Bone Morphogenetic Protein 2-Coated Porous Poly-L-lactic Acid Scaffolds: Release Kinetics and Induction of Pluripotent C3H10T1/2 Cells

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Abstract
Induction of a pluripotent C3H10T1/2 (Tl/2) stem cell line down the osteoblastic lineage was demonstrated in vitro using a three-dimensional, porous poly-L-lactic acid (PLLA) scaffold containing bone morphogenetic protein 2 (rhBMP-2). PLLA sponges (100 kDa) were fabricated using a solvent casting/particulate leaching method. C3H10T1/2 cells (5 × 105) and BMP (2.2–6.6 µg) were suspended in collagen I gel and seeded on the scaffold, or rhBMP-2 was precoated on the scaffold and followed by cell seeding. rhBMP-2 release from cell-free rhBMP-2-coated scaffolds, measured directly by enzyme-linked immunosorbant assay (ELISA) and indirectly by alkaline phosphatase assay of Tl/2 cells treated with rhBMP-2-conditioned media, showed release from scaffolds for up to 14 days, with no difference between suspension and precoat groups. Cell-seeded samples were evaluated at 7, 14, and 28 days by histology and scanning electron microscopy, which revealed morphological differences between rhBMP-2 and non-BMP groups. Cells remained viable throughout the study, and osteocalcin immunofluorescence, used to test for osteoinduction, showed a time- and dose-dependent osteocalcin expression in all rhBMP-2 groups, with no staining observed in non-BMP controls. In summary, a single initial dose of rhBMP-2 within a three-dimensional PLLA scaffold was sufficient to induce pluripotent Tl/2 cells to differentiate into the osteoblastic lineage. The availability of tissue-enginered substitutes would simplify bone-grafting procedures.