• Metformin and Mesenchymal Stem Cell Osteogenic Differentiation: Role of Organic Cation Transporters

      Aljofi, Faisal Egal; Schneider, Abraham; 000-0002-1652-5325 (2017)
      Objective: The long-term goal of these studies is to develop novel tissue engineering strategies to enhance craniofacial bone regeneration by combining human umbilical cord derived mesenchymal stem cells (UC-MSCs) and other potential MSC with either systemically or locally delivered metformin. Metformin is a first line, well-tolerated antidiabetic drug with potential osteogenic actions most likely mediated by the activation of the AMP-activated protein kinase (AMPK) signaling pathway. As a highly hydrophilic cationic drug, metformin requires active intracellular uptake via polyspecific cell membrane organic cation transporters (OCTs) encoded by the SLC22A gene family. Despite their critical involvement in hepatic and renal cellular transport, the role played by OCTs in metformin-induced AMPK pathway activation and osteogenic differentiation in UC-MSCs, remains largely unexplored. Here, we hypothesize that to effectively induce AMPK activation and osteogenic differentiation, metformin must gain intracellular access into functional OCT-expressing UC-MSCs. Methods: Immunoblotting was used to assess OCT expression in human-derived UCMSCs. UC-MSCs were treated in vitro with metformin to determine its intracellular uptake, AMPK pathway activation, mineralized nodule formation, and induction of osteogenic markers. Results: Immunoblotting and cellular uptake assays demonstrate that one or more of the OCT isoforms are highly expressed in UC-MSCs and mediate responses to metformin. Treatment of UC-MSCs with clinically relevant doses of metformin (10 μM) resulted in activation of the AMPK signaling pathway. Use of chemical inhibitors targeting OCT function (10 μM quinidine) or AMPK activation (10 μM compound C) markedly inhibited these responses. Metformin significantly enhanced UC-MSC mineralized nodule formation and increased expression and nuclear localization of the osteogenic transcription factor RUNX2. Collectively, these findings indicate that both OCTs and the AMPK signaling pathway play an important role in mediating metformin-induced UCMSC osteogenic differentiation. Conclusions: By gaining a mechanistic insight into the role played by OCTs on metformin-induced MSC osteogenic differentiation mediated by AMPK/RUNX2 signaling, our work may lead to future tissue engineering platforms where metformin together with functional, OCT expressing UC-MSCs may be used as a novel autogenous therapeutic option to enhance bone regeneration. In particular, these treatment strategies might benefit pediatric patients affected with congenital malformations that compromise orofacial skeletal tissues.
    • Methylsulfonylmethane: Possible Role in Bone Remodeling

      Aljohani, Hanan; Chellaiah, Meenakshi A. (2020)
      Methylsulfonylmethane (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.
    • The optimal size range of particulate demineralized bone matrix for osteoinduction

      Desverreaux, Robert William; Hiatt, James L., 1934- (1991)
      There have been several investigations to determine the optimal particle size range of demineralized bone matrix (DBM) for osteoinduction. Results from these investigations have not been in agreement. Particulate bone matrix preparations can be combined with biodegradable delivery systems to treat ablative bony wounds. Therefore, an optimization of the particle size range is important. The purpose of this study was to determine the optimal size range of particulate DBM for osteoinduction at both a heterotopic and an orthotopic site. DBM was prepared according to the method of Reddi-Huggins and sieved to 13 different size ranges. In the first phase of the experiment DBM (20 mg) was placed in gelatin capsules, sealed in nylon mesh baskets, and implanted in the pectoralis muscle of 210 Long-Evans male and female rats (27 to 35 days old). In the second phase DBM (20 mg) was implanted in an 8 mm critical sized calvarial defect in an additional 210 Long-Evans male and female rats. There were 15 animals per treatment group in each phase of the study. Specimens were retrieved at 28 days post surgery, and embedded, undecalcified in plastic, sectioned, and stained using von Kossa stain. Bone formation was quantitated by radiomorphometry and histomorphometry using a Quantimet 520 Image Analysis System. Moreover, alkaline phosphatase and calcium were quantitated by atomic absorption spectrophotometry in the heterotopic site. All implants demonstrated a high degree of biocompatibility and no significant inflammatory cell infiltrates were observed in any of the specimens. Results indicate that the 590-710 um range particles produced significantly more new bone than all other size ranges (p {dollar}<{dollar} 0.05). These results further narrow those obtained by Reddi and Huggins, who found the optimum size range for particles to be 420-850 um.
    • Potential Role of Metformin in Mesenchymal Stem Cell-based Osteogenic and Angiogenic Coupling

      Alshawaf, Nour; Schneider, Abraham (2017)
      Objective: Metformin is a first-line medication used to treat type 2 diabetes. Non-antidiabetic effects of metformin have recently included its osteogenic effect on bone marrow mesenchymal stem cells (BMSCs). The purpose of the present work was to study the potential effects of metformin in the osteogenic/angiogenic coupling. Results: Clinically relevant doses of metformin exerted both direct and indirect effects on endothelial cells capillary tube formation, a well-known assay used to analyze angiogenic effects in vitro. Also, metformin directly induced BMSCs to express and secrete vascular endothelial growth factor (VEGF). Metformin induced these responses in both BMSCs and endothelial cells due to the fact that they express functional organic cation transporters (OCTs), critical cellular determinants of metformin intracellular uptake and activity. This study provides preliminary data and further research is required to gain a better understanding on the role of metformin as a potential osteogenic/angiogenic factor in skeletal regeneration.