【作者】 杨帆；

【导师】 刘昌胜；

【作者基本信息】 华东理工大学 ， 材料科学与工程， 2012， 博士

【摘要】 组织修复材料的降解速率难以与体内组织的再生速率相匹配是生物材料面临的瓶颈问题。采用常用的改性方法,虽然可以通过分子设计调节材料的降解速率,但对于同一种材料,其降解时间相对固定,无法适应体内复杂多变的修复情况。由此引发一系列问题,有的材料降解速度过快,导致新生组织的生长、爬移失去必要的附着体,无法实现缺损的完整修复；有的则降解速度太慢或几乎不降解,使得新生组织的生长受到阻挡和抑制,同样导致修复效果不理想。设计具有响应特性的生物材料,借助体内微环境的变化作为触发开关,改变材料本身的性能,引起材料结构发生断链,导致降解发生并调控降解速率成为构建响应性降解材料的新思路,有望实现材料与机体组织生长速度相匹配,达到自适应性降解。本论文从生物响应性降解角度出发,设计具有氧化还原响应性的四臂聚乙二醇基水凝胶体系,考察这一体系材料的理化性能、生物相容性、尤其是生物可降解性能；同时以其为载体,与重组人骨形态发生蛋白-2(rhBMP-2)复合,制备用于骨组织修复的响应性凝胶体系。基于以上思路,本文开展了以下工作：(1)四臂聚乙二醇基PEG-TA水凝胶的合成与表征以四臂聚乙二醇为基体,通过两步法制得凝胶前驱物聚乙二醇硫代乙酸酯醚PEG-TA,并对其结构进行表征。在碱性条件下PEG-TA水解生成具有活性的硫负离子,经H202氧化形成以二硫键交联的水凝胶体系。考察了PEG-TA质量分数、H202浓度和用量、pH值和温度对凝胶化时间、凝胶含量和含水量、流变学、亲水性等性能的影响,凝胶化时间从15s到6min可调。同时,二硫键交联凝胶可对还原作用产生响应,切断凝胶网络中的二硫键从而发生降解。着重考察了凝胶在模拟人体细胞外基质浓度水平的谷胱甘肽(GSH,0.01mM)溶液中的响应性降解行为,降解时间为4d至75d。MTT法考察凝胶及其产物的细胞毒性,结果显示产物无毒,细胞毒性等级为1级。研究表明,PEG-TA水凝胶的形成和降解可通过氧化和还原作用进行调控,是一种氧化还原响应型水凝胶体系。(2)四臂聚乙二醇基可注射PEG-SH水凝胶的合成与表征以PEG-TA和甲醇为原料,通过酯交换反应得到具有活性的巯基化聚乙二醇PEG-SH。在H2O2氧化作用下PEG-SH能够发生二硫键交联形成水凝胶。与PEG-TA相比,反应条件更加温和,在中性介质中即发生凝胶化,可作为原位注射水凝胶使用。凝胶化时间从14s到26min可调,注射性良好。利用二硫键同时具有氧化性和还原性的特点,凝胶对模拟人体细胞外基质浓度水平的GSH (0.01mM)和病灶部位浓度水平的H2O2(0.3mM)均能发生二硫键响应性断链降解。在GSH中的降解时间最长达32d,H2O2中最长达70d。以牛血清白蛋白(BSA)为模型蛋白考察凝胶的缓释行为,释放前期存在突释效应,释放中期速率平缓,后期能够达到完全释放。MTT法评价PEG-SH可注射凝胶体系的细胞相容性,凝胶及其降解产物与小鼠成肌细胞C2C12共培养实验表明,材料的细胞毒性等级为1级。(3)可注射PEG-SH水凝胶的生物降解性及负载rhBMP-2异位诱导成骨通过细胞簇侵入实验考察细胞在凝胶中的行为,结果表明,包覆于凝血酶的细胞簇能够迁移至凝胶内部并完全铺展。注射于大鼠皮下的凝胶能够快速发生相转变,10d可观察到明显降解。将生长因子rhBMP-2与PEG-SH前驱液混合,制备负载rhBMP-2的可注射凝胶体系。异位成骨实验显示成骨量随时间延长而增加：未负载rhBMP-2凝胶无成骨现象发生,材料基本降解。成骨与凝胶的降解密切相关,材料在细胞的吞噬作用和骨基质包埋作用下降解,使rhBMP-2得以完全释放,持续诱导成骨作用；持续成骨导致更多骨基质的分泌,从而加速材料的降解,成骨与降解相互促进。水凝胶的降解为骨细胞和血细胞提供了生长空间和自由的养分传输,有利于细胞的增殖、铺展和迁移,也有利于血管发生。(4) PEG-SH凝胶冻干支架的生物降解性及负载rhBMP-2诱导异位成骨采用冷冻干燥方法将PEG-SH水凝胶制成多孔干凝胶支架。质量分数为3%、5%、10%、15%的干凝胶支架在细胞外浓度水平GSH(0.01mM)中响应性降解时间分别为3d.6d、6d、22d;在细胞内浓度水平GSH(3mM)中为0.5h、1h、3h、4h。与水凝胶相比,支架对GSH响应更加灵敏和迅速,溶胀率明显增加。负载rhBMP-2凝胶支架异位成骨实验显示,成骨量随时间延长而增加,高于同期注射型水凝胶的异位诱导成骨量;未负载rhBMP-2凝胶无成骨现象发生,支架材料基本降解。成骨与支架降解密切相关,材料通过红细胞免疫粘附作用、白细胞吞噬作用、成骨细胞分泌骨基质的包埋作用达到逐步降解。降解后期rhBMP-2得以完全释放,持续诱导成骨；持续成骨加速材料降解,成骨与降解相互促进。支架的降解为骨细胞和血细胞提供了生长空间,促进成骨和血管发生。(5) PEG-SH凝胶冻干支架负载rhBMP-2修复临界桡骨缺损制备生长因子与PEG-SH冻干凝胶支架复合体系,建立兔桡骨15mm节段骨缺损动物模型,评价自行修复、单纯支架修复、载rhBMP-2或RGD多肽复合支架对兔临界骨缺损的修复效果。实验结果表明因子的诱导作用、支架材料的传导作用和降解作用等因素均明显影响修复效果。随时间由2w延长至12w,各组成骨量逐渐增加,单纯支架组成骨量略高于自行修复组,rhBMP-2复合支架组明显高于单纯支架组,而rhBMP-2和RGD多肽同时复合支架组优于只加rhBMP-2组。12w新骨生物力学性能测试表明,rhBMP-2复合支架组、rhBMP-2和RGD多肽同时复合材料组最大弯曲载荷分别为232.9N和251.0N,接近正常健康兔骨256.4N的数值,远高于空白组和不含BMP组。通过组织学切片分析,自行修复组成骨速度较慢,单纯材料组略快,12w内两组均不能使骨髓腔再次连通,且出现骨不连；生长因子复合材料组能够完成缺损区域的修复,再造连通骨髓腔。成骨与支架的降解密切相关,相互促进。支架材料对成骨过程中血管发生起到促进作用,含有支架的三组均比自行修复组显现出更多血细胞和血管。负载rhBMP-2的PEG-SH凝胶支架可以用作临界骨缺损的修复。更多还原

【Abstract】 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.更多还原