Biological Effects of RGD-Grafted Thermoreversible Polymers On Primary Cultured Bone Marrow Stromal Cells

Objectives: to design thermoreversible polymer for enhanced adhesion of primary cultured bone marrow stromal cells (BMSCs) responsive to BMP-2. Methods: RGD containing peptides GRGDSPC with a concentration of 0.1 mg/mL were conjugated to N-isopropylacrylamide/ethylmethacrylate (NiPAM/EMA) polymers via amine-reactive N-acryloxysuccinimide (NASI) groups. BMSCs of rat were cultured and the adhesion of BMSCs to NiPAM/EMA and NiPAM/EMA/NASI (with and without GRGDSPC exposure) as well as to tissue culture polystyrene (TCPS) was assessed by MTT assay and DNA assay 4 hours or 2 days after seeding. A quantitative comparison of alkaline phosphatase (ALP) activity based on per cell number was done to test the osteogenesis 7 days later after exposed to 200 ng/mL BMP-2. Results: Cells observed on EMA and EMA/NASI and EMA GRGDSPC surfaces remained round 1 day after seeding while Cells on GRGDSPC -grafted EMA/NASI were spreading and their morphology was comparable to that observed on the un-coated TCPS surfaces. NiPAM/EMA/NASI treated with GRGDSPC had the highest cell attachment among the polymer surfaces (p<0.05) at both 4 hours and 2 days, and attachment to TCPS was equivalent to the GRGDSPC-grafted NiPAM/EMA/NASI (p>0.05) 2 days after seeding. Similar cells response of differentiation response to bone morphogenetic protein-2 (BMP-2) by ALP assay were found on RGD-grafted NiPAM/ EMA/NASI surface as on TCPS control surfaces. Conclusion:RGD-containing thermorevesible polymer allows strengthened BMSCs attachment and maintains the differentiation response to BMP-2.