Preparation Of A PEG-based Bioresponsively Degradable Hydrogel And Study On Its Bone Repair Properties

It is a bottleneck problem for biomaterials to make their degradation rate match the in vivo tissue regeneration rate. The regulation of degradation rate usually accheived using materials modification by molecular design. However, their degradation time is comparatively too static to adapt complicated in vivo enviornment. So problems arise. Degrading too fast results in the lost of the necessary host where the new tissue would attach to, thus complete repair of defect cannot be achieved, while too slow rate of degradation or nondegradable materials may cause obstacles for new tissue growing in, can not reach ideal repair effect neither.Therefore, to design responsive materials which are able to change their porperties by stimulus trigger in biological milieus and thus causes degradation provides a prospective way to make degradation rate suitable for tissue grwoth.A redox-responsive PEG-based hydrogel system was designed from biodegradation point of view. Its physical and chemical properties, biocompatibility, especially degradation properties were evaluated. It was applied as a carrier material for loading recombinant human bone morphogenetic protein (rhBMP-2) using in bone repair. Based on this thought, we carried out research work in the following aspects.(1) Preparation and characterization of four-arm PEG-TA hydrogels.The hydrogel precursors PEG thioacetate (PEG-TA) were synthesized via two-step reactions before structural characterization. PEG-TA was able to respond to oxidation in alkaline medium by hydrolytic release of sulfur anion which can be oxidized by H2O2to form disulfide hydrogels. The concentration of PEG-TA, the concentration and amount of H2O2. pH value and temperature of medium were found to have effect on gelation time, gel content, water uptake, rheology and hydrophilicity. Gelation took place from15s to6min. Besides, disulfide bonds were also sensitive to reduction, leading to bond broken and thus degrading. The degradation behavior was especially concerned by putting gels in GSH solution (0.01mM) that was mimicing GSH concentration in extracellular matrix (ECM) with degradation time ranging from4d to75d. MTT method was carried out to test its cell cytotoxicity, showing the cytotoxicity of Grade1. Based on these results, the redox regulated hydrogles forming and degradation realized a redox-responsive hydrogel system.(2) Preparation and characterization of four-arm PEG-SH hydrogels. Thiolated PEG (PEG-SH) was obtained from PEG-TA and methonal by tranesterification. PEG-SH was able to form disulfide crosslinked hydrogels by H2O2oxidation. Gelation occurred in neutral meduim. much gentler than the reaction of PEG-TA, which can be served as a injectable hydrogel system. The system had a good injectable ability with gelation time ranging from14s to26min. Degradation can be triggerd in both0.01mM GSH that was mimicing GSH level in ECM and0.3mM H2O2that was mimicing local concentration in lesions. The longest degradation time in GSH and H2O2were32d and70d respectively. Bovine serum albumin (BSA) was ultilized as a protein model in the release test. The release process witneesed a burst at the initial time, gentle release in the middle and complete release at last. The cell cytotoxicity test showed good cell biocompatibility of10%PEG-SH gels and degradation products with Grade1cytotoxicity.(3) Biodegradation of injectable PEG-SH hydrogels and their ectopic bone formation effect with rhBMP-2loaded.The cell cluster invasion test showed that cells embedded in a thrombin clot were capable of migrating into surrounding hysrogels and spreading within them. Phase transition occurred instantaneously after subcutaneous injection. Obvious degradation was observed in the time of10d.RhBMP-2loaded injectable hydrogels were prepared by mixing rhBMP-2with PEG-SH precursors. Ectopic bone formation experiment showed that bone weight increased with time increasing. While rhBMP-2free hydrogels did not induce any new bone and the materials almost degraded. Bone formation and degradation were closely related. Materials degraded by cell phagocytosis and bone matrix embedment resulted in complete release of rhBMP-2. Sustained bone formation secreted more bone matrix which accelerated materials degradation. Thus bone formation and materials degradation multually promoted. The degradation also provided room and nutrient delivery for both bone cells and blood cells, which were benefit for cell proliferation, spreading, migration, as well as vasculogenesis.(4) Biodegradation of PEG-SH gel scaffolds and their ectopic bone formation effect with rhBMP-2loaded.PEG-SH gel scaffolds were prepared by freeze-drying of PEG-SH hydrogels. The degradation time of scaffolds formed at the concentrations of3%,5%,10%and15%were0.5h,1h,3h and4h in solution of mimicing intracellular concentration of GSH. However, the time was3d,6d,16d and22d in0.01GSH solutions that was mimicing extracellular concentration of GSH. Scaffolds responded to GSH more sensitively and faster with obvious increase in swelling ratios compared with PEG-SH hydrogels. The coculture with C2C12cells showed cytotoxicity level of scaffolds and their degradation products was Grade1.Ectopic bone formation experiment showed that bone weight increased with time increasing, higher than that induced by hydrogel system. RhBMP-2free scaffolds did not induce and new bone and the scaffolds almost degraded. Bone formation and degradation were closely related. Scaffolds degraded gradually through the immune adhesion function of erythrocyte and phagocytosis of leukocyte, embedment of bone matrix. Sustained bone formation by scaffolds complete degradation and totally release of rhBMP-2. secreted more bone matrix which accelerated materials degradation. Thus bone formation and materials degradation multually promoted. Scaffolds degradation supported bone cells and blood cells with sufficient room to grow in, thus promoted bone formation and vasculogenesis.(5) Repair of radius defect with rhBMP-2loaded gel scaffoldsAn animal model of15mm rabbit radius defect was set up for evaluation of orthotopic bone formation induced by rhBMP-2loaded PEG-SH scaffolds. The materials in the experiment involved of four groups:self-repair witout any materials, scaffolds only, rhBMP-2loaded scaffolds, rhBMP-2and RGD pipetide loaded scaffolds.The induction of growth factor, conduction and degradation of scaffolds showed influence on repair effect. Bone weight increased with time increasing from2w to12w. The scaffolds only group induced more new bone than self-repari group. The bone weitht in group of rhBMP-2loaded scaffolds, rhBMP-2and RGD popetide loaded scaffolds was higher than the other two groups. The maximum bending load of defective radius of232.9N for rhBMP-2loaded group and251.0N for rhBMP-2and RGD popetide loaded group, which were close to the normal radius of256.4N, were much higer than the other two groups. From the histology results, bone formation rate was slow in self-repair group and relatively fast in materials only group. But the defective radius did not reach the repatency of marrow cavity. Also nonunions can be observed. While the growth factor and pipetide loaded scaffolds groups were able to reach complete repair of defect with reconnection of marrow cavity. Bone formation and scaffolds degradation were of close relation and mutual promotion. The three groups with materials showed more blood cells and blood vessels compared with self-repair group. In this way. rhBMP-2loaded PEG-SH scaffolds may be applied in the bone defect repair of critical zise.