Research In Method Of The Surface Of Allograft Bone Modified With RGD Polypeptides

Objective: 1. To investigate the method with application ofultraviolet radiation and the chemical coupling to graftpolypeptide containing ROD series on allograft bone throughcovalent bond in order to enhance the adhesion ability of thematerial. 2. To choose the best combination of experimentalfactor and level to affect the graft the polypeptide containingROD series on allograft bone fragment by orthogonal experimentdesign.Methods: The carboxyl group on the surface of allograft bonewas activated by ultraviolet radiation. Using EDC as thecoupling agent, the synthetic RGD containing octapeptide(Glu-Pro-irg-Gly-isp-Asn-Tyr-Arg, EPRGDNYR) was bonded to thesurface of allograft bone through covalent bond. Blank bonefragment was used as control, morphological change on theallograft bone surface was observed by the environmentalscanning electronic microscope. The grafting of RGDpolypeptide was inspected by the change of element content onthe surface analyzed by X-ray photoelectronspectroscopy(XPS). By orthogonal tests, the concentration ofRGD was subjected to be 0.1mg/ml, 0.5mg/ml and 1.0mg/ml, theduration for ultraviolet radiation, 30, 60 and 90 minutes, andthe concentration of EDC, 1ï¼…, 2ï¼…and 3ï¼…respectively, and theorthogonal experiment was conducted using above factors. Theresults of 9 experiments were analyzed, using the content of nitrogen as the assessment indicator so as to determine theeffect of experimental factors to the results and the optimumlevels of each factorsto find out the best combination of thesefactors.Results: By observing under the environmental scanningelectronic microscope, we found out that the surface asperityof allograft bone fragments bonded with RGD polypeptide waslower than the blank ones. The frame structure of bone saltwas also sparse, with the holes filled with numerous groundsubstances. Hole separation density was decreased. Changeswere seen on the morphology of surface of polypeptide bondedbone fragments. Examined by XPS, the content of nitrogen atomsin polypeptide bonded bone fragments was higher than the blankcounterparts in various degrees. The amido bond in the bonefragment was strengthened, showing that polypeptide wassuccessfully bonded to the surface of allograft bone. Theresults of orthogonal tests are: the sum of level 1,2 and 3of factor A are 12,16.7 and 10.3 respectively, with level 2wasbetter. The sum of level 1,2 and 3 of factor B are 12.5,14.8and 11.7 respectively, with level 2 was better. The sum of level1,2 and 3 of factor C are 11.7,10.6 and 16,7 respectively, withlevel 3 was better. From the results, it can be seen that theconcentration of RGD is the most important among the threefactors, seconded by concentration of EDC, with the durationof ultraviolet radiation being the least important. The bestcombination is then A₂B₂C₃.Conclusion: By activating the carboxyl group with ultravioletradiation, and coupling EDC and RGD, we successfully adherethe RGD polypeptide onto the surface of allograft bone bycovalent bond. From the orthogonal analyses, it can be seen that the factors affecting this experiment are, ranking fromthe most important one, RGD concentration, EDC concentration,and ultraviolet radiation time. The best experiment solutionwould be: RGD concentration, 0.5mg/ml, EDC concentration, 3%and ultraviolet radiation time, 60 minutes.