In vitro cellular responses to bioerodible particles loaded with recombinant human bone morphogenetic protein-2

Porous 50:50 poly(d,l lactide-co-glycolide) microspheres containing varying amounts of "free" recombinant human bone morphogenetic protein-2 (rhBMP-2) were evaluated for their ability to induce/enhance expression of osteoblastic characteristics by pluripotent mesenchymal cells in vitro. "Free" protein (Fp) is defined as protein present on the surface and within the porous matrix of the microspheres. Four preparations of bioerodible particles (BEP) were used: blank--without rhBMP-2; low Fp--24 microg of free rhBMP-2 per g of particles; medium Fp--403 microg/g; and high Fp--884 microg/g. C3H10T1/2 cells (C3H) and bone marrow stromal cells (BMC) were cultured with 1 mg of BEP for up to 4 weeks, and cell growth and expression of osteogenic responses were determined weekly. For both cell types, control cultures (neither BEP nor rhBMP-2) and cultures with blank BEP exhibited no or minimal osteoblastic characteristics. Compared to control and blank BEP cultures, C3H cells responded to particles having medium and high amounts of free rhBMP-2 with increased cell growth and alkaline phosphatase activity, but osteocalcin secretion and mineralization were not markedly influenced. Low Fp BEP enhanced only the alkaline phosphatase activity of C3H cells. In contrast, although growth was not affected, rhBMP-2-loaded BEP increased alkaline phosphatase activity, osteocalcin secretion, and mineralization in BMC cultures in a dose-dependent manner (i.e., blank < low < medium < high Fp).