Osteogenic Differentiation of Mesenchymal Stem Cells on Pregenerated Extracellular Matrix Scaffolds in the Absence of Osteogenic Cell Culture Supplements

Abstract

This study utilized a full-factorial design to investigate the effect of four factors: presence of whole bone marrow cells, presence of in vitro-generated mineralized extracellular matrix (ECM), presence of dexamethasone, and variations in culture duration, on the proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) cultured on a polymer scaffold. Electrospun poly(e-caprolactone) (PCL) fiber mesh scaffolds were seeded with rat MSCs and cultured in complete osteogenic medium for 12 days to generate constructs containing mineralized ECM. MSCs or MSCs and whole bone marrow cells were seeded onto decellularized ECM constructs (PCL=ECM) or plain PCL scaffolds and cultured statically for 4, 8, and 16 days in medium either with or without dexamethasone. After each culture period, the cell number was determined by DNA analysis, and the osteogenic differentiation state of the cells was determined by alkaline phosphatase activity and calcium assays. MSCs seeded onto PCL=ECM constructs and cultured in medium either with or without dexamethasone demonstrated similar amounts of calcium deposition after 16 days. A significant increase in cell number over time compared with all other groups was observed when whole bone marrow cells were cocultured with MSCs on PCL scaffolds in medium without dexamethasone. This study establishes that the osteogenic differentiation of MSCs seeded onto ECM-containing constructs is maintained even in the absence of dexamethasone and that the coculture of MSCs and whole bone marrow cells without dexamethasone and ECM enhances the proliferation of a cell population (or populations) present in the whole bone marrow.

Biomimetic Extracellular Matrix-Incorporated Scaffold Induces Osteogenic Gene Expression in Human Marrow Stromal Cells

“…In general, our construct exhibited more robust changes in the expression of osteoblast-specific genes that regulate osteoblast differentiation than other constructs reported thus far. 7,8 We believe that our construct has the potential to replace the existing surgical aids such as demineralized bone matrix (DBM) and freeze-dried bone. DBM has been shown to possess intact BMP2, but growth factor concentration and behavior varies between manufacturers and does not perform consistently even between batches from the same source.…”

“…The native ECM is a complex entity comprising of structural proteins (primarily collagen) that provide support, proteoglycans and hyaluronan that can bind to growth factors, and specialized multi-adhesive proteins that bind cells to the matrix. [3][4][5][6][7][8] Although ECM proteins can be categorized, a majority of them are multifunctional and they can also transmit biochemical signals that influence cell migration, proliferation, and differentiation. The composition of the ECM is unique for each cell type and tissue.…”

“…6 Recent studies have also shown that the ECM of MSCs cultured in osteogenic medium influence the differentiation of these stem cells toward an osteoblastic lineage. 7,8 Therefore, it is reasonable to expect that engineered native ECM scaffolds will play a significant role in future tissue engineering applications.…”

Enhanced Osteogenesis in Cocultures with Human Mesenchymal Stem Cells and Endothelial Cells on Polymeric Microfiber Scaffolds

“…31 Electrospun PCL scaffolds with 5-or 10-mm fiber diameters used in our laboratory have been shown to successfully support MSC proliferation and osteogenic differentiation. 32,33 In this study, it was hypothesized that cocultures of human mesenchymal stem cells (hMSCs) and/or osteogenically precultured hMSCs and human umbilical vein endothelial cells (HUVECs) on 3D scaffolds would lead to enhanced osteogenesis over hMSCs or their derivatives alone. To test this hypothesis, hMSCs were cocultured with HUVECs on electrospun PCL microfiber scaffolds under osteogenic culture conditions, and the effect on osteogenic differentiation was quantified with a variety of biochemical assays.…”

Osteogenesis of Mesenchymal Stem Cells Derived from Bone Marrow in vitro

“…The osteogenic induction medium led to a morphological change of the MSCs from an elongated fibroblast-like cell type to shorter, cuboidal cells [16] Fig. (4C) [17] Fig.(5). In conclusions, bone marrow derived mesenchymal stem cells had osteogenic potential and could be a suitable option for cell-based tissue engineering therapies.…”