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Bone formation in the lab is aided by Vitamin D, Vitamin K1, and Vitamin K2 – meta-analysis Nov 2017

Effects of Vitamin D, K1, and K2 Supplementation on Bone Formation by Osteoblasts In Vitro: A Meta-analysis

Journal of Biometrics & Biostatistics 2017, 8:4 DOI: 10.4172/2155-6180.1000365
Charlene E Lancaster and Rene E Harrison
Dept of Cell and Systems Biology and the Dept. of Biological Sciences, University of Toronto Scarborough, Toronto, Canada


This is a meta-analysis of scores of studies of bone formation in the lab (not the body)
Noet that the effect size on the vertical axis of the charts is non-linear and varies with the vitamin.
It appears that Vitamin D produces the most bone formation.
I am not clear about how much benefit K1 or K2 provide to bone formation by Vitamin D in the lab
Note: Vitamin K1 and K2 have both been proven to provide many other benefits to the human body

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Bone loss is a major health problem that many aging individuals will face and thus research focusing on enhancing bone formation is of great importance. Cell biology or in vitro studies are particularly useful in exploring the exact effects a vitamin, supplement or drug has on particular processes within a certain cell type. Although there have been many cell biology articles focusing on the effects of vitamin D, K1 or K2 addition on bone formation in vitro, there has yet to be a consensus amongst the literature. The purpose of this article is to determine the effects of vitamin D, K1 and K2 supplementation on osteoblast maturation parameters through meta-analysis of past cell biology literature. A Hedges d effect size was calculated for each experiment extracted from past literature and the experiments were grouped by experiment and cell type. Homogeneity was assessed by the Cochran's Q test, while the effect sizes' departure from zero was assessed by a 95% confidence interval and a non-directional test. Supplementation with vitamin D, K1 and K2, along with the combination of vitamin K2+ 1,25-dihydroxyvitamin D, increased bone mineralization, while not consistently affecting all of the other parameters associated with bone formation. Vitamin K2 and D addition had variable effects on bone formation using different cell types, which calls into question the suitability of particular cell lines as models for clinical trials. Therefore, the conditions and parameters in which bone formation is studied in vitro must be considered carefully before running a vitamin supplementation or drug-testing experiment.

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Discussion and Conclusion

Our work represents the first cell biology meta-analysis of the effects of vitamin supplementation on parameters related to bone formation. The experiment type grouped meta-analyses revealed that the addition of vitamin K;, 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 significantly increased with vitamin K2 or vitamin D addition, but the effects of vitamin K; supplementation were inconclusive given that the confidence interval and non-directional tests did not agree. The levels of osteocalcin increased with the addition of vitamin D, but did not change when vitamin K2 was supplemented.
Surprisingly, vitamin K2 addition increased the DNA levels within the culture, but significantly decreased the amount of proliferation within the culture. Supplementation with vitamin D also resulted in increased collagen and osteopontin production by osteoblasts. 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 and vitamin D addition had inconclusive effects on the osteoblast maturation characteristics in the Other Experiments group.

Interestingly, the effect of the combination of vitamin K2+ 1,25D supplementation compared to the effect of vitamin K2 addition alone resulted in increased bone formation characteristics in the Other Experiments group, but not when compared to the effects of 1,25D addition alone.

The lack of consistency amongst the meta-analyses concerning the Other Experiments group could be because there were a variety of experiments delegated to this group for each meta-analysis.
Altogether this indicated that the addition of vitamin K1, K2 and D as well as the combination of vitamin 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.
The cell type categorized meta-analyses revealed that both vitamin K2 and vitamin D supplementation increased bone cell parameters in murine osteoblast cell lines and human primary osteoblasts. In addition, only supplementation of vitamin K2 resulted in an increase in maturation parameters in murine primary osteoblast cell cultures, while vitamin D addition had no effect. In human osteoblast cell lines, 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 depending on cell type tested.
The vitamin K2 meta-analysis revealed that the addition of vitamin K2 to osteoblasts decreased proliferation. Since the first stage of osteoblast maturation is the proliferative phase [39], this suggests that vitamin K2 addition impairs osteoblast maturation. However, decreased human primary osteoblast proliferation after a week of 1,25D supplementation has also been linked with increased late stage mineralization [32]. The addition of 1,25D may prolong the time spent in the differentiation and mineralization phases, which explains the increased mineralization in the supplemented cultures as compared to the untreated cultures. However, our vitamin K2 meta-analysis also revealed that vitamin K2 supplementation increased DNA levels, which is another indicator of cell proliferation. This contradictory data suggests that these experimental assays should be more closely analyzed to determine their predictive value in measuring osteoblast maturation.

Homogeneity of the experiments within a subgroup of the metaanalysis 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). Some of the subgroups that were analyzed within these meta-analyses were heterogeneous and thus one cannot be completely certain 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.

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 metaanalyses 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 therefore more likely to result in nonsignificance when the sample size is small and the variance is large [40]. Simultaneously, the confidence interval test is more affected by outliers than the robust non-directional test [41] 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. Therefore the definition of a significant result within meta-analyses in general might need to be re-evaluated [40].

A species specific effect of 1,25D on mineralization was observed in past literature, where 1,25D supplementation resulted in increased mineralization in human osteoblast cultures [17,32,35,42,43], but had primarily negative effects on mineralization in the mouse MC3T3 cell line [25,44]. In addition, the supplementation of 1,25D to mice resulted in increased levels of pyrophosphate and therefore decreased mineralization of bones within the mouse [45], as there needs to be low pyrophosphate levels for mineralization to occur in vitro and within the body [46]. Increased pyrophosphate levels also leads to adverse effects within cell culture, including autophagic cell death observed in inorganic pyrophosphatase-mutated yeast cells under fermentative conditions [47]. Altogether, this could help explain why vitamin D addition had differential effects on osteoblast maturation parameters in murine or human osteoblasts.
Clinical meta-analyses have previously revealed that supplementation with vitamin D and vitamin K resulted in increased bone mineral density at the femoral neck and lumbar spine, respectively [48], which agrees with the significant increase in mineralization we observed using supplementation with vitamin D and K. Similar to our meta-analyses, the results of both of these studies must be treated with caution due to heterogeneity. However, we observed variable results of the effects of these vitamins on parameters indicative of bone formation depending on the type of cells used, which calls into question the use of particular cell lines and animal models to test drugs/supplements as a precursor to clinical studies.

Although there have been widespread publications on the effect of vitamin D, K1 and K2 supplementation on bone formation, a definite conclusion concerning their effect in vitro on cell lines or primary cells 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 grouped 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. Ultimately, this work indicates that the conditions in which bone formation is studied must be considered carefully to determine the effect of vitamin K;, vitamin K2 and vitamin D supplementation on osteoblasts in vitro. In addition, meta-analysis is an extremely useful tool that has yet to be fully utilized in the field of cell biology.

Created by admin. Last Modification: Wednesday November 22, 2017 02:53:22 UTC by admin. (Version 8)

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8738 Bone K2.jpg admin 14 Nov, 2017 19:03 42.69 Kb 41
8737 Bone K1.jpg admin 14 Nov, 2017 19:03 43.82 Kb 46
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8734 Bone D K1 K2.pdf PDF 2017 admin 14 Nov, 2017 18:21 1.05 Mb 16
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