Three-dimensional culture of differentiating marrow stromal osteoblasts in biomimetic poly(propylene fumarate-co-ethylene glycol)-based macroporous hydrogels

This study assesses the ability of biomimetic poly(propylene fumarate-co-ethylene glycol)-based hydrogels to sustain the differentiation of marrow stromal cells (MSCs) to the osteoblastic phenotype and to produce a mineralized matrix in vitro. Macroporous hydrogels based on poly(propylene fumarate-co-ethylene glycol) with and without covalently linked RGD cell-adhesive peptide were synthesized and seeded with rat MSCs suspended in media or in a type I collagen solution. Cells suspended in media were found to adhere to RGD-modified but not to unmodified hydrogels. Cells suspended in a collagen solution were entrapped after collagen gelation and proliferated independent of the peptide modification of the hydrogel. Hydrogel modification with RGD peptide was sufficient to allow for the adhesion and differentiation of MSCs to the osteoblastic phenotype in the presence of osteogenic culture supplements. MSCs seeded with a collagen gel onto RGD-modified macroporous hydrogels after 28 days of culture showed a significant increase in cell numbers, from 15,200 +/- 2,000 to 208,600 +/- 69,700 cells (p < 0.05). Moreover, significant calcium deposition was apparent after 28 days of culture in RGD-modified hydrogels for cells suspended in a collagen gel in comparison to cells suspended in media, 3.47 +/- 0.26 compared to 0.82 +/- 0.20 mg Ca(2+) per scaffold (p < 0.05). Confocal microscopy revealed that MSCs suspended in a collagen gel and cultured on RGD-modified hydrogels for 28 days were adhered to the surface of the hydrogel while MSCs suspended in a collagen gel and cultured on unmodified hydrogels were located within the pores of and not in direct contact with the hydrogel surface. The results demonstrate that these biomimetic hydrogels facilitate the adhesion and support the differentiation of MSCs to the osteoblastic phenotype in the presence of osteogenic culture media.