Growth and differentiation potential of human bone marrow-derived mesenchymal stem cells in respons to dexamethasone and selection for collagen adherence
Weissman, Alexander Clemens
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Osteoporosis (OP) is a chronic and progressive disease characterized by a decrease in bone mineral density (BMD), loss of bone tissue microstructure, and increased incidence of fracture. OP currently affects more than 10 million people in the United States, and its prevalence is expected to increase in the coming decades. This rising trend will add to the already $17 billion dollar per year healthcare industry which treats and rehabilitates those suffering from fragility related fractures. The causes of OP are multifactorial, with genetic and sex-dependent traits related to BMD and osteogenic cells having the largest effect on outcome. This study explored effective methods for culturing and inducing osteogenic differentiation in human bone marrow-derived mesenchymal stem cells (MSCs). MSCs are multipotent progenitors cells, and of particular importance to the study of OP because of their contribution to bone and cartilage repair and regeneration throughout life. Specifically, our goals were to examine the relationships between osteoinductive medium containing dexamethasone (Dex) and collagen culture plate pretreatment on human bone marrow-derived MSC growth, differentiation, and mineralization. Bone marrow was obtained from the acetabular reamings of 10 patients (age range 51-68; sex: 6 female and 4 male) undergoing total hip arthroplasty. After isolation, cells were cultured in a basal medium of Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum and 10% penicillin/streptomycin. Plates were further designated as ±collagen pretreatment, and +collagen plates were treated with a thin layer of collagen prior to cell seeding. On day 6 post seeding, the basal medium was replaced with an osteoinductive medium of α-Modified Eagle Medium, 10% fetal bovine serum, 1% penicillin/streptomycin, 12.5µg/mL ascorbic acid and 8mM β-Glycerophosphate. Osteogenic conditions were designated as ±10-8M Dex. After a 21 day culture in osteoinductive medium, cells were fixed and biochemical assays were assessed for DNA, alkaline phosphatase (ALP) and mineralization content. Plain photographic and phase-contrast microscopy examinations of cell cultures showed an increased tendency of +Dex cultures to promote earlier proliferation, and greater osteogenic nodule formation as quantified by alizarin red staining. There were no apparent qualitative differences in culture plates pretreated with collagen. Biochemical assays showed no significant difference in DNA content between either the -Dex or collagen conditions compared to the control (p>0.05). However, there was a significant difference in DNA content between patients (p<0.01). The addition of Dex had a significant effect on the DNA normalized ALP content of cell cultures (p<0.05). There was no significant difference in ALP content between groups with collagen pretreatment (p>0.05), or across patients for either the -Dex group or collagen group as compared to the control (p>0.05). Neither Dex as an osteoinductive component nor collagen coating tissue culture plates had a significant effect on mineralization as measured by DNA normalized alizarin red stain content (p>0.05). There was also no significant difference in mineralization between patients for either the -Dex or collagen group as compared to the control (p>0.05). Our findings demonstrate the potential of Dex to induce in vitro MSC proliferation and differentiation, while cell culture plate pretreatment with collagen did not appear to have significant effects. Further study and larger sample sizes are needed to produce more statistically significant results and allow for examination of underlying patient genetics and co-morbidities.