plate. The appropriate solvent (ethanol or water) was used as
a vehicle control for vitamin or calcium treated wells. In order to determine how much calcium
solution we needed to add to the cells to produce our desired final calcium concentration, we had
to account for the calcium levels present within the media and FBS. We looked at the chemical
analyses of the media run by the manufacturers and at several chemical analyses of FBS run by
several different companies. This allowed us to estimate the calcium concentration present in the
media supplemented with 10% FBS.
2.4 Treatment with AA and von Kossa (VK) Staining
Given that vitamin C or AA is critical for the hydroxylation of collagen, we wondered if the
effect of vitamin or calcium addition on bone formation in vitro would differ under either
continual supplementation of AA (Continual-AA Treatment) or under vitamin C-stressed
conditions (AA-Primed Treatment).
2.4.1 AA-Primed Treatment
Preosteoblasts were grown in 6-well plates with 50 µg/mL AA for the first 5 days and 10 mM β-
glycerophosphate from day 5 until day 23. Simultaneously the cells were treated with various
concentrations of the vitamins/calcium starting on day 1 and continuing until day 23 (only day
22 for the combination VK experiments). The vitamins/calcium, AA and/or β-glycerophosphate
were replenished with each 2 day media change.
2.4.2 Continual-AA Treatment
Unlike the AA-Primed treatment, AA was supplemented throughout the 22 day assay (slightly
shorter assay period), where its addition began on day 1 and was replenished with every 2 day
media change until day 22. However, β-glycerophosphate was still only added beginning on day
5.
2.4.3 von Kossa Staining with Silver Nitrate Solution
On day 22 or 23 of the mineralization experiment (depending on AA treatment), PBS-washed
osteoblasts were fixed in 4% PFA for 15 minutes and subsequently washed with distilled water.
Silver nitrate solution was added to each well and incubated under a bright light for 30 minutes.
15
Silver nitrate solution stains the inorganic or mineral portion of the extracellular matrix black.
Cells were washed with distilled water and imaged using an inverted bright-field microscope.
Locations of images were randomly chosen across each well and a total of 20 pictures were
obtained for each replicate. The exposure time varied depending on cell type and the AA
treatment used.
2.4.4 Quantification
Total mineralized area was determined using Image J by first applying an intensity threshold
followed by a size threshold to the images (Supplemental Figure 1). The intensity threshold
converted any pixel with an intensity over a set intensity threshold to black and any pixel with an
intensity under the threshold to white. The size threshold removed any bone nodule that was
below a set area and the areas of the bone nodules over this size threshold were summed to
determine the total mineralized area of the image. The intensity and size threshold values used
varied depending on the exposure time utilized to capture the images and were chosen such that
background noise (i.e. cell outlines) at that specific exposure time was removed.
2.5 Collagen Production
Preosteoblasts were treated with AA and vitamins/calcium for five days, where the AA and
vitamins/calcium were replenished every 2 days. On day 5 of the collagen experiment, PBS-
washed osteoblasts were fixed in Bouin’s fluid for 1 hour and subsequently washed with distilled
water. The cells were stained with 1 mg/mL Sirius red dye in aqueous picric acid for 1 hour and
washed with 0.01 N HCl. The stain was extracted with 0.1 N NaOH and the absorbance read at
528 nm. The collagen concentration of each sample was determined through the interpolation of
a collagen standard curve. Briefly, various concentrations of soluble collagen in 0.05 N HCl
were incubated with 1 mg/mL Sirius red dye in picric acid for 1 hour. The pellets were washed
with 0.01 N HCl and extracted with 0.1 N NaOH. The absorbance of each of the standards,
which was read by the plate reader, was used to create a collagen standard curve. The collagen
concentration values for the treatments were normalized to the appropriate vehicle controls.
16
2.6 Statistical Analysis of Experiments
2.6.1 Individual Vitamin/Calcium VK Statistics
The data was presented graphically as the mean ± SEM of the total mineralized area, which was
associated with a certain concentration of vitamin/calcium, for each trial point and the mean
trendline of the three trials. Linear mixed effects analysis was performed by fitting a linear and
quadratic model to the data, in order to determine if there was a relationship between total
mineralized area and calcium/vitamin concentration. In both the linear and quadratic models,
total mineralized area and calcium/vitamin concentration were considered fixed effects, while
trial was considered a random effect (allows one to characterize variation due to trial
differences). The models were statistically compared using a likelihood ratio test. Since
likelihood ratio tests tend to favour more complex or parameter-rich models, we decided to also
compare the models using Akaike’s Information Criterion (AIC), which punishes more complex
models. A significant difference between the models using the likelihood ratio test, as well as a
lower AIC value for the quadratic model, indicated that the quadratic model fit the data better
than the linear model. If there was no statistical difference between the models and the AIC was
not lower for the quadratic model compared to the linear model, then the simpler model (linear
model) was chosen. In the case where the linear model was selected, the slope of the line was
analyzed to determine if it was significantly different than zero (i.e. no relationship between
mineralized area and vitamin/calcium concentration). In any of the statistical tests performed,
values of p < 0.05 were considered significant.
2.6.2 Combination Vitamin/Calcium VK Statistics
The mean ± SEM of triplicate experiments represented the combination VK data. A one-way
ANOVA with a Tukey’s posthoc test was performed, where the combination was considered
significant when p < 0.05 for all comparisons of the combination against the appropriate singular
vitamin or calcium control.
17
2.6.3 Collagen Statistics
The data was displayed graphically as points representing the mean ± SEM of the three trials and
the regression line. Simple linear regression analysis was performed and when p < 0.05 we
concluded that the slope of the line was considered significantly different than zero.
18
3 RESULTS
3.1 Meta-analysis
We were interested in how vitamin supplementation influences bone health and began our study
with a survey of the cell biology literature that looked at the effects of vitamins on bone
formation by osteoblasts. After an exhaustive search, peer-reviewed articles were chosen that
tested the effects of vitamins K1, K2, D and combinations of vitamins on osteoblast bone
formation. Importantly, these reports contained all the information (i.e. number of replicates) that
is necessary for articles included in a meta-analysis. The final journal articles chosen are listed in
Supplemental Table 1. A meta-analysis was then performed for each of the vitamins and for the
experiments from a single study that used a combination of vitamins, where their effect on bone
formation was investigated, and the protocol in the paper entitled “Dispersal and Species
Diversity: A Meta-analysis” (Cadotte, 2006) was followed.
Two separate subanalyses on the same experiments were performed within the meta-analyses
based on: 1) type of experiment performed and 2) cell type used (see Figure 1 for an overview of
the meta-analysis section). The experiments in the vitamin K2, vitamin D, vitamin K1 and K2 +
1,25D meta-analyses were experiment type subanalyzed by grouping them into subgroups
relating to the type of experiment they represent. Unfortunately, only the experiments in the
vitamin K2 and D meta-analyses were subanalyzed by cell type because there was an insufficient
number of experiments within each of the cell type groups for the vitamin K1 and K2 + 1,25D
meta-analyses. The results of the homogeneity test for each meta-analysis were summarized into
two tables (more detail to come in the following section), depending on if they were subanalyzed
by experiment or cell type. In addition, the effect of each vitamin or combination of vitamins on
bone formation (measured as effect size) in different cell types was displayed graphically in
several figures. Each figure contained the experiment or cell type subanalyzed meta-analysis of
one vitamin or combination of vitamins (more detail to come in section 3.1.2).
19
Meta-analysis:
Experiment Type Subanalysis:
Homogeneity Table . . . . Table 1
Vitamin K1 . . . . . . . . . . . Figure 2
Vitamin K2 . . . . . . . . . . . Figure 3
Vitamin D . . . . . . . . . . . Figure 4
K2 + 1,25D . . . . . . . . . . . Figure 5
Summary of Results . . . . Table 2
Cell Type Subanalysis:
Homogeneity Table . . . . Table 3
Vitamin K2 . . . . . . . . . . . Figure 6
Vitamin D . . . . . . . . . . . Figure 7
Summary of Results . . . . Table 4
Figure 1. Overview of meta-analyses performed. Cell biology articles were acquired that
contained experiments where the effect of vitamin K1, K2, D and K2 + 1,25D on osteoblast
maturation characteristics in different cell types were tested. A meta-analysis was performed for
each vitamin and two separate meta-analyses were executed for the combination such that the
combination could be compared against each of the single vitamin effects. The meta-analyses
were subanalyzed first by type of experiment and then by cell type, where cell type subanalysis
was only performed if there was enough experiments in each subtype. The homogeneity within
each meta-analysis was analyzed and summarized into a table, while the effect sizes, a
measurement of the effect of the vitamin on maturation, were displayed graphically in a series of
figures. The overall results of the meta-analyses were summarized into Tables 2 and 4.
20
3.1.1 Discussion of the homogeneity within the meta-analyses that were subanalyzed by the type of experiment.
In order to choose the model that was used to calculate the grand mean effect sizes (the average
of effect sizes in that group) for each subgroup as well as the overall grand mean, the
homogeneity of the experiments used in each meta-analysis was tested (Table 1). Homogeneity
of the experiments increases the confidence that the overall grand mean represents any study
looking at the same phenomenon. If the experiments were found to be homogeneous using the
fixed-effects model (i.e. the test was not significant), then the fixed-effects model was used to
calculate the overall grand mean effect size. However, if the homogeneity test using the fixed-
effects model was significant (indicating heterogeneity), the mixed-effects model was used to
calculate the grand mean.
The experiments used in the vitamin K1, K2, D and K2 + 1,25D meta-analyses, before sub-
analysis (in bold in Table 1), were heterogeneous using the fixed model and thus the mixed-
effects model was used to calculate the overall grand mean effect sizes. Only the experiments
used for the combination of K2 + 1,25D meta-analyses was homogeneous when using the mixed-
effects model, which suggested that the experiments within the other meta-analyses should be
subanalyzed in smaller, more homogeneous groups.
To observe the effect of the vitamins and the combination on specific experiment types
performed, the experiments within the meta-analyses were first subanalyzed by type of
experiment (Table 1). For the vitamin D meta-analysis most of the experiment type groups were
heterogeneous and thus the mixed-effects model was used to calculate the grand mean for each
group. However, for the majority of the groups within the vitamin D meta-analysis, the mixed
model was still not homogeneous. Unlike the vitamin D meta-analysis, the vitamin K1
experiment groups were all heterogeneous using the fixed-effects model and the grand means
were determined using the mixed model. In this case however, the experiment groups were
considered homogeneous using the mixed model. Most of the grand means calculated for each
experiment type group for the vitamin K2 meta-analysis used the mixed-effects model and the
majority of the experiments within the groups were considered homogeneous using this model.
In contrast to the other meta-analyses, where a control untreated group was compared to a
21
vitamin treatment group, the K2 + 1,25D data was used to run two meta-analyses. In this first
meta-analysis the combination group was considered the treated group and the group where only
vitamin K2 was added was the control group, while in the second meta-analysis the group
supplemented with only 1,25D was considered the control group. It should be noted that all the
experiments used for the combination meta-analyses originated from the same paper (Koshihara
et al., 1996). In the K2 + 1,25D compared to K2 alone meta-analysis, the data was heterogeneous
within both of the experiment groups, which indicated that a mixed-effects model needed to be
used to calculate the grand mean effect sizes. The use of the mixed model also resulted in
homogeneity within the groups. The experiment type group called Mineralization within the K2 +
1,25D compared to 1,25D alone meta-analysis was homogeneous, while the Other Experiments
group was heterogeneous using a fixed-effects model and thus the fixed and mixed models were
used to calculate the grand means, respectively. Homogeneity was obtained in the group called
Other Experiments through the use of the mixed effects model.
22
Table 1. The homogeneity test results for the fixed-effects (Qf) and mixed-effects (Qm)
models of each vitamin meta-analysis, where the meta-analyses are subanalyzed by type of
experiment. Cochran’s Q tests were used to determine the homogeneity of the treatment
responses from k # of experiments, where * p < 0.05 indicates that the responses are
heterogeneous and NS (no significance) indicates that the responses are homogeneous. The
homogeneity of the mixed-effects model was only determined if the fixed-effects model was
considered heterogeneous (p < 0.05).
Test k Qf Qm
Vitamin D Mineralization ALP Activity Osteocalcin Levels Collagen Levels Osteopontin Levels Other Experiments
128 29 28 18 31 10 12
651.92* 183.22* 60.20* 119.84* 131.86* 33.88*
13.21 NS
310.82* 79.78*
32.11 NS 52.10* 61.38* 21.03*
···
Vitamin K1 Mineralization ALP Activity Other Experiments
27 15 7 5
122.78* 65.28* 16.30* 39.77*
44.68* 20.41 NS 6.43 NS 5.45 NS
Vitamin K2 Mineralization ALP Activity Proliferation DNA Levels Osteocalcin Levels Other Experiments
98 29 33 10 11 6 9
566.58* 138.71* 151.01* 32.48*
17.30 NS 28.88* 49.49*
167.75* 42.35*
41.62 NS 9.42 NS
··· 6.14 NS 13.08 NS
K2 + 1,25D Compared to K2 Alone Mineralization Other Experiments
11 6 5
28.78* 16.73* 10.05*
10.89 NS 4.94 NS 3.71 NS
K2 + 1,25D Compared to 1,25D Alone Mineralization Other Experiments
11 6 5
62.31* 3.56 NS 37.28*
11.70 NS ···
5.78 NS
23
3.1.2 For the vitamin K1, K2, D and K2 + 1,25D meta-analyses, most of the overall grand mean effect sizes and the experiment type grand mean effect sizes were significantly greater than zero.
Within the graphical representations of the meta-analyses that were subanalyzed by type of
experiment (Figures 2, 3, 4 and 5), each unlabelled bar represents a single experiment extracted
from one of the papers listed within Supplemental Table 1. For each of the experiments, effect
sizes were calculated and the measurement indicated if the vitamin(s) changed a parameter of
osteoblast maturation, including mineralization and osteocalcin levels, compared to an untreated
group. The overall effect of the vitamin(s) on osteoblast maturation parameters was determined
by calculating the overall grand mean effect size (bar labelled in Figures 2, 3, 4 and 5).
Additionally, the experiments were sorted into experiment type subgroups and colour-coded
according to these groups. The grand mean of each subgroup was used to determine the effect of
the vitamin(s) on particular osteoblast maturation characteristics (bars labelled and a short
form for each experiment name in Figures 2, 3, 4 and 5).
Within the vitamin K1 meta-analysis, the experiments were grouped into the following
subgroups: Mineralization, ALP Activity and Other Experiments (Figure 2), where Other
Experiments contained experiment types that were not in large enough quantities to have their
own group. The effect sizes of the majority of experiments analyzed within the vitamin K1 meta-
analysis were positive with confidence intervals (CIs) that did not intersect zero, which indicated
that these effect sizes were significantly greater than zero. This trend was mirrored when looking
at the overall grand mean, which suggested that the addition of vitamin K1 significantly and
positively increased osteoblast maturation parameters. A non-directional test also indicated
significance for the overall grand mean’s departure from zero (χ2
= 62.865, df = 27, p = 0.0001),
which agreed with the CI test. The grand mean effects of the Mineralization and ALP experiment
groups were also positive and their CIs did not intersect zero. However, the non-directional tests
indicated significance for the Mineralization group (χ2
= 36.988, df = 15, p = 0.0013) and non-
significance for the ALP group (χ2
= 13.570, df = 7, p = 0.0594). This and any other discrepancy
between the CI test and non-directional test will be examined within the Discussion section.
Unlike the Mineralization and ALP groups, the Other Experiment group had a grand mean effect
size that was not significantly different than zero (χ2
= 5.4496, df = 5, p = 0.3635).
24
The experiments in the vitamin K2 meta-analysis were sorted into the following experiment type
subgroups: Mineralization, ALP Activity, Proliferation, DNA Levels, Osteocalcin Levels and
Other Experiments (Figure 3). Most of the experiments in the vitamin K2 meta-analysis had
significantly positive effect sizes, which translated to a positive and significant overall grand
mean (χ2
= 228.17, df = 98, p < 0.0001). A significantly positive grand mean effect was also
observed for the following experiment type groups: Mineralization (χ2
= 90.324, df = 29, p <
0.0001), ALP (χ2
= 62.008, df = 33, p = 0.0016), DNA Levels (χ2
=104.31, df = 11, p < 0.0001)
and Other Experiments (χ2
= 34.038, df = 9, p < 0.0001). This indicated that the addition of
vitamin K2 significantly increased mineralization, ALP activity, DNA levels and other osteoblast
experiments as compared to an untreated group. In contrast, a negative grand mean with a CI that
did not intersect zero was found for the Proliferation group, which was confirmed by the results
of a non-directional test being significant (χ2
= 28.927, df = 10, p = 0.0013). The Osteocalcin
group in the vitamin K2 meta-analysis had a grand mean effect size that was not significantly
different than zero (χ2
= 7.0894, df = 6, p = 0.3127).
Within the vitamin D meta-analysis, the experiments were arranged into the following
experiment type subgroups: Mineralization, ALP Activity, Osteocalcin Levels, Collagen Levels,
Osteopontin Levels and Other Experiments (Figure 4). The overall grand mean for the vitamin D
meta-analysis had a significantly positive effect size (χ2
= 393.84, df = 128, p < 0.0001), which
corresponded to the majority of the effect sizes being positive and significant for the
experiments. A significantly positive grand mean effect size was also observed for the
Mineralization (χ2
= 107.93, df = 29, p < 0.0001), ALP Activity (χ2
= 55.487, df = 28, p =
0.0015), Osteocalcin Levels (χ2
= 79.047, df = 18, p < 0.0001), Collagen Levels (χ2
= 78.398, df
= 31, p < 0.0001) and Osteopontin Levels (χ2
= 42.346, df = 10, p < 0.0001) groups. Conversely,
the group called Other Experiments had a negative grand mean with a confidence interval that
did not include zero. However, the non-directional test indicated that the grand mean effect was
not significantly different than zero (χ2
= 20.546, df = 12, p = 0.0574).
The experiments used in both of the K2 + 1,25D meta-analyses were sorted into two subgroups:
Mineralization and Other Experiments (Figure 5A and 5B). All of the experiments had a
significantly positive effect size within the K2 + 1,25D compared to K2 alone meta-analysis
(Figure 5A), which corresponded to a significantly positive overall grand mean effect size
25
(χ2
= 65.293, df = 11, p < 0.0001). Likewise, both the grand means were positive and significant
for the Mineralization (χ2
= 31.541, df = 6, p < 0.0001) and Other Experiments groups (χ2
=
29.062, df = 5, p < 0.0001). For the K2 + 1,25D compared to 1,25D meta-analysis (Figure 5B),
the majority of the experiments had effect sizes that were positive and significant. This trend
agreed with the overall grand mean being positive with a confidence interval that does not
include zero. However, the non-directional test was not significant, meaning that the overall
grand mean might not be significantly different than zero (χ2
= 18.522, df = 11, p = 0.0702). The
Mineralization group of the K2 + 1,25D compared to 1,25D meta-analysis had a significantly
positive grand mean (χ2
= 53.000, df = 6, p < 0.0001), while the grand mean effect size of the
Other Experiment group was not significantly different than zero (χ2
= 5.9901, df = 5, p =
0.3072). We summarized the results of the vitamin K1, K2, D and K2 + 1,25D meta-analyses,
which was subanalyzed by type of experiment, into Table 2. In conclusion, the addition of
vitamin K1, K2 and D to osteoblasts resulted in a significant increase to several bone cell
parameters, while the addition of K2 + 1,25D increased mineralization within osteoblast cultures.
26
Figure 2. The overall grand mean effect size and the experiment type grand mean effect sizes for the vitamin K1 meta-analysis
were significantly greater than zero. In the graphical representation of the vitamin K1 meta-analysis that is subanalyzed by
experiment type, each unlabelled bar represents the effect size, as measured using the Hedges’ d method, of an experiment and the
error bars are the 95% confidence intervals. The bars are coloured according to the type of experiment they are representing. The
experiments used in the K1 meta-analysis were extracted from 5 articles. The colour-coded grand means for each experiment type is
signified by and a short form for each experiment name and the overall grand mean is signified by .
27
Figure 3. The majority of the experiment type grand means and the overall grand mean effect size were significantly positive
for the vitamin K2 meta-analysis. In the graphical representation of the vitamin K2 meta-analysis that is subanalyzed by experiment
type, each unlabelled bar represents the effect size, as measured using the Hedges’ d method, of an experiment and the error bars are
the 95% confidence intervals. The bars are coloured according to the type of experiment they are representing. The experiments used
in the K2 meta-analysis were extracted from 8 articles. The colour-coded grand means for each experiment type is signified by and a
short form for each experiment name and the overall grand mean is signified by .
28
Figure 4. The overall grand mean effect size and most of the experiment type grand mean effect sizes were significantly
greater than zero for the vitamin D meta-analysis. In the graphical representation of the vitamin D meta-analysis that is
subanalyzed by experiment type, each unlabelled bar represents the effect size, as measured using the Hedges’ d method, of an
experiment and the error bars are the 95% confidence intervals. The bars are coloured according to the type of experiment they are
representing. The experiments used in the vitamin D meta-analysis were extracted from 15 articles. The colour-coded grand means for
each experiment type is signified by and a short form for each experiment name and the overall grand mean is signified by .
29
Figure 5. The overall grand mean effect sizes for the meta-analyses comparing the results of the combination of vitamin K2 +
1,25D against vitamin K2 and 1,25D alone were significantly positive. In the graphical representation of the experiment type
subanalyzed meta-analyses for K2 + 1,25D compared to (A) vitamin K2 alone and (B) 1,25D alone, each unlabelled bar represents the
effect size, as measured using the Hedges’ d method, of an experiment and the error bars are the 95% confidence intervals. The bars
are coloured according to the type of experiment they are representing. The experiments used in the both K2 + 1,25D meta-analyses
were extracted from a single article. The colour-coded grand means for each experiment type is signified by and a short form for
each experiment name and the overall grand mean is signified by .
30
Table 2. Summary of the results of the vitamin K1, K2, D and K2 + 1,25D meta-analyses
that were subanalyzed by type of experiment. For each experiment type subgroup the grand
mean effect sizes that were considered to be significantly greater or smaller than zero based on
the results of the CI and non-directional test were indicated as Increased or Decreased,
respectively. If the effect sizes were not significantly different than zero, based on both statistical
tests, the result was labelled No Effect. When the results of non-directional test did not agree
with those of the CI test, the result was displayed as Inconclusive. If we were unable to sort the
experiments from a meta-analysis into a particular subgroup, we indicated by two consecutive
dashes.
Experiment
Type
Vitamin K1
Meta-
analysis
Vitamin K2
Meta-
analysis
Vitamin D
Meta-
analysis
K2 + 1,25D
Compared to
K2 Alone
K2 + 1,25D
Compared to
1,25D Alone
Mineralization Increased Increased Increased Increased Increased
ALP Activity Inconclusive Increased Increased -- --
Proliferation -- Decreased -- -- --
DNA Levels -- Increased -- -- --
Osteocalcin -- No Effect Increased -- --
Collagen Levels -- -- Increased -- --
Osteopontin -- -- Increased -- --
Other
Experiments
No Effect Increased Inconclusive Increased No Effect
31
3.1.3 Discussion of the homogeneity within the meta-analyses that were subanalyzed by cell type.
In addition to subanalyzing the osteoblast experiments by experiment type, the data within the
vitamin K2 and D meta-analyses was also subanalyzed by the cell type utilized for the
experiments (Table 3) and the homogeneity within each of these subgroups also had to be
assessed. The overall grand mean for each of the vitamin meta-analyses was calculated using
either the fixed or mixed-effects models, as discussed in a previous section (3.1.1; also in bold in
Table 3). The cell type groups within both meta-analyses were heterogeneous using the fixed
model and thus the grand means for each group was calculated using the mixed model. Only the
Murine Primary Cells group within the vitamin K2 meta-analysis was homogeneous using the
mixed-effects model, while the rest of the groups were heterogeneous. In the vitamin D meta-
analysis, both murine groups were homogenous, while the human groups were heterogeneous
using the mixed-effect model.
32
Table 3. The fixed-effects (Qf) and mixed-effects (Qm) models’ homogeneity test results of
each vitamin meta-analysis, where the meta-analyses are subanalyzed by cell type. Cochran’s Q tests were used to determine the homogeneity of the treatment responses from k #
of experiments, where * p < 0.05 indicates that the responses are heterogeneous and NS (no
significance) indicates that the responses are homogeneous. The homogeneity of the mixed-
effects model was only determined if the fixed-effects model was considered heterogeneous
(p < 0.05).
Test k Qf Qm
Vitamin D Murine Cell Line Human Cell Line Murine Primary Cells Human Primary Cells
128 36 34 14 44
651.92* 130.60* 125.35* 44.82* 240.58*
310.82* 62.74* 65.03*
20.69 NS 93.51*
Vitamin K2 Murine Cell Line Human Cell Line Murine Primary Cells Human Primary Cells
98 29 15 19 35
566.58* 138.79* 138.94* 57.36* 213.40*
167.75* 34.82 NS
26.65* 18.02 NS
52.49*
33
3.1.4 The overall grand mean effect size and most of the cell type grand mean effect sizes for the vitamin K2 and D meta-analyses were significantly greater than zero.
Within the graphical representations of the meta-analyses subanalyzed by cell type (Figures 6
and 7), each unlabelled bar represents a single experiment from one paper and its effect size
indicated if the vitamin changed a measurement of osteoblast maturation compared to an
untreated group. The overall grand mean effect size (bar labelled in Figures 6 and 7) for each
meta-analysis was the same information that was displayed in the experiment type subanalyzed
graphs for vitamin K2 and D (Figures 3 and 4, respectively). The experiments were colour-coded
according to what cell type they used and the grand means for each of the cell type subgroups
was utilized to determine if the vitamin affects osteoblast maturation parameters differently in
various cell types (bars labelled and a short form for each cell type name in Figures 6 and 7).
The experiments in the vitamin K2 meta-analysis were also sorted into the following cell type
subgroups: Murine Cell Line, Human Cell Line, Murine Primary Cells and Human Primary Cells
(Figure 6). As previously stated, the overall grand mean effect size for the vitamin K2 meta-
analysis was positive and significant. The cell type grand means for the following groups were
significantly positive: Murine Cell Line (χ2
= 47.663, df = 29, p = 0.0159), Murine Primary Cells
(χ2
= 62.543, df = 19, p < 0.0001) and Human Primary Cells (χ2
= 84.061, df = 35, p < 0.0001).
The Human Cell Line group had a grand mean that was not significantly different than zero
based on the confidence interval test, but was significantly greater than zero using the non-
directional test (χ2
= 27.936, df = 15, p = 0.0220). This could indicate that the addition of vitamin
K2 to human cell line osteoblasts might not result in any change to maturation characteristics as
compared to an untreated control.
Within the vitamin D meta-analysis, the experiments were also grouped into the following cell
type subgroups: Murine Cell Line, Human Cell Line, Murine Primary Cells and Human Primary
Cells (Figure 7). The overall grand mean effect for the vitamin D meta-analysis was significantly
positive, as mentioned earlier. Significantly positive grand means were obtained for the Murine
Cell Line (χ2
= 69.703, df = 36, p = 0.0006), Human Cell Line (χ2
= 85.626, df = 34, p < 0.0001)
and Human Primary Cells (χ2
= 189.04, df = 44, p < 0.0001) groups. However, the grand mean
effect size of the group called Murine Primary Cells was not significantly different than zero
34
(χ2
= 20.693, df = 14, p = 0.1098). The results of the vitamin K2 and D meta-analyses that were
subanalyzed by cell type were summarized into Table 4. Altogether this indicated that the
addition of vitamin D to murine and human osteoblast cell lines, as well as human primary
osteoblasts, led to an increase in osteoblast maturation/bone formation parameters, while vitamin
K2 supplementation led to an increase in osteoblast maturation characteristics within murine cell
lines, murine primary cells and human primary cells.
35
Figure 6. The overall grand mean effect size and most of the cell type grand mean effect sizes were significantly greater than
zero for the vitamin K2 meta-analysis. In the graphical representation of the vitamin K2 meta-analysis that is subanalyzed by cell
type, each unlabelled bar represents the effect size, as measured using the Hedges’ d method, of an experiment and the error bars are
the 95% confidence intervals. The bars are coloured according to the cell type they are representing. The experiments used in the K2
meta-analysis were extracted from 8 articles. The colour-coded grand means for each cell type is signified by and a short form for
each cell type name and the overall grand mean is signified by .
36
Figure 7. The majority of the cell type grand mean effect sizes and the overall grand mean effect were significantly positive for
the vitamin D meta-analysis. In the graphical representation of the vitamin D meta-analysis that is subanalyzed by cell type, each
unlabelled bar represents the effect size, as measured using the Hedges’ d method, of an experiment and the error bars are the 95%
confidence intervals. The bars are coloured according to the cell type they are representing. The experiments used in the vitamin D
meta-analysis were extracted from 15 articles. The colour-coded grand means for each cell type is signified by and a short form for
each cell type name and the overall grand mean is signified by .
37
Table 4. Summary of the results of the vitamin K2 and D meta-analyses that were
subanalyzed by cell type. For each cell type subgroup the grand mean effect sizes that were
considered to be significantly greater or smaller than zero based on the results of the CI and non-
directional test were indicated as Increased or Decreased, respectively. If the effect sizes were
not significantly different than zero, based on both statistical tests, the result was labelled No
Effect. When the results of non-directional test did not agree with those of the CI test, the result
was displayed as Inconclusive.
Cell Type Vitamin K2 Meta-analysis
Vitamin D Meta-analysis
Murine Cell Line Increased Increased
Human Cell Line Inconclusive Increased
Murine Primary Cells Increased No Effect
Human Primary Cells Increased Increased
38
3.2 Effects of vitamin/calcium supplementation on in vitro bone formation using a mouse and human osteoblast cell line.
After performing the meta-analyses, we decided to run our own tests on the effects of vitamins
and calcium on mineralization and collagen levels of a mouse and human osteoblast cell line (see
Figure 8 for an overview of the experiments performed). MC3T3-E1, a murine preosteoblast cell
line, was chosen as it is a well-characterized cell line and is a commonly used model within bone
research (Czekanska et al., 2012; Nabavi, Pustylnik, & Harrison, 2012; Nabavi, Urukova,
Cardelli, Aubin, & Harrison, 2008; Pustylnik et al., 2013). The human osteosarcoma cell line,
Saos-2, was chosen as these cells form mineralized bone (Czekanska et al., 2012) and secrete
collagen that is similar in structure to collagen produced by human primary osteoblast cells
(Fernandes, Harkey, Weis, Askew, & Eyre, 2007). These cell lines were used to assess the effect
of calcium, vitamin K1, vitamin K2, 25D and 1,25D on bone mineralization under different
conditions of ascorbic acid (AA) supplementation, as well as early collagen production. In
addition, the effects of combinations of calcium, the vitamins and AA on bone nodule formation
were determined.
39
Experiments:
1) Single Vitamin/Calcium Mineralization
Continual-AA:
MC3T3:
Calcium . . . . . . . . . . . Figure 9
Vitamin K1 . . . . . . . . . Figure 10
Vitamin K2 . . . . . . . . . Figure 11
25D . . . . . . . . . . . . . . . Figure 12
1,25D . . . . . . . . . . . . . Figure 13
Summary of Results . . Table 5
Saos-2:
Calcium . . . . . . . . . . . Figure 14
Vitamin K1 . . . . . . . . . Figure 15
Vitamin K2 . . . . . . . . . Figure 16
25D . . . . . . . . . . . . . . . Figure 17
1,25D . . . . . . . . . . . . . Figure 18
Summary of Results . . Table 6
AA-Primed:
MC3T3:
Calcium . . . . . . . . . . . Figure 19
Vitamin K1 . . . . . . . . . Figure 20
Vitamin K2 . . . . . . . . . Figure 21
25D . . . . . . . . . . . . . . . Figure 22
1,25D . . . . . . . . . . . . . Figure 23
Summary of Results . . Table 7
2) Combinations Mineralization
Continual-AA:
MC3T3 . . . . . . . . . . . . . Figure 24
Saos-2 . . . . . . . . . . . . . . Figure 26
AA-Primed:
MC3T3 . . . . . . . . . . . . . Figure 25
Saos-2 . . . . . . . . . . . . . . Figure 27
3) Collagen Concentration
MC3T3 . . . . . . . . . . . . . Figure 28
Saos-2 . . . . . . . . . . . . . . Figure 29
Figure 8. Overview of experiments executed. A series of experiments were performed to look
at the effect of calcium, vitamin K1, vitamin K2, 25D and 1,25D on mineralization and early
collagen production in a mouse, MC3T3, and human, Saos-2, osteoblast cell lines.
Mineralization for both cell types was assessed under continual-AA conditions, where ascorbic
acid (AA) was added throughout the experiment, while the mineralization under AA-primed
conditions, where AA was added only for the first five days of the experiment, was only
performed using the MC3T3 cells. The amount of mineralization produced by the combination of
calcium and vitamins was also assessed under both AA conditions and using both cell types.
40
3.2.1 Increasing concentrations of calcium resulted in increased bone mineralization in continual-AA MC3T3 cultures, while bone nodule formation decreased with increasing vitamin K2 and 1,25D levels. In contrast, mineralization did not change in mouse osteoblast cultures upon vitamin K1 and 25D supplementation.
First we decided to look at the effect of calcium and vitamin supplementation on bone formation
by murine osteoblasts under continual-AA conditions, where AA was added throughout the
entire experimental period, as this condition was comparable to the studies used in the meta-
analyses. AA and β-glycerophosphate addition to MC3T3 cultures throughout the 22 day
experiment lead to bone mineralization, as detected by the black, von Kossa stained bone
nodules, compared to the control cultures (Figures 9A, 10A, 11A, 12A and 13A). Based on the
von Kossa staining pattern of the MC3T3 cultures, the mineral deposited around the cell layers
was similar to what has been seen previously in MC3T3 and primary human osteoblasts
(Welldon, Findlay, Evdokiou, Ormsby, & Atkins, 2013; Yamauchi, Yamaguchi, Kaji, Sugimoto,
& Chihara, 2005). Bone nodule formation, visualized with von Kossa staining, was increased as
the concentration of calcium was increased for the continual-AA MC3T3 cultures (Figure 9A).
This trend was confirmed by quantification of the total mineralized area of each image through
the use of Image J (Figure 9B). Linear mixed effects analysis of the relationship between total
mineralized area and calcium concentration was performed to determine if the slope of the line
was significantly different than zero. The slope of the line between the total mineralized area and
calcium concentration was considered positive and significantly non-zero (t-value = 7.576, df =
236, p < 0.05). This suggested that increasing concentrations of calcium resulted in increasing
bone mineralization within the continual-AA MC3T3 cultures. In contrast to calcium, bone
nodule formation did not change with increasing levels of vitamin K1 and 25D in continual-AA
treated MC3T3 cells (Figure 10A and 12A, respectively), which corresponded to the slopes
being not significantly different than zero for the K1 (t-value = -0.256, df = 236, p > 0.05; Figure
10B) and 25D (t-value = 1.038, df = 236, p > 0.05; Figure 12B) lines. However, the addition of
increasing concentrations of vitamin K2 led to decreased mineralization in treated MC3T3
cultures (Figure 11A), where the slope of the line was both negative and significantly non-zero
(t-value = -2.651, df = 236, p < 0.05; Figure 11B).
41
Similar to vitamin K2, bone mineralization by MC3T3 cells decreased with increasing
concentrations of 1,25D (Figure 13A), but unlike the other vitamins and calcium, where there
was no significant difference between the linear model and the quadratic model, we detected a
significant difference between the 1,25D quadratic and linear models using a likelihood ratio test
(χ2
= 6.6571, df = 1, p < 0.05; Figure 13B). In addition, the AIC value for the quadratic model
that was fit to the 1,25D data was smaller than the linear model fit to the same data. This
indicated that the quadratic model better fit the 1,25D data than the linear model and thus the
amount of mineralization decreased quadratically with increasing concentrations of 1,25D. We
summarized the results of the bone formation experiments using the vitamin/calcium
supplemented, continual-AA treated MC3T3 cultures into Table 5. In conclusion, the amount of
bone mineralization for continual-AA treated MC3T3 cultures increased with increasing levels
of calcium and decreased with increasing concentrations of vitamin K2 and 1,25D.
42
Figure 9. Increasing the concentration of calcium lead to increased bone mineralization in
continual-AA MC3T3 cultures. MC3T3 cells were supplemented with AA and calcium
throughout the 22 day experiment, while β-glycerophosphate was added from day 5 to day 22.
(A) Brightfield images were taken of the von Kossa stained cells and the total mineralized area
of each image was determined using Image J. (B) The graph displays the mean ± SEM of total
mineralized area for 20 images for each trial point and the mean trendline of the three trials.
Linear mixed effects analysis of the relationship between total mineralized area and calcium
concentration was performed and * p < 0.05 when the slope of the line was considered
significantly different than zero. Scale bar represents 100 µm.
43
Figure 10. Bone mineralization did not change with increasing levels of vitamin K1 in
continual-AA MC3T3 cultures. MC3T3 cells were supplemented with AA and vitamin K1
throughout the 22 day experiment, while β-glycerophosphate was added from day 5 to day 22.
(A) Brightfield images were taken of the von Kossa stained cells and the total mineralized area
of each image was quantified using Image J. (B) The graph displays the mean ± SEM of total
mineralized area for 20 images for each trial point and the mean trendline of the three trials.
Linear mixed effects analysis of the relationship between total mineralized area and vitamin K1
concentration was performed and * p < 0.05 when the slope of the line was considered
significantly different than zero. Scale bar represents 100 µm.
44
Figure 11. Increasing vitamin K2 concentration in continual-AA MC3T3 cultures resulted
in decreased bone nodule formation. MC3T3 cells were supplemented with AA and vitamin
K2 throughout the 22 day experiment, while β-glycerophosphate was added from day 5 to day
22. (A) Brightfield images were taken of the von Kossa stained cells and the total mineralized
area of each image was quantified using Image J. (B) The graph displays the mean ± SEM of
total mineralized area for 20 images for each trial point and the mean trendline of the three trials.
Linear mixed effects analysis of the relationship between total mineralized area and vitamin K2
concentration was performed and * p < 0.05 when the slope of the line was considered
significantly different than zero. Scale bar represents 100 µm.
45
Figure 12. Bone mineralization of continual-AA treated MC3T3 cultures did not change
with increasing 25D concentration. MC3T3 cells were supplemented with AA and 25D
throughout the 22 day experiment, while β-glycerophosphate was added from day 5 to day 22.
(A) Brightfield images were taken of the von Kossa stained cells and the total mineralized area
of each image was quantified using Image J. (B) The graph displays the mean ± SEM of total
mineralized area for 20 images for each trial point and the mean trendline of the three trials.
Linear mixed effects analysis of the relationship between total mineralized area and 25D
concentration was performed and * p < 0.05 when the slope of the line was considered
significantly different than zero. Scale bar represents 100 µm.
46
Figure 13. Total mineralized area of continual-AA MC3T3 cultures decreased with
increasing levels of 1,25D. MC3T3 cells were supplemented with AA and 1,25D throughout the
22 day experiment, while β-glycerophosphate was added from day 5 to day 22. (A) Brightfield
images were taken of the von Kossa stained cells and the total mineralized area of each image
was quantified using Image J. (B) The graph displays the mean ± SEM of total mineralized area
for 20 images for each trial point and the mean quadratic trendline of the three trials. The
quadratic model is considered a better fit as compared to the linear model, when * p < 0.05 using
a likelihood ratio test and when the AIC of the quadratic model is lower than the linear model.
Scale bar represents 100 µm.
47
Table 5. Summary of the results of the calcium or vitamin supplemented bone formation
experiments run on the continual-AA treated MC3T3 cultures. The total mineralized area of
von Kossa-stained cultures was quantified for each supplement and the relationship between the
mineralized area and supplement concentration was determined. For each supplement, we
indicated the type of model (Quadratic or Linear) that best fit the data and the directionality of
that trend (Positive or Negative). In the cases where the slope of the linear trend was not
significantly different than zero, we displayed this as No Trend with two consecutive dashes in
the directionality column.
Vitamin or Calcium
Added
Linear, Quadratic
or No Trend
Positive or Negative
Trend
Calcium Linear Positive
Vitamin K1 No Trend --
Vitamin K2 Linear Negative
25D No Trend --
1,25D Quadratic Negative
48
3.2.2 Total mineralized area increased with increasing levels of calcium in continual-AA Saos-2 cultures, but did not change with the addition of increasing concentrations of vitamin K1, vitamin K2, 25D and 1,25D.
As previously mentioned in section 3.2.1, the supplementation of AA and β-glycerophosphate,
under continual-AA conditions, to Saos-2 cultures lead to bone nodule formation compared to
the control cultures (Figures 14A, 15A, 16A, 17A and 18A). The Saos-2 mineralization was
different than that of the MC3T3 cells, in that von Kossa stained mineral was only deposited
around clusters of cells, similar to what has been described (Czekanska et al., 2012). Over time
the cells that were not mineralized detached from the well, due to Saos-2’s sensitivity to high
levels of inorganic phosphate (Czekanska et al., 2012). Increasing levels of calcium resulted in
increasing bone mineralization within continual-AA Saos-2 cultures (Figure 14A), which was
confirmed through quantification (Figure 14B), as explained in the previous section. Linear
mixed effects analysis showed that the slope of the line representing the relationship between the
total mineralized area and calcium concentration was positive and significantly different than
zero (t-value = 3.483, df = 236, p < 0.05). Therefore, increasing the calcium concentration within
continual-AA Saos-2 cultures resulted in increased mineralization levels. As for the addition of
the vitamin K’s to the culture, both increasing levels of vitamin K1 and K2 did not change the
amount of bone nodule formation (Figures 15A and 16A, respectively) and thus led to the slopes
being not significantly different than zero for the vitamin K1 (t-value = 1.150, df = 236, p > 0.05;
Figure 15B) and K2 (t-value = -0.004, df = 236, p > 0.05; Figure 16B) lines. Likewise, bone
mineralization did not change with increasing concentrations of either 25D (Figure 17A) or
1,25D (Figure 18A). The slopes of the 25D (t-value = -0.124, df = 238, p > 0.05; Figure 17B)
and 1,25D (t-value = 0.141, df = 238, p > 0.05; Figure 18B) lines were not significantly different
than zero. The results of the bone formation experiments using the vitamin/calcium
supplemented, continual-AA treated Saos-2 cultures were summarized into Table 6. In summary,
increasing concentrations of calcium lead to increased bone mineralization in continual-AA
Saos-2 cultures.
49
Figure 14. Increasing levels of bone mineralization were associated with increasing
concentrations of calcium in continual-AA Saso-2 cultures. Saos-2 cells were supplemented
with AA and calcium throughout the 22 day experiment, while β-glycerophosphate was added
from day 5 to day 22. (A) Brightfield images were taken of the von Kossa stained cells and the
total mineralized area of each image was determined using Image J. (B) The graph displays the
mean ± SEM of total mineralized area for 20 images for each trial point and the mean trendline
of the three trials. Linear mixed effects analysis of the relationship between total mineralized
area and calcium concentration was performed and * p < 0.05 when the slope of the line was
considered significantly different than zero. Scale bar represents 100 µm.
50
Figure 15. Increasing concentrations of vitamin K1 in continual-AA Saos-2 cultures did not
lead to increased bone nodule formation. Saos-2 cells were supplemented with AA and
vitamin K1 throughout the 22 day experiment, while β-glycerophosphate was added from day 5
to day 22. (A) Brightfield images were taken of the von Kossa stained cells and the total
mineralized area of each image was determined using Image J. (B) The graph displays the mean
± SEM of total mineralized area for 20 images for each trial point and the mean trendline of the
three trials. Linear mixed effects analysis of the relationship between total mineralized area and
vitamin K1 concentration was performed and * p < 0.05 when the slope of the line was
considered significantly different than zero. Scale bar represents 100 µm.
51
Figure 16. Total mineralized area did not change with increasing levels of vitamin K2 in
continual-AA Saos-2 cultures. Saos-2 cells were supplemented with AA and vitamin K2
throughout the 22 day experiment, while β-glycerophosphate was added from day 5 to day 22.
(A) Brightfield images were taken of the von Kossa stained cells and the total mineralized area
of each image was determined using Image J. (B) The graph displays the mean ± SEM of total
mineralized area for 20 images for each trial point and the mean trendline of the three trials.
Linear mixed effects analysis of the relationship between total mineralized area and vitamin K2
concentration was performed and * p < 0.05 when the slope of the line was considered
significantly different than zero. Scale bar represents 100 µm.
52
Figure 17. Increasing levels of 25D in continual-AA Saos-2 cultures resulted in no change in
bone mineralization. Saos-2 cells were supplemented with AA and 25D throughout the 22 day
experiment, while β-glycerophosphate was added from day 5 to day 22. (A) Brightfield images
were taken of the von Kossa stained cells and the total mineralized area of each image was
determined using Image J. (B) The graph displays the mean ± SEM of total mineralized area for
20 images for each trial point and the mean trendline of the three trials. Linear mixed effects
analysis of the relationship between total mineralized area and 25D concentration was performed
and * p < 0.05 when the slope of the line was considered significantly different than zero. Scale
bar represents 100 µm.
53
Figure 18. Bone nodule formation did not change with increasing concentrations of 1,25D
in continual-AA Saos-2 cultures. Saos-2 cells were supplemented with AA and 1,25D
throughout the 22 day experiment, while β-glycerophosphate was added from day 5 to day 22.
(A) Brightfield images were taken of the von Kossa stained cells and the total mineralized area
of each image was determined using Image J. (B) The graph displays the mean ± SEM of total
mineralized area for 20 images for each trial point and the mean trendline of the three trials.
Linear mixed effects analysis of the relationship between total mineralized area and 1,25D
concentration was performed and * p < 0.05 when the slope of the line was considered
significantly different than zero. Scale bar represents 100 µm.
54
Table 6. Summary of the results of the calcium or vitamin supplemented bone formation
experiments run on the continual-AA treated Saos-2 cultures. The total mineralized area of
von Kossa-stained cultures was quantified for each supplement and the relationship between the
mineralized area and supplement concentration was determined. For each supplement, we
indicated the type of model (Quadratic or Linear) that best fit the data and the directionality of
that trend (Positive or Negative). In the cases where the slope of the linear trend was not
significantly different than zero, we displayed this as No Trend with two consecutive dashes in
the directionality column.
Vitamin or Calcium
Added
Linear, Quadratic
or No Trend
Positive or Negative
Trend
Calcium Linear Positive
Vitamin K1 No Trend --
Vitamin K2 No Trend --
25D No Trend --
1,25D No Trend --
55
3.2.3 Bone nodule formation increased with increasing concentrations of calcium, vitamin K1 and vitamin K2 in AA-primed MC3T3 cultures, while increasing 1,25D levels lead to decreasing bone mineralization. Conversely, the addition of increasing concentrations of 25D had no effect on bone mineralization.
Next, we decided to look at the effect of the vitamins and calcium on bone mineralization under
AA-primed conditions, where AA was only added for the first 5 days. This treatment allowed us
to see how supplementation helped bone growth under vitamin C-stressed conditions. Due to
time constraints, these experiments were only performed using the MC3T3 cell line.
The addition of AA to the MC3T3 cells for the first 5 days, along with β-glycerophosphate from
day 5 to day 23, resulted in bone nodule formation, when compared to the control cultures
(Figures 19A, 20A, 21A, 22A and 23A). The amount of mineralization increased as the
concentration of calcium was increased for the AA-primed cultures (Figure 19A) and this trend
was confirmed through quantification (Figure 19B), as previously described. Linear mixed
effects analysis revealed that the slope of the line between the total mineralized area and calcium
concentration was considered positive and significantly non-zero (t-value = 3.774, df = 356, p <
0.05), indicating that increasing calcium concentration within the AA-primed MC3T3 cultures
led to increasing bone nodule formation. We also found that increasing levels of vitamin K1 and
vitamin K2 led to increased bone mineralization of the AA-primed MC3T3 cultures (Figure 20A
and 21A, respectively), which corresponded to positive, significantly non-zero slopes for the
vitamin K1 line (t-value = 4.719, df = 416, p < 0.05; Figure 20B) and the vitamin K2 line (t-value
= 3.131, df = 416, p < 0.05; Figure 21B). Unlike calcium, vitamins K1 and K2, the
supplementation of increasing concentrations of 25D did not lead to changed bone nodule
formation (Figure 22A) and thus the slope of the line was not significantly different than zero (t-
value = 1.818, df = 416, p > 0.05; Figure 22B).
As opposed to the other vitamins and calcium, increasing concentrations of 1,25D led to
decreased total mineralized area (Figure 23A). Similar to the 1,25D supplemented, continual-AA
MC3T3 data, the quadratic model fit the 1,25D data better than the linear model (χ2
= 13.671, df
= 1, p < 0.05; Figure 23B). However, the quadratic model was still not a good fit for the 1,25D
data given that even a tiny amount of 1,25D in the AA-primed MC3T3 culture resulted in a sharp
56
drop in bone mineralization. We summarized the results of the bone formation experiments using
the vitamin/calcium supplemented, AA-primed MC3T3 cultures into Table 7. Altogether this
suggested that increasing levels of calcium, vitamin K1 and K2 resulted in increased bone
mineralization, while bone nodule formation decreased with increasing concentrations of 1,25D
within AA-primed MC3T3 cultures.
57
Figure 19. Bone mineralization increased with increasing concentrations of calcium, when
MC3T3 cells were primed with AA. MC3T3 cells were stimulated with or without AA and
calcium for the first 5 days and β-glycerophosphate and calcium from day 5 to day 23.
(A) Brightfield images were taken of the von Kossa stained cells and the total mineralized area
of each image was determined using Image J. (B) The graph displays the mean ± SEM of total
mineralized area for 20 images for each trial point and the mean trendline of the three trials.
Linear mixed effects analysis of the relationship between total mineralized area and calcium
concentration was performed and * p < 0.05 when the slope of the line was considered
significantly different than zero. Scale bar represents 100 µm.
58
Figure 20. Increasing levels of vitamin K1 resulted in greater bone nodule formation in AA-
primed MC3T3 cultures. MC3T3 cells were stimulated with or without AA and vitamin K1 for
the first 5 days and β-glycerophosphate and vitamin K1 from day 5 to day 23. (A) Brightfield
images were taken of the von Kossa stained cells and the total mineralized area of each image
was determined using Image J. (B) The graph displays the mean ± SEM of total mineralized area
for 20 images for each trial point and the mean trendline of the three trials. Linear mixed effects
analysis of the relationship between total mineralized area and vitamin K1 concentration was
performed and * p < 0.05 when the slope of the line was considered significantly different than
zero. Scale bar represents 100 µm.
59
Figure 21. Total mineralized area of AA-primed MC3T3 cultures increased with increasing
concentrations of vitamin K2. MC3T3 cells were stimulated with or without AA and vitamin
K2 for the first 5 days and β-glycerophosphate and vitamin K2 from day 5 to day 23.
(A) Brightfield images were taken of the von Kossa stained cells and the total mineralized area
of each image was determined using Image J. (B) The graph displays the mean ± SEM of total
mineralized area for 20 images for each trial point and the mean trendline of the three trials.
Linear mixed effects analysis of the relationship between total mineralized area and vitamin K2
concentration was performed and * p < 0.05 when the slope of the line was considered
significantly different than zero. Scale bar represents 100 µm.
60
Figure 22. Increasing concentrations of 25D did not lead to increased bone mineralization
of AA-primed MC3T3 cells. MC3T3 cells were stimulated with or without AA and 25D for the
first 5 days and β-glycerophosphate and 25D from day 5 to day 23. (A) Brightfield images were
taken of the von Kossa stained cells and the total mineralized area of each image was determined
using Image J. (B) The graph displays the mean ± SEM of total mineralized area for 20 images
for each trial point and the mean trendline of the three trials. Linear mixed effects analysis of the
relationship between total mineralized area and 25D concentration was performed and * p < 0.05
when the slope of the line was considered significantly different than zero. Scale bar represents
100 µm.
61
Figure 23. Bone mineralization of AA-primed MC3T3 cultures decreased with increasing
levels of 1,25D. MC3T3 cells were stimulated with or without AA and 1,25D for the first 5 days
and β-glycerophosphate and 1,25D from day 5 to day 23. (A) Brightfield images were taken of
the von Kossa stained cells and the total mineralized area of each image was quantified using
Image J. (B) The graph displays the mean ± SEM of total mineralized area for 20 images for
each trial point and the mean quadratic trendline of the three trials. The quadratic model is
considered a better fit as compared to the linear model, when * p < 0.05 using a likelihood ratio
test and when the AIC of the quadratic model is lower than the linear model. Scale bar represents
100 µm.
62
Table 7. Summary of the results of the calcium or vitamin supplemented bone formation
experiments run on the AA-primed MC3T3 cultures. The total mineralized area of von
Kossa-stained cultures was quantified for each supplement and the relationship between the
mineralized area and supplement concentration was determined. For each supplement, we
indicated the type of model (Quadratic or Linear) that best fit the data and the directionality of
that trend (Positive or Negative). In the cases where the slope of the linear trend was not
significantly different than zero, we displayed this as No Trend with two consecutive dashes in
the directionality column.
Vitamin or Calcium
Added
Linear, Quadratic
or No Trend
Positive or Negative
Trend
Calcium Linear Positive
Vitamin K1 Linear Positive
Vitamin K2 Linear Positive
25D No Trend --
1,25D Quadratic Negative
63
3.2.4 The addition of 25D + K2 lead to decreased bone mineralization of continual-AA treated MC3T3 cells as compared to both vitamin K2 and 25D alone. The other combinations, under AA-primed and continual-AA conditions, resulted in no change to the level of mineralization obtained from all the singular vitamin or calcium controls.
Next, combinations of calcium, vitamin K1, vitamin K2, 25D and 1,25D were added to MC3T3
and Saos-2 cultures under AA-primed or continual-AA conditions in order to assess their effect
on mineralization. It was hypothesized that combinations of vitamins and calcium should further
enhance the level of mineralization observed when cultures were treated with one
vitamin/calcium alone. Ideally we would have liked to use a range of concentrations for each
vitamin/calcium and looked for any interactions among all of the possible combinations.
However, we did not have time to complete a full interaction model and thus looked only at the
combinations using a single concentration of each vitamin/calcium.
The combinations of vitamins and calcium, with the exception of 25D + K2, did not change the
amount of bone mineralization obtained by the controls of vitamin or calcium alone within the
continual-AA MC3T3 cultures (see Supplemental Figure 2A - 2F and 2H for representative
brightfield images). This pattern was confirmed by quantification of the total mineralized area by
Image J (Figure 24A – 24F and 24H). A one-way ANOVA with a Tukey’s posthoc test was
performed to analyze the data. The combinations were only considered significantly different
than the singular vitamin or calcium controls if all the comparisons of the combination against
the relevant controls had a p-value less than 0.05. For all of the other continual MC3T3 cultures
besides 25D + K2, bone nodule formation for the combinations of vitamins and calcium were not
significantly different than that of the singular vitamin/calcium controls. In contrast, the
combination of 25D + K2 in continual-AA MC3T3 cultures resulted in less mineral than either
the 25D or K2 controls (Supplemental Figure 2G). This trend was confirmed through statistical
analysis of the total mineralized area of the images (Figure 24G), where the combination of 25D
and K2 was significantly lower than both the 25D and K2 controls.
The combinations used to treat the AA-primed MC3T3 cultures had no effect on bone nodule
formation as compared to the singular controls (Supplemental Figure 3A - 3H). This was
corroborated by the lack of a statistical difference between the combinations and relevant
64
controls (Figure 25A - 25H). In summary, the addition of 25D + K2 to continual-AA treated
MC3T3 cultures resulted in decreased bone mineralization compared to the 25D and K2 controls,
while all of the other combinations had no effect on bone nodule formation in both continual-AA
or AA-primed MC3T3 cultures.
65
Figure 24. The supplementation of 25D + K2 resulted in significantly decreased bone
mineralization of continual-AA treated MC3T3 cells as compared to both vitamin K2 and
25D alone. The other combinations caused no change to the level of mineralization obtained
from all the singular vitamin or calcium controls. MC3T3 cells were supplemented with AA,
vitamins and/or calcium throughout the 22 day experiment, while β-glycerophosphate was added
from day 5 to day 22. Image quantification was performed for von Kossa stained, combination
treated cultures (with appropriate singular vitamin/calcium controls) including (A) Ca + K1,
(B) Ca + K2, (C) Ca + K1 + K2, (D) K1 + K2, (E) Ca + D, (F) D + K1, (G) D + K2 and
(H) Ca + K1 + K2 + D. Data is represented as mean ± SEM of triplicate experiments, where 20
images were quantified for each trial. The combination was considered significant (*) when p <
0.05 for all comparisons of the combination against the appropriate singular controls, as
determined by a one-way ANOVA with Tukey’s posthoc test.
66
Figure 25. Amount of MC3T3 mineralization was unchanged for combinations of vitamins
and calcium under AA-primed conditions as compared to all the appropriate singular
vitamin or calcium controls. MC3T3 cells were treated with AA for the first five days and β-
glycerophosphate from day 5 to day 22, while vitamins and/or calcium were added throughout
the experiment. Image quantification was performed for von Kossa stained, combination treated
cultures (with appropriate singular vitamin/calcium controls) including (A) Ca + K1, (B) Ca +
K2, (C) Ca + K1 + K2, (D) K1 + K2, (E) Ca + D, (F) D + K1, (G) D + K2 and (H) Ca + K1 + K2 +
D. Data is represented as mean ± SEM of triplicate experiments, where 20 images were
quantified for each trial. The combination was considered significant (*) when p < 0.05 for all
comparisons of the combination against the appropriate singular controls, as determined by a
one-way ANOVA with Tukey’s posthoc test.
67
3.2.5 Bone mineralization levels of Saos-2 cultures supplemented with combinations of vitamins and calcium under AA-primed and continual-AA conditions were unchanged as compared to all of the singular vitamin or calcium controls.
Bone nodule formation of the combinations of vitamins and calcium in the continual-AA Saos-2
cultures did not look different than the amount of mineralization obtained by the controls of
vitamin or calcium alone (Supplemental Figure 4A - 4H) and this was confirmed by
quantification (Figure 26A - 26H) and statistical analysis, as previously described. Thus, there
was no significant difference between the amount of mineralization caused by the combination
treatments and the appropriate singular vitamin controls. Similarly for the AA-primed Saos-2
cultures, the bone nodule formation of the combination treatments was not different than that of
the vitamins or calcium alone based on both observation (Supplemental Figure 5A - 5H) and
statistics (Figure 27A - 27H). In summary, the combinations of vitamins and calcium did not
alter the amount of bone mineralization within AA-primed or continual-AA Saos-2 cultures, as
compared to the singular vitamin/calcium controls.
68
Figure 26. Combinations of vitamins and calcium caused no change to the level of
mineralization obtained from all the singular vitamin/calcium supplemented, continual-AA
treated, Saos-2 cultures. Saos-2 cells were treated with AA, vitamins and/or calcium
throughout the 22 day experiment, while β-glycerophosphate was added from day 5 to day 22.
Image quantification was performed for von Kossa stained, combination treated cultures (with
appropriate singular vitamin/calcium controls) including (A) Ca + K1, (B) Ca + K2,
(C) Ca + K1 + K2, (D) K1 + K2, (E) Ca + D, (F) D + K1, (G) D + K2 and (H) Ca + K1 + K2 + D.
Data is represented as mean ± SEM of triplicate experiments, where 20 images were quantified
for each trial. The combination was considered significant (*) when p < 0.05 for all comparisons
of the combination against the appropriate singular controls, as determined by a one-way
ANOVA with Tukey’s posthoc test.
69
Figure 27. Mineralization levels of Saos-2 cells treated with combinations of vitamins and
calcium under AA-primed conditions were unchanged as compared to all of the singular
vitamin or calcium controls. Saos-2 cultures were treated with AA for the first five days and β-
glycerophosphate from day 5 to day 22, while vitamins and/or calcium were supplemented
throughout the experiment. Image quantification was performed for von Kossa stained,
combination treated cultures (with appropriate singular vitamin/calcium controls) including
(A) Ca + K1, (B) Ca + K2, (C) Ca + K1 + K2, (D) K1 + K2, (E) Ca + D, (F) D + K1, (G) D + K2
and (H) Ca + K1 + K2 + D. Data is represented as mean ± SEM of triplicate experiments, where
20 images were quantified for each trial. The combination was considered significant (*) when p
< 0.05 for all comparisons of the combination against the appropriate singular controls, as
determined by a one-way ANOVA with Tukey’s posthoc test.
70
3.2.6 Increasing concentrations of calcium or vitamin did not lead to increased collagen concentrations within MC3T3 or Saos-2 cultures.
Collagen is a major component of the organic, fibrillar extracellular network, in that the
hydroxyapaptite crystals (mineral) are deposited onto this network (Long, 2012). In order to
assess if the changing levels of mineralization due to supplementation was the result of different
collagen levels, the concentration of collagen in early MC3T3 and Saos-2 cultures was assessed
(Figures 28 and 29, respectively). A five day assay was chosen because collagen secretion
increases rapidly during this time period (Nabavi et al., 2008). The concentrations were
determined by finding the absorbance of the Sirius red dye extracted from each well and
interpolating the concentration from a prepared collagen standard curve. The concentrations were
finally normalized to the appropriate controls before linear regression analysis. Increasing
concentrations of calcium had no effect on the relative collagen concentrations of the MC3T3 (F
= 0.06564, df = 1, 10, r2 = 0.006522, p = 0.8030; Figure 28A) and Saos-2 (F = 2.002, df = 1, 10,
r2 = 0.1668, p = 0.1875; Figure 29A) cells and thus the slopes of the regression lines were not
significantly different than zero. Similarly, the slope of the regression lines between the collagen
concentration and levels of vitamin K1 was not significantly different than zero for the MC3T3
(F = 2.092, df = 1, 10, r2 = 0.1167, p = 0.2772; Figure 28B) and Saos-2 cell cultures (F = 1.321,
df = 1, 10, r2 = 0.1668, p = 0.1875; Figure 29B). The collagen concentration was unchanged,
when vitamin K2 concentration was increased in MC3T3 (F = 0.02412, df = 1, 10, r2 = 0.002406,
p = 0.8797; Figure 28C) and Saos-2 cell cultures (F = 0.4383, df = 1, 10, r2 = 0.04199, p =
0.5229; Figure 29C). In addition, the slope of the lines between the relative collagen
concentration and the concentration of 25D was not significantly different than zero for the
cultures of MC3T3 (F = 1.525, df = 1, 10, r2 = 0.1323, p = 0.2451; Figure 28D) and Saos-2 (F =
1.863, df = 1, 10, r2 = 0.1570, p = 0.2022; Figure 29D) cells. Finally, increasing concentrations
of 1,25D had no effect on the collagen concentrations of MC3T3 (F = 3.943, df = 1, 10, r2 =
0.2828, p = 0.0752; Figure 28E) and Saos-2 (F = 2.998, df = 1, 10, r2 = 0.2307, p = 0.1140;
Figure 29E) cells. In conclusion, the relative collagen concentrations within MC3T3 and Saos-2
cultures did not change with increasing concentrations of calcium, vitamin K1, vitamin K2, 25D
or 1,25D.
71
Figure 28. Increasing concentrations of calcium or vitamin did not lead to increased
collagen concentrations within MC3T3 cultures. MC3T3 cultures were supplemented with
AA and calcium or vitamins for five days. Cells were stained with picrosirius dye and the dye
was extracted from each well using NaOH. The absorbance of the extracted dye solution was
read at 528 nm using a plate reader and the collagen concentrations were normalized to the
appropriate controls, after interpolation of a collagen standard curve. Graphical representation of
the normalized collagen concentrations of MC3T3 cultures treated with (A) calcium, (B) vitamin
K1, (C) vitamin K2, (D) 25D and (E) 1,25D. Data was presented as points representing the mean
and SEM of 3 independent experiments and a regression line, where * p < 0.05 indicates that the
slope of the regression line is significantly different than zero.
72
Figure 29. Collagen levels within Saos-2 cultures did not change with increasing
concentrations of calcium or vitamin. AA and calcium or vitamins were added for five days to
Saos-2 cultures. Cells were stained with picrosirius dye and the dye was extracted from each well
using NaOH. The absorbance of the extracted dye solution was read at 528 nm using a plate
reader and the collagen concentrations were normalized to the appropriate controls, after
interpolation of a collagen standard curve. Graphical representation of the normalized collagen
concentrations of Saos-2 cultures treated with (A) calcium, (B) vitamin K1, (C) vitamin K2, (D)
25D and (E) 1,25D. Data was presented as points representing the mean and SEM of 3
independent experiments and a regression line, where * p < 0.05 indicates that the slope of the
regression line is significantly different than zero.
73
4 DISCUSSION
4.1 Inferences that can be made from the results of the meta-analyses.
As far as we know, this thesis includes the first cell biology meta-analysis that has ever been run
on the effects of vitamins on parameters related to bone formation. The experiment type
subanalyzed meta-analyses revealed that the addition of vitamin K1, K2 or D to osteoblasts
resulted in increased mineralization within the culture. Enhanced mineralization was also
observed for the combination of vitamin K2 + 1,25D against both of the singular vitamin
controls. ALP activity was found to significantly increase with the addition of vitamin K2 or
vitamin D, but the effects of vitamin K1 supplementation are inconclusive given that the
confidence interval and non-directional tests do not agree. The levels of osteocalcin increased
with the addition of vitamin D to the osteoblast cultures, but did not change with
supplementation with vitamin K2. Surprisingly, vitamin K2 increased the DNA levels within the
culture, but significantly decreased the amount of proliferation within the culture. This
discrepancy will be discussed further in section 4.2. Supplementation with vitamin D also
resulted in increased collagen and osteopontin levels within the osteoblast cultures. The addition
of vitamin K2 significantly increased the bone formation parameters measured in the group
called Other Experiments, but vitamin K1 supplementation had no effect on the osteoblast
maturation characteristics in the Other Experiments group. The effect of vitamin D on the
parameters measured within the Other Experiments group is inconclusive, since the results of the
confidence interval test and the non-directional test do not agree. Interestingly, the effect of the
combination of K2 + 1,25D compared to the effect of K2 alone resulted in increased bone
formation characteristics in the Other Experiments group, but the combination did not increase
maturation parameters in the Other Experiments group when compared to the effects of 1,25D
alone. The lack of consistency amongst the meta-analyses concerning the Other Experiments
group could be because there were different types of experiments included within the group for
each meta-analysis. Altogether this indicated that the addition of vitamin K1, K2 and D as well as
the combination of K2 + 1,25D increases bone mineralization within osteoblast cultures, but
might not consistently increase all of the other osteoblast maturation parameters that are thought
to be indicative of bone formation.
74
The cell type subanalyzed meta-analyses revealed that both vitamin K2 and vitamin D
supplementation increased bone cell parameters within osteoblast cultures made of murine cell
lines and human primary cells. In addition, only supplementation of vitamin K2 resulted in an
increase in maturation parameters within murine primary cell cultures, while vitamin D addition
had no effect. In human cell line cultures, vitamin D addition increased bone formation
measurements, but vitamin K2 supplementation had inconclusive effects. In summary, addition
of vitamin K2 or D has variable effects on bone cell parameters within cultures of different cell
types.
4.2 Uncertainty in the use of proliferation measurements within the K2 meta-analysis.
Previously the vitamin K2 meta-analysis revealed that the addition of vitamin K2 to osteoblasts
decreased proliferation, as seen through a significantly negative grand mean effect size for the
proliferation subgroup. In the meta-analysis this result was interpreted as vitamin K2
supplementation will impair the maturation of osteoblast cultures, since the first stage of
osteoblast maturation is the proliferative phase (Neve et al., 2011). However, decreased
osteoblast proliferation after a week of 1,25D supplementation has also been linked with
increased human primary cell mineralization later in the culture period (Atkins et al., 2007). The
addition of 1,25D could have decreased the amount of time that the osteoblast cells were in the
proliferative phase, leading to a decreased number of cellular divisions. Simultaneously, 1,25D
supplementation could also increase the time spent in the differentiation and mineralization
phases, which could indicate why there was increased mineralization in the supplemented
cultures as compared to the untreated cultures. However, the meta-analysis also revealed that
vitamin K2 supplementation increased DNA levels, which can be used as a measure of
proliferation. Altogether this suggests that proliferation measurements might not be a clear
indicator of osteoblast maturation.
75
4.3 Considerations for the interpretation of the meta-analyses.
4.3.1 Homogeneity issues within the experiment and cell type subgroups.
Homogeneity of the experiments within a subgroup of the meta-analysis will increase the
confidence that the grand mean effect size will represent any study looking at the same
phenomenon under the same conditions (i.e. same cell type if the subgroup was from the cell
type subanalyzed meta-analysis). Some of the subgroups that were analyzed within these meta-
analyses were heterogeneous and thus one cannot be completely sure that the results seen will
represent every vitamin supplementation cell biology paper. Ultimately it would be ideal to
continue further subanalysis of all of the heterogeneous subgroups until each group is
homogeneous. However, further subanalysis on all of the subgroups was not possible with the
already small number of experiments in some of the groups.
4.3.2 Conflict between the results of the non-directional test and the confidence interval test.
Both the confidence interval (CI) test and the non-directional test were used to assess if the grand
mean effect sizes were significantly different than zero. However, there were times within the
meta-analyses when the results of the confidence interval test did not agree with that of the non-
directional test. The non-directional test is more conservative than the CI test and is more likely
to result in nonsignificance when the sample size is small and the variance is large (Cadotte,
2006). Simultaneously, the confidence interval test is more affected by outliers than the robust
non-directional test (Mchugh, 2013) and could lead to nonsignificance when there are outliers
present. Altogether, this could indicate why the results of both statistical tests did not agree with
each other in every scenario. In the cases where the tests do not agree, the effect sizes could still
be significantly different than zero and thus the definition of a significant result within meta-
analyses in general might need to be re-evaluated (Cadotte, 2006).
76
4.4 Mineralization in calcium or vitamin supplemented mouse, MC3T3, cell cultures compared to human, Saos-2, cell cultures under continual-AA conditions.
4.4.1 Adverse effects of 1,25D supplementation on continual-AA treated mouse osteoblasts as compared to human osteoblasts.
The addition of 1,25D had negative effects on mineralization in continual-AA (ascorbic acid is
added throughout the experiment) treated mouse osteoblast cultures, while 1,25D had no effect
on mineralization in the human osteoblast cultures. The species specific effect of 1,25D on
mineralization was also seen in past literature, where 1,25D supplementation resulted in
increased mineralization with human osteoblast cultures (Atkins et al., 2007; Koshihara et al.,
1996; van Driel et al., 2006; Woeckel et al., 2010; Zhou et al., 2012) and primarily negative
effects on von Kossa and Alizarin red S stained mineralization within mouse MC3T3 cultures
(Shi et al., 2007; Masayoshi Yamaguchi & Weitzmann, 2012). This reported decrease in
mineralization resulting from 1,25D addition to MC3T3 cultures was in agreement with our
findings. However, there was a single article that observed a positive effect of continual 1,25D
supplementation on calcium levels within mouse MC3T3 cultures (Matsumoto et al., 1991),
which does not agree with any of other literature or our study. It is likely that the 1,25D increase
in human osteoblast mineralization was not seen within our experiments since the concentration
of vitamin used was very small (0.01-0.1 nM), as compared to the previously mentioned human
osteoblast papers that used 1 nM up to 100 nM. In addition, the supplementation of 1,25D to
mice resulted in increased levels of pyrophosphate and therefore decreased mineralization of
bones within the mouse (Lieben et al., 2012), as there needs to be low pyrophosphate levels for
mineralization to occur in vitro and within the body (Russell, Bisaz, Donath, Morgan, & Fleisch,
1971). Increased pyrophosphate levels also leads to adverse effects within cell culture, including
autophagic cell death as seen in fermenting yeast (Serrano-Bueno et al., 2013). This is consistent
with the cell morphology changes and increased cell death that was observed in the MC3T3
cultures, when 1,25D was added at a concentration greater than 1 nM (only added in these high
concentrations under AA-primed conditions). Altogether, this indicates that 1,25D has adverse
effects on mouse osteoblasts that impair bone nodule formation, while sufficient concentrations
of 1,25D stimulates bone mineralization within human osteoblast cultures.
77
4.4.2 Vitamin K2 addition has different effects on continual-AA treated mouse osteoblasts in comparison to human osteoblasts.
The supplementation of vitamin K2 to continual-AA treated mouse osteoblasts resulted in
decreased mineralization, while vitamin K2 addition had no effect on mineralization in human
osteoblast cultures. Although, there were no cell biology papers that looked at the effect of
vitamin K2 supplementation on mouse osteoblasts, vitamin K2 addition to human osteoblasts
resulted in increased mineralization levels in previous literature (Atkins et al., 2009; Koshihara et
al., 1996; Sugimoto et al., 2007). Since past studies have shown that vitamin K2 addition
increases mineralization within other human osteoblast cultures (did not use the Saos-2 cell line),
the lack of an increase in bone formation within our Saos-2 cultures could be attributed to a cell
line specific effect or could be due to the higher concentration used in the past literature (1 µM
up to 10 µM), as compared to our experiments (maximal concentration of 1 µM). In addition,
there is no past literature that looked at the effect of vitamin K2 on MC3T3 cultures and thus the
vitamin K2 -induced decrease on mineralization within continual-AA treated MC3T3 cultures
could also be related to this specific cell line.
4.4.3 Human and mouse osteoblast mineralization increases with calcium supplementation under continual-AA treatment.
Unlike the confusing species specific mineralization response induced by the vitamins we
discussed earlier, previous literature has shown that calcium addition to both human and mouse
cell lines results in increased mineralization (Adluri et al., 2010; Dvorak et al., 2004; Maeno et
al., 2005; Welldon et al., 2013; Yamauchi et al., 2005). In particular our observed increase in
mineralization due to calcium supplementation within MC3T3 cultures was confirmed by a
study, which observed the calcium-induced increase in mineralization through von Kossa
staining and the quantification of the absorbance of Alizarin red stain (stains mineral) extracted
from the treated MC3T3 cultures (Yamauchi et al., 2005). This was consistent with the increased
bone mineralization that we observed within the continual-AA treated mouse and human
osteoblast cultures. Increased calcium supplementation has previously been linked with
increased transcript level of osteoblast differentiation markers, including osteocalcin, osteopontin
and collagen type I (Dvorak et al., 2004). When the function of the calcium-sensing receptor,
which detects extracellular calcium levels, is abolished within osteoblasts, osteocalcin
78
expression, ALP activity and mineralization is decreased within cell culture (Yamauchi et al.,
2005). Thus it is thought that extracellular calcium levels can alter the differentiation and
mineralization of osteoblasts through the calcium-sensing receptor (Marie, 2010) and calcium
supplementation will lead to increased bone nodule formation within cell culture.
4.4.4 The addition of vitamin K1 or 25D has no effect on the amount of mineralization within the continual-AA treated MC3T3 and Saos-2 cultures.
Previously it was shown that bone mineralization levels in continual-AA treated human and
mouse cultures did not change with supplementation with either vitamin K1 or 25D. Unlike what
we found, past literature has observed increased mineralization in human cell culture (no papers
using the Saos-2 cell line) with addition of 25D (Atkins et al., 2007) or vitamin K1 (Koshihara et
al., 1996). However, vitamin K1 supplementation in human primary cells was variable, leading
either to an increase or no change in mineralization levels depending on the donor (Atkins et al.,
2009). At this point in time the lack of an increase in bone nodule formation with the addition of
vitamin K1 to the MC3T3 and Saos-2 cell lines appears to be a cell line specific effect and
studies need to be completed using these cell lines in order to confirm this. However, the paper
by Koshihara et al. (1996) used 2500 nM vitamin K1 supplementation, which is more than twice
the maximum concentration we used in our mineralization experiments and could also have
affected mineralization in their human osteoblast cultures. In regards to 25D supplementation,
25D is not the active form of vitamin D and both human and mouse primary osteoblasts express
the enzyme required for the conversion from 25D to the active form, 1,25D (Howard, Turner,
Sherrard, & Baylink, 1981; F. Ichikawa et al., 1995). The mouse osteoblasts thus might have the
ability to limit the amount of the cytotoxic 1,25D in their environment, which could help to
prevent the 1,25D-induced decrease in mineralization. The mineralization levels within Saos-2
cultures supplemented with 25D might not have exhibited the increase observed by Atkins et al.
(2007) using human osteoblasts, due to a cell specific effect or since they used a different source
of inorganic phosphate, monopotassium phosphate, as compared to our use of β-
glycerophosphate. Therefore, the addition of vitamin K1 or 25D does not change mineralization
levels under continual-AA conditions within mouse and human osteoblast cultures.
79
4.5 Implications of collagen production on mineralization within human and mouse cell culture.
The amount of collagen within the extracellular matrix places a limit on the amount of
mineralization that can occur both in vitro and within the body (Landis, Silver, & Freeman,
2006). Thus it was surprising that we found that early collagen production was not changed with
calcium, vitamin K1, vitamin K2, 25D or 1,25D supplementation within MC3T3 and Saos-2
cultures. This indicated that early collagen production was not linked with mineralization levels,
given that addition of the vitamins and calcium had variable effects on the amount of
mineralization in vitro and also indicated that vitamin or calcium addition did not affect the
initial rate of collagen deposition. This suggests that the amount of collagen production does not
appear to be a limiting factor to the levels of mineralization we observed in both the AA-primed
MC3T3 cultures and continual-AA treated Saos-2 and MC3T3 cultures.
It is expected that collagen production cannot continue without the presence of ascorbic acid, as
it serves as a reducing agent (in the anion form called ascorbate) in the hydroxylation of
peptides. It is also expected that mineralization levels would be limited by the lack of collagen
produced under AA-primed conditions, where AA is only added for the first 5 days. However,
we observed that day 23 collagen levels under AA-primed conditions were greater than that of
the day 5 levels based on picrosirius staining (data not shown). This indicated that collagen
production is still occurring despite the lack of ascorbic acid. It is possible that there is another
suitable reducing agent within the cell media or FBS, as tetrahydropteridines, tetrahydrofolate
and dithiothreitol have all been reported to replace ascorbate in the hydroxylation reactions,
albeit less effectively than ascorbate (Hutton, Tappel, & Udenfriend, 1967; Rhoads &
Udenfriend, 1970). It is also possible that there is a small supply of ascorbic acid within the cells
after the supplementation period. In addition, the hydroxylation reactions of proline and lysine
do not use ascorbic acid stoichiometrically when there are sufficient procollagen substrates
present within the ER, which means that many hydroxylation reactions will occur in the absence
of ascorbic acid (Kivirikko & Myllylä, 1985). Altogether, this signifies that collagen production
is not limiting the amount of vitamin/calcium-induced mineralization in MC3T3 and Saos-2
cultures under various ascorbic acid treatments.
80
4.6 Vitamin and calcium-induced bone nodule formation under AA-primed conditions as compared to continual-AA conditions in murine MC3T3 cultures.
4.6.1 Mineralization levels resulting from the addition of vitamin K1 or K2 varies depending on the amount of AA within the MC3T3 culture.
Vitamin K1 or K2 supplementation resulted in increased mineralization under AA-primed
conditions, while under continual-AA conditions, mineralization was unchanged or decreased,
respectively. Given that ascorbic acid is an antioxidant (Padayatty et al., 2003), the AA-primed
conditions might set-up an environment that is richer in reactive oxygen species (ROS) than the
continual-AA environment, leading to greater cell stress under the AA-primed conditions.
Although the vitamin K’s are not considered classical antioxidants, they were found to inhibit
cell death due to oxidative stress within primary neuronal cultures (Li et al., 2003). Vitamin K1
and K2 could stand in for ascorbic acid under AA-primed conditions to help to prevent oxidative
damage to the cells and the proteins within the extracellular matrix, thus leading to increased
mineralization under the AA-primed conditions.
4.6.2 Calcium supplementation leads to increased mineralization in AA-primed and continual-AA treated MC3T3 cultures.
It was previously found that the addition of calcium to osteoblasts under continual-AA
conditions and AA-primed conditions resulted in increased mineralization, which was not
inhibited by extracellular collagen levels. Assuming that the lack of ascorbic acid under the AA-
primed conditions leads to oxidative stress in cell culture, then calcium must find a way to
combat this stress. Extracellular calcium levels increase osteoblast differentiation through the
calcium-sensing receptor (Dvorak et al., 2004). If calcium increases the levels of the protein
EB1, which is known to increase when osteoblasts are differentiating, then more β-catenin will
be stabilized at the cell cortex and not degraded by the destruction complex (Pustylnik et al.,
2013). Increased β-catenin levels enhance Forkhead box O (FOXO) transcriptional activity,
where FOXO transcribed proteins have been linked to protecting cells and proteins against
oxidative stress (Essers et al., 2005). Therefore, greater calcium levels might increase the amount
of mineralization under AA-primed conditions due to the production of antioxidant proteins.
81
4.6.3 The addition of 25D or 1,25D has the same effect on mineralization in AA-primed and continual-AA treated MC3T3 cultures.
Supplementation of 25D under AA-primed and continual-AA conditions had no effect on the
amount of mineralization observed within MC3T3 cultures, while 1,25D addition decreased bone
nodule formation under AA-primed and continual-AA conditions. Although vitamin D has
antioxidant properties (Wiseman, 1993), the production or addition of cytotoxic 1,25D could
have prevented the potential antioxidant-induced increase in mineralization under AA-primed
conditions in MC3T3 cultures that has previously been seen with calcium or the vitamin K’s.
4.7 Most combinations of vitamins and calcium did not have an effect on mineralization in cultures of the mouse cell line, MC3T3, and the human cell line, Saos-2.
The majority of combinations of vitamins and calcium under both AA-primed and continual-AA
conditions did not change the level of mineralization obtained from the single vitamin/calcium
controls within the human and mouse osteoblast cultures. The only vitamin combination that
affected mineralization was 25D + K2 in continual-AA treated MC3T3 cultures, where the
combination significantly lowered the amount of mineralization seen by both the 25D and
vitamin K2 singular controls. There have been previous cell biology or clinical studies that
looked at the effects of combinations of vitamins and calcium on bone formation (Adluri et al.,
2010; Bolton-Smith et al., 2007; Dawson-Hughes, Harris, Krall, & Dallal, 1997; Harwood,
Sahota, Gaynor, Masud, & Hosking, 2003; Inoue, Sugiyama, Matsubara, Kawai, & Furukawa,
2001; J. Iwamoto, Takeda, & Ichimura, 2000; Je et al., 2011; Kärkkäinen et al., 2010; Koshihara
et al., 1996; Y. Sato, Kanoko, Satoh, & Iwamoto, 2005; Somekawa et al., 1999; Sugimoto et al.,
2007; Ushiroyama et al., 2002; Yonemura et al., 2004; Zhu, Devine, Dick, Wilson, & Prince,
2008). However, the majority of these studies either fail to include the proper singular controls or
fail to perform the statistical comparisons between these controls and the combinations. Thus
they were unable to ascertain if the combination had changed bone nodule formation beyond that
of the singular effects of the vitamin/calcium, which meant that parallels could not be drawn
between our combination data and the majority of the previous literature. In contrast to the
decrease in mineralization that we observed with the combination of 25D + K2, there were three
82
clinical studies that found that the combination of vitamin D and vitamin K2 had either a positive
effect or no effect on bone mineral density compared to the singular controls (J. Iwamoto et al.,
2000; Ushiroyama et al., 2002; Yonemura et al., 2004). Given the lack of studies focusing on the
effect of combinations of vitamins and calcium on bone formation in vitro and in vivo with the
appropriate controls, more work needs to be done in order to conclusively decide the effect of
combinations on bone formation.
4.8 Limitations within the mineralization and collagen experiments.
Random pictures were taken of the von Kossa stained mineralized nodules within each treatment
culture, but this does not eliminate the possibility that an area of the culture was not imaged.
Thus it might be advantageous to take a single photograph of the entire mineralized culture and
quantify the total mineralized area of the treatment based on those images. The levels of collagen
determined for each treatment directly depended on the amount of picrosirius dye that was
extracted from the stained cultures using NaOH. If some of the dye was not removed from the
cell layers after the NaOH extraction step, then it is possible that the collagen levels within the
treatments could be different than one another. This could be avoided by directly growing the
cells in a smaller vessel that could be placed within the plate reader immediately after being
stained.
4.9 Comparison of our mineralization and collagen experiments with our meta-analyses.
The results of the meta-analyses can only be compared with the continual-AA mineralization
experiments we ran, as well as the collagen experiments, since all of the experiments used within
the meta-analyses were using continual supplementation of AA. Vitamin K1 supplementation had
no effect on mineralization in both the human Saos-2 and mouse MC3T3 cultures, but increased
mineralization in the meta-analysis. However, none of the mineralization experiments included
within the vitamin K1 meta-analysis utilized the Saos-2 or MC3T3 cell lines (Atkins et al., 2009;
Koshihara et al., 1996). In addition, the vitamin K1 meta-analysis did not include any collagen
experiments and thus there is no data from the meta-analysis to compare with our collagen
experiments.
83
Neither addition of vitamin K2 to human, Saos-2, nor mouse, MC3T3, cultures within our
experiments resulted in the increased mineralization that was observed within the meta-analysis.
Similar to the vitamin K1 meta-analysis, the vitamin K2 meta-analysis did not contain any
mineralization experiments that used the cell lines MC3T3 and Saos-2 (Atkins et al., 2009;
Koshihara et al., 1996; M Yamaguchi et al., 2001). Also, the vitamin K2 meta-analysis did not
include any collagen experiments, which could be compared to our collagen data.
The meta-analysis showed that vitamin D addition to cell culture resulted in increased bone
nodule formation, which was not seen with either supplementation of 1,25D or 25D in our
experiments. There were no experiments included within the vitamin D meta-analysis that
utilized the Saos-2 cell line or supplemented the cultures with 25D, but there were several
experiments that used MC3T3 cells. Mineralization experiments derived from the paper by
Masayoshi Yamaguchi & Weitzmann (2012) had significantly negative effect sizes when 1,25D
was added to MC3T3 cultures, which agreed with the results of our 1,25D mineralization data
within MC3T3 cultures. However, mostly significantly positive effect sizes were obtained for
MC3T3 mineralization experiments that were extracted from three other papers included in the
vitamin D meta-analysis. This is in direct contrast with our vitamin D MC3T3 mineralization
experiments. However, there were some experimental differences when comparing our
experiments to the experiments from within these three papers. In one paper, they used the
prohormone vitamin D, which could have different effects on mineralization than utilizing 1,25D
or 25D (Widaa et al., 2014). In the paper by Chen et al. (2013) they transiently exposed the
MC3T3 cultures with 1,25D for 15 minute periods three times a week and thus they might not
have observed the potentially cytotoxic effects of 1,25D. In the last paper, they used a different
source of phosphate ions, monopotassium phosphate, and used a lower concentration of FBS in
their media (Matsumoto et al., 1991), both of which could have affected mineralization. In
addition, vitamin D resulted in increased collagen levels as found in the meta-analysis, which we
did not see in either MC3T3 or Saos-2 cultures. However, the vitamin D meta-analysis did not
include collagen experiments that used Saos-2 cells. The majority of the experiments within the
vitamin D meta-analysis that did look at the effect of 1,25D on collagen synthesis in MC3T3
cultures had effect sizes that were not significantly different than zero (Matsumoto et al., 1991),
indicating that 1,25D addition has no effect on collagen levels in MC3T3 cultures. This result
from the meta-analysis concurs with the lack of change in collagen levels that we observed in
84
1,25D supplemented MC3T3 cultures. Overall, the results of the meta-analyses do not agree with
our experimental findings.
4.10 Advantages and disadvantages of our meta-analyses and bone formation experiments.
There are many advantages and disadvantages to performing our own bone formation
experiments in vitro and meta-analyses of past cell biology literature. The meta-analyses allowed
us to form conclusions about the effects of vitamins on several bone formation parameters and
could allow one to investigate the effect of unstudied cell culture variables (FBS concentration
and media type) on vitamin supplemented osteoblast characteristics. However, the lack of
homogeneity in subgroups within the meta-analyses prevents definite overall conclusions from
being formed that will represent any future vitamin supplementation cell biology study. This
heterogeneity could be due to the inclusion of experiments using different methodologies and
cell culture conditions. Running our own vitamin supplementation cell biology experiments
allows us to control growth conditions and thus limits some of the variability seen within the
meta-analyses caused by culture differences. In addition, we were able to include all of the
necessary controls that are required for appropriate statistical analysis of the combination
experiments. In contrast, our own experiments do not allow us to make major conclusions about
vitamin supplementation in vitro based solely on our research. Each approach has its own merits
and limitations, but together they allow us to form a more complete picture on the effect of
vitamin supplementation on bone formation parameters in vitro.
4.11 Future Directions
4.11.1 Additional mineralization experiments and the utilization of other measurements of mineralization.
Measuring mineralization within cell culture has previously been considered the in vitro
substitute of measuring the osteoblast mineralization in vivo (Atkins et al., 2007). The majority
of the previous experiments we ran used a single measurement of mineralization, where the bone
nodules were stained using the histological von Kossa method. However, there are many ways to
measure bone in cell culture, including looking at calcium or phosphate concentrations within the
mineralized nodules (Koshihara et al., 1996). In order to confirm the vitamin and calcium
85
supplementation trends obtained using the von Kossa stain, other methods should be utilized. In
addition, mouse and human primary cells should be used to confirm that the cell lines’ response
to vitamin and calcium represents the response of osteoblasts within mice and humans.
4.11.2 Effect of vitamin and calcium on other bone cell parameters.
In addition to the mineralization experiments, other osteoblast parameters could be measured to
confirm the results of the meta-analysis. These include immunostaining against ALP and looking
at the transcript levels of many osteoblast differentiation markers, like osteocalcin and collagen
type I. This will allow one to understand the effect of vitamin and calcium supplementation on a
variety of osteoblast parameters.
4.12 Conclusion
Although there has been much published literature about the effect of vitamin D, K1 and K2
supplementation on bone formation, a definite conclusion concerning their effect in vitro on cell
lines has yet to be made. We found through performing meta-analyses of the previous literature
that the addition of vitamin K1, K2 or D, as well as the addition of K2 + 1,25D, to osteoblasts
increased bone mineralization, but did not consistently change all of the osteoblast maturation
parameters that are associated with bone formation. When the experiments were subanalyzed by
cell type, it was revealed that vitamin K2 or D supplementation had variable effects on bone cell
parameters within cultures of different cell types. Through our own experiments, we found that
the effect of calcium, vitamin K1, vitamin K2, 25D and 1,25D supplementation on bone
mineralization varied depending on the presence of ascorbic acid or the organism from which the
cell lines were derived. The change in mineralization observed from the addition of the vitamins
or calcium was not due to differences in early collagen production. In addition, supplementation
with combinations of vitamins and calcium for the most part had no effect on mineralization in
osteoblasts cultures compared to singular vitamin or calcium controls. Ultimately, this work
indicates that the conditions in which bone formation is studied must be considered carefully to
determine the effect of calcium, vitamin K1, vitamin K2 and vitamin D supplementation on
osteoblasts in vitro and that meta-analysis is an extremely useful tool that has yet to be fully
utilized in the field of cell biology.
86
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Appendices
Supplemental Table 1. Articles used in the vitamin K1, K2, D and K2 + 1,25D meta-
analyses.
Vitamin K1 Vitamin K2 Vitamin D K2 + 1,25D
(Atkins et al., 2009) (Urayama et al., 2000)
(Masayoshi Yamaguchi & Weitzmann, 2012)
(Koshihara et al., 1996)
(Koshihara et al., 1996) (Akedo et al., 1992)
(Chen et al., 2013)
(Koshihara, Hoshi, Okawara, Ishibashi, & Yamamoto, 2003)
(M Yamaguchi et al., 2001)
(F. Sato et al., 1991)
(Gigante et al., 2008) (Ichiro Iwamoto et al., 2002)
(Matsumoto et al., 1991)
(Notoya et al., 1995) (Atkins et al., 2009)
(Widaa et al., 2014)
(Koshihara et al., 1996)
(R Narayanan et al., 2002)
(Koshihara et al., 2003)
(Franceschi et al., 1988)
(Notoya et al., 1995)
(Atkins et al., 2007)
(Kunisada, Kawai, Inoue, & Namba, 1997)
(Adluri et al., 2010)
(Koshihara et al., 1996)
(Zhou et al., 2012)
(Gigante et al., 2008)
(Kumei et al., 2004)
(Lynch, Stein, Stein, & Lian, 1995)
103
Supplemental Methods. Meta-analysis Equations
The following equations were adapted from Cadotte (2006). For the ith experiment extracted
from a paper, we calculated an unbiased standardized mean difference using the Hedges’ d
method, which we referred to as an effect size:
[1]
where was the mean experimental osteoblast maturation measurement of the experimental
vitamin treatment (e) and the control untreated condition (c), si was the pooled standard
deviation and J was the correction term for small sample size bias. The pooled standard
deviation was computed as follows:
[2]
where N was the sample size of the vitamin treated (e) and control (c) for the ith experiment. J,
the correction, was calculated as
[3]
and as The sampling variance for the ith experiment was computed as
[4]
which allowed us to calculate the confidence interval (CI) for each experiment’s effect size:
[5]
where experiments with significant vitamin treatment effects had a CI that did not overlap with
zero.
104
Fixed-Effects Model
The Hedges’ d effect sizes from k experiments were combined into a grand mean effect size for
the fixed-effects model:
[6]
where . The variance of this grand mean was calculated as
[7]
and allowed us to calculate the grand mean 95% confidence interval for the fixed-effects model:
[8]
It was assumed that the experiments used within the meta-analyses had homogeneous responses
to the vitamin treatments. In order to test the homogeneity of the treatment responses, we utilized
a Cochran’s Q test:
[9]
which is analogous to the within-class variation in an ANOVA test and has a distribution.
If the effects were considered homogeneous (Cochran’s Q was not significant), a fixed-effects model
was used to calculate the grand mean effect [6]. However, if the effects were considered
heterogeneous, a mixed-effects model was utilized to calculate the grand mean effect [11]. As
another assessment of the grand mean effect size’s difference from zero, a non-directional test,
similar to a x2 test was employed:
[10].
105
Mixed-Effects Model
The grand mean effects size calculations using the mixed-effects model were very similar to
those calculations performed for the fixed-effects model, except the variances were adjusted. For
the grand mean effect,
[11]
where
. The variance of this grand mean was calculated as
[12].
Unlike the fixed-effects model, the sampling variance for the ith experiment was adjusted to
account for the between-experiments variance (now ):
[13]
where
[14]
and the constant, c, was
[15].
The variance in the grand mean effect size for the mixed-effects model allowed us to calculate
the grand mean 95% confidence interval:
[16].
In order to determine if the experiments had homogeneous responses to the vitamin treatments
using the mixed-effects model, we again used Cochran’s Q test:
[17].
106
Beyond using the CI test to assess the grand mean effect size’s difference from zero for the mixed-
effects model we used a non-directional test:
[18].
107
Supplemental Figure 1. Overview of the quantification method used to determine the total
mineralized area for each image. Brightfield images were first thresholded for intensity, in
order to remove any pixels under a certain value, using Image J. Next, a size threshold was used
to remove any bone nodules under a set area. The areas of the bone nodules remaining after the
size threshold were summed to determine the total mineralized area of the image.
108
Supplemental Figure 2. The supplementation of 25D + K2 resulted in decreased bone
mineralization of continual-AA treated MC3T3 cells as compared to both vitamin K2 and
25D alone. The other combinations caused no change to the level of mineralization obtained
from all the singular vitamin or calcium controls. MC3T3 cells were supplemented with AA,
vitamins and/or calcium throughout the 22 day experiment, while β-glycerophosphate was added
from day 5 to day 22. (Continued on next page)
109
Supplemental Figure 2 continued
Representative brightfield images of von Kossa stained, combination treated cultures (with
appropriate singular vitamin/calcium controls) included (A) Ca + K1, (B) Ca + K2, (C) Ca + K1 +
K2, (D) K1 + K2, (E) Ca + D, (F) D + K1, (G) D + K2 and (H) Ca + K1 + K2 + D. Scale bars
represent 100 µm.
110
Supplemental Figure 3. Amount of MC3T3 mineralization was unchanged for
combinations of vitamins and calcium under AA-primed conditions as compared to all the
appropriate singular vitamin or calcium controls. MC3T3 cells were treated with AA for the
first five days and β-glycerophosphate from day 5 to day 22, while vitamins and/or calcium were
added throughout the experiment. Representative brightfield images of von Kossa stained,
combination treated cultures (with appropriate singular vitamin/calcium controls) included (A)
Ca + K1, (B) Ca + K2, (C) Ca + K1 + K2, (D) K1 + K2, (E) Ca + D, (F) D + K1, (G) D + K2 and
(H) Ca + K1 + K2 + D. Scale bars represent 100 µm.
111
Supplemental Figure 3 continued
112
Supplemental Figure 4. Combinations of vitamins and calcium caused no change to the
level of mineralization obtained from all the singular vitamin/calcium supplemented,
continual-AA treated, Saos-2 cultures. Saos-2 cells were treated with AA, vitamins and/or
calcium throughout the 22 day experiment, while β-glycerophosphate was added from day 5 to
day 22. Representative brightfield images of von Kossa stained, combination treated cultures
(with appropriate singular vitamin/calcium controls) included (A) Ca + K1, (B) Ca + K2, (C) Ca
+ K1 + K2, (D) K1 + K2, (E) Ca + D, (F) D + K1, (G) D + K2 and (H) Ca + K1 + K2 + D. Scale
bars represent 100 µm.
113
Supplemental Figure 4 continued
114
Supplemental Figure 5. Mineralization levels of Saos-2 cells treated with combinations of
vitamins and calcium under AA-primed conditions were unchanged as compared to all of
the singular vitamin or calcium controls. Saos-2 cultures were treated with AA for the first
five days and β-glycerophosphate from day 5 to day 22, while vitamins and/or calcium were
supplemented throughout the experiment. Representative brightfield images of von Kossa
stained, combination treated cultures (with appropriate singular vitamin/calcium controls)
included: (A) Ca + K1, (B) Ca + K2, (C) Ca + K1 + K2, (D) K1 + K2, (E) Ca + D, (F) D + K1, (G)
D + K2 and (H) Ca + K1 + K2 + D. Scale bars represent 100 µm.
115
Supplemental Figure 5 continued
