Combined macromolecule biomaterials together with fluid shear stress promote the osteogenic differentiation capacity of equine adipose-derived mesenchymal stem cells.

BACKGROUND: Combination of mesenchymal stem cells (MSCs) and biomaterials is a rapidly growing approach in regenerative medicine particularly for chronic degenerative disorders including osteoarthritis and osteoporosis. The present study examined the effect of biomaterial scaffolds on equine adipose-derived MSC morphology, viability, adherence, migration, and osteogenic differentiation. METHODS: MSCs were cultivated in conjunction with collagen CultiSpher-S Microcarrier (MC), nanocomposite xerogels B30 and combined B30 with strontium (B30Str) biomaterials in osteogenic differentiation medium either under static or mechanical fluid shear stress (FSS) culture conditions. The data were generated by histological means, live cell imaging, cell viability, adherence and migration assays, semi-quantification of alkaline phosphatase (ALP) activity, and quantification of the osteogenic markers runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) expression. RESULTS: The data revealed that combined mechanical FSS with MC but not B30 enhanced MSC viability and promoted their migration. Combined osteogenic medium with MC, B30, and B30Str increased ALP activity compared to cultivation in basal medium. Osteogenic induction with MC, B30, and B30Str resulted in diffused matrix mineralization. The combined osteogenic induction with biomaterials under mechanical FSS increased Runx2 protein expression either in comparison to those cells cultivated in BM or those cells induced under static culture. Runx2 and ALP expression was upregulated following combined osteogenic differentiation together with B30 and B30Str regardless of static or FSS culture. CONCLUSIONS: Taken together, the data revealed that FSS in conjunction with biomaterials promoted osteogenic differentiation of MSCs. This combination may be considered as a marked improvement for clinical applications to cure bone defects.

Osteogenic differentiation of equine adipose tissue derived mesenchymal stem cells using CaCl2.

Adipose tissue derived mesenchymal stem cells (ASCs) may be used to cure bone defects after osteogenic differentiation. In this study we tried to optimize osteogenic differentiation for equine ASCs using various concentrations of CaCl2 in comparison to the standard osteogenic protocol. ASCs were isolated from subcutaneous adipose tissue from mixed breed horses. The osteogenic induction protocols were (1) the standard osteogenic medium (OM) composed of dexamethasone, ascorbic acid and ß-glycerol phosphate; (2) CaCl2 based protocol composed of 3, 5 and 7.5mM CaCl2. Differentiation and proliferation were evaluated at 7, 10, 14 and 21days post-differentiation induction using the alizarin red staining (ARS) detecting matrix calcification. Semi-quantification of cell protein content, ARS and alkaline phosphatase activity (ALP) were performed using an ELISA reader. Quantification of the transcription level for the common osteogenic markers alkaline phosphatase (ALP) and Osteopontin (OP) was performed using RT-qPCR. In the presence of CaCl2, a concentration dependent effect on the osteogenic differentiation capacity was evident by the ARS evaluation and OP gene expression. We provide evidence that 5 and 7mM CaCl2 enhance the osteogenic differentiation compared to the OM protocol. Although, there was a clear commitment of ASCs to the osteogenic fate in the presence of 5 and 7mM CaCl2, cell proliferation was increased compared to OM. We report that an optimized CaCl2 protocol reliably influences ASCs osteogenesis while conserving the proliferation capacity. Thus, using these protocols provide a platform for using ASCs as a cell source in bone tissue engineering.