| The ultimate goal of this thesis was to design a biomaterial to enhance cell adhesion while retaining BMP-2 at a coinjected site, resulting in increased local osteoinductive activity. Peptides containing the Arginine-Glycine-Aspartic Acid sequence were conjugated to thermoreversible N-isopropylacrylamide (NiPAM) copolymers via protein-reactive N-acryloxysuccinimide (NASI) groups. 1H-NMR was used to confirm successful conjugation. The peptide/polymer yield in the conjugates was directly correlated to the concentration of peptide in the conjugation reaction, decreasing from 3.8 to 0.7 peptides/polymer chain as the peptide concentration decreased from 2.5 to 0.5 mg/mL. The conjugation efficiency was determined to be ∼8.5% using Reverse Phase High Pressure Liquid Chromatography (RP-HPLC). C2C12 cell adhesion to RGDconjugated polymer surfaces was significantly higher than adhesion on NiPAM/NASl surfaces and was morphologically comparable to adhesion on TCPS control surfaces. The increased adhesion on the RGD-conjugated surfaces was directly correlated to the peptide surface density (max. ∼3.4 nmol/cm2). BMP-2 treatment induced osteoblastic differentiation in C2C12 cells (as assessed by the induction of ALP activity) in a dose dependent manner. In the absence of BMP-2 treatment, cells cultured on RGDconjugated surfaces expressed higher levels of ALP activity than those cells cultured on TCPS surfaces, indicating that the conjugated RGD sequence induces osteoblast activity in C2C12 cells without the need for BMP-2 treatment. The RGD-containing polymers offer a viable biomaterial for local bone regeneration in vivo. |
