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
10 Most-visited pages in Vitamin K category in VitaminDWiki
Pages in both of the categories Vitamin K and Cardiovascular
- Vitamin K, cardiovascular health, and stroke - many studies
- Bone loss results in blood vessel plaque if low Vitamin K2, less bone loss if high K2– April 2021
- Cardiovascular Disease prevented by Vitamin K2-4 when enough is used – RCT review Sept 2020
- Calcium Supplementation is OK provided you also take Vitamin K – Feb 2019
- Vitamin K reduces calcification (reported yet again) – Feb 2019
- Vitamin K (across all dose sizes and types) decrease Vascular Stiffness – meta-analysis - Dec 2018
- Vitamin D and Vitamin K together fight CVD Part 1- Pizzorno
- Vitamin K, Cardiovascular and interactions with Vitamin D and Vitamin A – Pizzorno July 2018
- Mortality associated with Vitamin K insufficiency (PREVEND Study) – Nov 2017
- Intracranial arterial calcification in 85 percent of ischemic strokes (Vitamin K and Vitamin D should help) – Oct 2017
- Decalcify Aortic Valve – 3 year trial with 1 mg of Vitamin K and 5,000 IU of Vitamin D – 2021
- Cardiovascular problems reduced by low dose aspirin and perhaps Omega-3 (also Vit K) – Sept 2017
- Athletes maximal cardiac output increased 12 percent with Vitamin K2 – RCT July 2017
- Fast blood flow 6.8 X more likely if high vitamin D AND high vitamin K – Aug 2017
- Low Vitamin K2 is as risky as smoking for heart disease - Oct 2016
- Cardiovascular death: 9 percent due to hypertension or air pollution, 7 percent: low Vitamin K2 or smoking – Oct 2016
- Decreased need for warfarin after Vitamin D levels optimized – RCT May 2016
- Cardiovascular calcification prevented by Omega-3, Magnesium, Vitamin K, and Vitamin D – April 2015
- The health benefits of vitamin K – Oct 2015
- If you must take statins and want to avoid hardening of arteries, take vitamin K2 – RCT May 2015
- Vitamin K2-7 decreases arterial stiffness (cleans arteries) – RCT Feb 2015
- Hypothesis: Vitamin K will reduce prostate blood vessel problems – Jan 2015
- Increased Vitamin K2 reduces the problems of excess Calcium – Nov 2013
- Cholesterol, Vitamins D3 and K2, heart disease, sulfates, LDL, – Masterjohn Interview Jan 2013
- Soft Bones, Hard Arteries, Vitamin D, Vitamin K2 and antibiotics – Sept 2012
- Low Vitamin D and Vitamin K: brittle bones and hardened arteries – LEF Sept 2010
Pages in bothof the categories Vitamin K and Calcium
- Which supplements are often taken for healing hairline bone fractures
- Calcium and Vitamin K2 - many studies
- Excellent reviews of supplements at ConsumerLab
- Lowering Calcium Risk when having High Dose Vitamin D3 – Cawley Dec 2019
- Hypercalcemia in critically ill patients taking 10,000 IU of vitamin D (many solutions) – Oct 2019
- Calcium Supplementation is OK provided you also take Vitamin K – Feb 2019
- Calcium supplements go to muscle, not bone, unless have enough Vitamin K – Feb 2019
- Drugs which create deficiencies in Vitamin D, Vitamin K, Magnesium, Zinc, Iron, etc. – Sept 2017
- Vitamin D Cofactors in a nutshell
- Vascular calcification greatly reduced by 3 per week 1000 ug of Vitamin K2 MK-7 – Dec 2013
- Increased Vitamin K2 reduces the problems of excess Calcium – Nov 2013
- Review of Micronutrients such as vitamin D for women and childhood – Aug 2013
- Healthy bones need Ca, Silicon, Vitamins B, C, D, and K – Dec 2012
- Interview of Vitamin K2 and Calcium Paradox author by Dr. Mercola – Dec 2012
- Vitamin K2 and the Calcium Paradox – 2012 book
- Low cost cofactors for vitamin D
- Postmenopausal women should supplement vitamins C D K and Calcium – June 2010
Pages in both of the categories Bone and Vitamin K
- Which supplements are often taken for healing hairline bone fractures
- Vitamin K2-7 helps bone, blood vessels, cancer, diabetes, etc. – June 2022
- Role of Vitamin K in Bones and Muscles - Feb 2022
- Bone loss results in blood vessel plaque if low Vitamin K2, less bone loss if high K2– April 2021
- Bone increased : Stiffness (Vitamin D), Flexibility (Vitamin K2) – Sept 2020
- Bone quality improved 2X by Vitamin D plus Vitamin K2 (trend) – meta-analysis March 2020
- Vitamin D and Calcium do not increase bone density (also need exercise, Mg, K2, protein etc.) – RCT Aug 2019
- Vitamin K (any amount and any kind) reduced bone fractures by 24 percent – meta-analysis – May 2019
- Calcium Supplementation is OK provided you also take Vitamin K – Feb 2019
- Hard bones, soft arteries, rather than vice versa (Vitamin D and Vitamin K) – March 2016
- Many seniors do not get enough protein, Vitamin D, Mg, etc. needed for bones – Feb 2019
- Adding just vitamin D again failed to add bone density (also need Magnesium, Vitamin K, etc) – RCT Aug 2018
- Osteocalcin – overview of the hormone needed to build bones, etc. Jan 2018
- Vitamin K and bone – review Oct 2017
- Bone formation in the lab is aided by Vitamin D, Vitamin K1, and Vitamin K2 – meta-analysis Nov 2017
- Better bones again associated with higher vitamin K intake – Nov 2015
- Vitamin K-2 – bone biomarkers indicate at least 600 ug of MK-4 are needed daily – Sept 2014
- Vitamin K2 (as MK-7) is needed for bone quality – Review Feb 2013
- Increased Vitamin K2 reduces the problems of excess Calcium – Nov 2013
- Vitamin K and bone health – need more research Oct 2013
- Vitamin K-2 (180 ug MK-7) helped both bone density and strength – RCT March 2013
- Healthy bones need Ca, Silicon, Vitamins B, C, D, and K – Dec 2012
- Increasing bone mineral density increases breast cancer by at least 2X – Aug 2012
- Healthy bones need: Calcium, Vitamin D, Magnesium, Silicon, Vitamin K, and Boron – 2012
- Vitamin D, K2, Magnesium, etc increase bone density when taking together– Jan 2012
- BONE SPURS not produced if have enough Vitamin D3, Vitamin K2, etc. – Nov 2011
- Vitamin K1 reduced hip fracture but Vitamin K2 did not – Aug 2011
- Women with hip fractures very low on vitamins D3 and K – Mar 2011
- Vitamin K2 from natto improved bone mineral density – March 2011
Pages in both the categories Bone and Magnesium
- Which supplements are often taken for healing hairline bone fractures
- Magnesium helps bones a bit – meta-analysis Jan 2022
- Magnesium increases Vitamin D, which increases Mg. Both increase bone – Oct 2019
- Vitamin D and Calcium do not increase bone density (also need exercise, Mg, K2, protein etc.) – RCT Aug 2019
- Many seniors do not get enough protein, Vitamin D, Mg, etc. needed for bones – Feb 2019
- More Magnesium makes more bone when there is enough Vitamin D (petri dish) – Jan 2019
- Diagnosis and treatment of osteopenia – Holick 2010
- Adding just vitamin D again failed to add bone density (also need Magnesium, Vitamin K, etc) – RCT Aug 2018
- MAGNESIUM IN MAN - IMPLICATIONS FOR HEALTH AND DISEASE – review 2015
- Stronger bones after 3 generations of tap water (more Ca and Mg) vs bottled water – March 2015
- Bones grow better with high level of magnesium: rat study – Dec 2013
- 20 percent fewer male hip fractures if more Magnesium in the water – July 2013
- Magnesium may be more important to kids’ bone health than calcium – May 2013
- Healthy bones need: Calcium, Vitamin D, Magnesium, Silicon, Vitamin K, and Boron – 2012
- Vitamin D, K2, Magnesium, etc increase bone density when taking together– Jan 2012
- 400 IU of vitamin D Magnesium and Calcium helped Twin bones – Feb 2011
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Abstract
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.
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.