Bone generation on PHBV matrices: an in vitro study

Bone formation was investigated in vitro by culturing rat marrow stromal osteoblasts in biodegradable, macroporous poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) matrices over a period of 60 days. Foams were prepared after solvent evaporation and solute leaching. PHBV solutions with different concentrations were prepared in chloroform: dichloromethane (1:2, v/v). In order to create a matrix with high porosity and uniform pore sizes, sieved sucrose crystals (300-500 microm) were used. PHBV foams were treated with rf-oxygen plasma (100 W 10 min) to modify their surface chemistry and hydrophilicity with the aim of increasing the reattachment of osteoblasts. Osteoblasts were isolated from rat bone marrow and seeded onto PHBV foams. The cell density on and in the foams was determined with MTS assay. MTS results showed that osteoblasts proliferated on PHBV. Twenty-one days after seeding of incubation, growth of osteoblasts on matrices and initiation of mineralization were observed by confocal laser scanning microscopy. Increasing ALP and osteocalcin secretion during 60 days confirmed the osteoblastic phenotype of the derived stromal cells. SEM, histological evaluations and confocal laser scanning microscopy showed that osteoblasts could grow inside the matrices and lead to mineralization. Cells exhibited spindle-like morphology and had a diameter of 10-30 microm. Based on these, it could confidently be stated that PHBV seems to be a promising polymeric matrix material for bone tissue engineering.