AdvisorChellaiah, Meenakshi A.
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AbstractMethylsulfonylmethane (MSM) is a popular dietary supplement to assist with various conditions. The anti-inflammatory and osteogenic capabilities of MSM makes it an excellent material for inducing bone formation and promoting osteointegration. MSM is a non-toxic, naturally occurring sulfur-containing compound. This thesis investigated the effect of MSM on osteogenesis in vitro and in vivo. We first used oral stem cells derived from the human exfoliated deciduous teeth (SHED) to elucidate the effect of MSM on osteogenic differentiation using MC3T3-E1 and UMR-106 cells as positive controls. MSM reproduced the results of the osteogenic medium in the osteogenic differentiation of SHED cells. Osteogenic differentiation of SHED cells was determined by an increase in the expression of differentiation markers such as osterix, RUNX2, osteopontin (OPN), and collagen type 1 (Col 1), at both mRNA and protein levels. Moreover, MSM increased the activity of the alkaline phosphatase enzyme, which is vital in the maturation of the extracellular matrix and the formation of mineralized nodules. Very interestingly, the addition of mineralized bone particles enhanced the MSM’s effect on mineralization compared with MSM alone or MSM with demineralized bone particles. Therefore, MSM can act as a cost-effective osteoinductive material for reinforcing bone regeneration. Secondly, we determined the role of transglutaminase-2 (TG2) enzyme in the calcification process via cross-linking of matrix proteins. TG2 is a multifunctional enzyme implicated in matrix stabilization and maturation. MSM treated SHED cells showed a time-dependent increase in TG2 protein expression from 7 to 21 days. Furthermore, immunoprecipitation and immunostaining analyses showed an increase in TG2 colocalization with two prominent osteogenic markers (OPN, Col 1) in a time- dependent manner. An inhibitor to TG2 reduced not only the differentiation of SHED cells but also the mineralization processes by reducing the interaction of TG2 with OPN and Col 1. Our studies demonstrated the effect of MSM on osteogenesis in vitro in TG2 mediated cross-linking of matrix proteins. Thirdly, we identified the effect of MSM on osteogenesis in vivo using aging mice model. We injected aging C57BL/6 female mice (36 weeks old) subcutaneously with MSM and PBS for 13 weeks. Micro-computed tomography (Micro-CT), histological, and immunohistochemistry analyses were done extensively in the bone sections of mandibles isolated from aging mice injected with PBS and MSM. Comparative studies were also done in the tibial and femoral bones of long bones. An increase in the mandibular bone density at the inter-radicular area was observed in mice injected with MSM. The increase was either little or not seen in the femoral or tibial bones analyzed by Micro-CT or in bone sections stained with H&E and TRAP-stains. Immunohistochemistry analyses demonstrated an increase in osteocalcin (OCN) staining in osteoblast-like cells and a decrease in CD105, which is a marker for stem cells. Additionally, we found that MSM has an osteogenic effect via not only increasing the osteogenesis potential of osteoblast- like cells but also the differentiation potential of stem cells into osteoblast-like cells. More experiments are needed to further confirm whether the increase in bone density is a result of the induction of bone formation by osteoblasts or reduction of bone resorption by osteoclasts. MSM is a sulfur-containing non-toxic natural nutrient found in small quantities in many foods. It is commonly used as a supplement to treat arthritis and other inflammatory conditions. This is the first study to show the in vivo effect of MSM on bone remodeling in an aging mice model. We trust, our results may ultimately impact the treatment of other bone loss-associated diseases, including rheumatoid arthritis and periodontitis, which share several pathologic features with osteoporosis.
Biomedical Sciences-Dental School
University of Maryland, Baltimore