Osteogenesis in Marrow-Derived Mesenchymal Cell Porous Ceramic Composites Transplanted Subcutaneously: Effect of Fibronectin and Laminin on Cell Retention and Rate of Osteogenic Expression

Abstract

Cultured-expanded rat marrow-derived mesenchymal cells differentiate into osteoblasts when combined with a porous calcium phosphate delivery vehicle and subsequently implanted in vivo. In this study, the effects of ceramic pretreatment with the cell-binding proteins fibronectin and laminin on the osteogenic expression of marrow-derived mesenchymal cells were assessed by scanning electron microscopy, [3H]-thymidine-labeled cell quantitation, and histological evaluation of bone formation. Scanning electron microscopic observations showed that marrow-derived mesenchymal cells rapidly spread and attach to both fibronectin- or laminin-adsorbed ceramic surfaces but retain a rounded morphology on untreated ceramic surfaces. Quantitation of [3H]-thymidine labeled cells demonstrated that laminin and fibronectin preadsorbed ceramics retain approximately double the number of marrow-derived mesenchymal cells than do untreated ceramics harvested 1 wk postimplantation. Histological observations indicate that the amount of time required to first detect osteogenesis was shortened significantly by pretreatment of the ceramic with either fibronectin or laminin. Fibronectin- and laminin-coated ceramic composite samples were observed to contain bone within 2 wk postimplantation, while in untreated ceramic the earliest observation of bone was at 4 wk postimplantation. A comparison was made of the initial cell-loading, in vivo cell retention characteristics, and rate of osteogenesis initiation of marrow-derived mesenchymal cells on two types of ceramic with different pore structure and chemical composition, with and without preadsorption with fibronectin or laminin. “Biphasic” ceramics contain randomly distributed pores 200-400 μm in diameter, and “coral-based” ceramics have continuous pores of approximately 200 μm in diameter. Laminin or fibronectin preadsorption significantly increases the number of cells retained in all ceramic test groups by day 7 postimplantation. In addition, by day 7 postimplantation, the biphasic ceramics retain a significantly greater number of cells for all test groups than do coral-based ceramics. The biphasic ceramics consistently have more specimens positive for bone with the identical cell-loading conditions used throughout this study. These results indicate that the retention of cells within the ceramic is an important factor for optimization of marrow mesenchymal cell initiated bone formation. The retention of cells within ceramics is augmented by the adsorption of the cell-binding proteins laminin and fibronectin, but this effect varies depending on ceramic pore structure and/or chemical composition.