Method: C3H10T1/2-derived progenitor cells genetically engineered to express the differentiation inducer rhBMP-2, under control of the Doxycycline (Dox)-repressible promoter, Tet-Off, were plated on spun cast films of Polybutadiene (PB) and partially Sulfonated polystyrene (PSS28) polymers, whose moduli are 1.4MPa and 3 GPa, respectively. In order to probe the influence of mechanical cues, PB films, 20nm and 200nm, whose moduli vary by a factor 4, were used. The role of chemistry was probed by plating on PSS28 and Tissue Culture Petri-Dish. The cells were grown in DMEM, supplemented with 10% fetal-bovine serum, 0.2mM L-ascorbic acid 2-phosphate, 2mm glutamine, 10mM beta-glycerol phosphate 100 U/ml Penicillin, and 100ug/ml Streptomycin with or without 1 µg/mL Dox. The substrates were prepared by spin casting monodispersed PB or PSS28 films on HF treated Si wafers, which were then annealed in at P~10-6 and P=10 -3 Torr at T=170 °C. Differentiation was monitored over a 21 day period and biomineralization was characterized by SEM, EDAX, and qPCR.
Result: We show that the C3H10T1/2-derived progenitor cells expressing rhBMP-2, when grown on PB (20 or 200 nm), fail to show significant biomineralization even up to 21 days and express aggrecan typical of chondrogenesis. In contrast, those grown on SPS (200 nm) biomineralize as early as day 14. These cells do not express aggrecan but rather the osteogenic marker, bone sialoprotein. Addition of Doxycycline prevented biomineralization on all surfaces. Cells expressing rhBMP-2 had increased mRNA expression of collagen X, osteocalcin, alkaline phosphatase and osterix, as compared to the C3H10T1/2 and those grown with Doxycycline, regardless of the polymer substrate surface.
Conclusion: Surface chemistry can modify the response to rhBMP-2 in C3H10T1/2-derived progenitor-cells, and participate in the control of endochondral bone formation.