Osteogenic differentiation of adipose-derived stromal cells treated with GDF-5 cultured on a novel three-dimensional sintered microsphere matrix

Abstract

It is well known that under the proper conditions multipotential bone marrow stromal cells are capable of osteogenic differentiation. Recently studies have demonstrated that an analogous subpopulation of cells exist within adipose tissue. Although early studies characterizing these adipose-derived stromal (ADS) cells in culture exist, investigations exploring the characteristics and viability of these cells cultured on a three-dimensional sintered microsphere matrix are absent. To characterize and investigate the viability of ADS cells cultured on bioengineered three-dimensional sintered microsphere matrices (SMM). Basic science, laboratory study. Sixty SMM total. Six underwent examination by scanning electron microscopy, 18 for cellular viability, 18 for biochemical assay, and 18 for evaluation by gene expression. The SMM were examined under scanning electron microscopy to evaluate for adherence, migration, and proliferation at 7, 14, and 28 days. Cellular viability was assessed using colorimetric assay for mitochondrial dehydrogenases activity in viable cells (MTS [3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay) at each corresponding time point. Osteoblastic differentiation was determined using biochemical assays for alkaline phosphatase activity and gene expression for alkaline phosphatase (ALP), osteocalcin (OC), and core binding factor alpha-1 (Cbfa1). Multipotential ADS cells from adult Sprague Dawley rats were isolated and maintained in media. Sintered microsphere matrices of poly(lactide-co-glycolide) [85:15] were prepared using solvent evaporation technique followed by mechanical sieving and fabricated by heating in metal molds. ADS cells were then seeded on the SMM and cultured in media with growth and differentiation factor-5 (GDF-5). Treated samples and controls were evaluated at 7, 14, and 28 days. Statistical significance was set at p<.05. Multipotential ADS cells were capable of being isolated from adipose tissue. Scanning electron microscopy evaluation revealed cells adherent to the scaffold surface in a monolayer by 7 days. Cytoplasmic extensions were seen linking the cells on adjacent microspheres. Migration and proliferation resulting in extension of the cellular elements into the scaffold was apparent by 14 days. MTS confirmed cell viability within the scaffold throughout the 28-day study. Osteoblastic differentiation was confirmed using biochemical assays for alkaline phosphatase activity and gene expression for ALP, OC, and Cbfa1. This is the first study to investigate the fate of ADS seeded on a three-dimensional sintered microsphere matrix. The results of this study confirm that ADS cells, when treated with GDF-5, are not only capable of adhering to the bioengineered scaffold, but also remain viable and demonstrated the ability to migrate, proliferate, and subsequently undergo osteogenic differentiation under the conditions described. These early findings support the concept that ADS cells cultured on a SMM may serve as a viable alternative to more traditional methods of bone graft materials.