【作者】 刘欢叶；

【导师】 艾红军；

【作者基本信息】 中国医科大学 ， 口腔临床医学， 2010， 博士

【摘要】 目的随着年龄的增长,由感染、肿瘤、创伤、外科手术、关节病变和骨不连等造成的骨缺损越来越多。而面对不规则骨缺损和逐渐增长的微创技术,尤其是颅颌面区域的应用,寻找一种有良好的生物相容性和较快的成骨性能的可注射性的生物降解的支架材料变成骨组织工程的热点。可注射性骨水泥可分成四类：磷酸钙化合物,丙烯酸骨水泥,硫酸钙及其合成物。在这些材料中,作为骨移植的替代物,硫酸钙因其在缺损部位的原位自固化性能、无炎性反应、以及促进骨愈合等优良性能被临床上广泛应用。可用于牙周病、牙髓病、牙槽骨丧失、上颌窦充填等疾病。然而,硫酸钙与自体骨的骨缺损修复相比有一定的劣势。硫酸钙骨水泥由于其较低的生物活性,其在治疗早期与骨组织缺乏较好的化学键结合力,这些显著的缺点限制了其在临床的应用。而理想的骨支架应能促进骨的早期矿化,并能与此同时促进新骨的形成。基于生物矿化原理的纳米基胶原合成材料,由于其与天然骨相似的结构,并添加了Ⅰ型矿化胶原,而受到广泛的关注。在这个材料中,胶原分子和纳米羟基磷灰石组装成与天然骨的成分和空间结构及其相似的矿化纤维,并有大量的研究证明nHAC且有良好的生物活性和生物相容性。为了提高硫酸钙的骨再生性能,有文献报道可以将羟基磷灰石添加到硫酸钙中,变成可注射性的骨水泥,可替代自体骨用于骨再生的应用。本文研究了不同配比的nHAC和CSH的nHAC/CSH复合水泥的凝固时间,及其机械性能,体外的生物活性和降解性,体外及体内的生物相容性,及体内成骨性能等,旨在推进用于无负荷的整形外科的骨缺损修复,以及推进骨肿瘤切除术和骨质疏松症的研究。1、通过对不同配比的nHAC和CSH的nHAC/CSH复合水泥的凝固时间,及其机械性能,体外的生物活性和降解性,体外及体内的生物相容性研究,初步评价复合材料的生物相容性和生物活性,筛选出适合临床应用的nHAC和CSH比例。2、通过体外材料与骨髓基质细胞共培养来定性和定量地研究材料的生物相容性。在条件培养基的诱导下,将骨髓间充质干细胞向骨髓源成骨细胞诱导,并通过检测细胞的成骨性能的表达,评价骨髓源成骨细胞的成骨性能。将骨髓源成骨细胞用于MTT细胞毒性试验和流式细胞仪检测与材料共培养后,材料对细胞周期的影响,并通过细胞在两种材料表面的培养的扫描电镜结果,观察细胞在材料表面的粘附情况,同时对于材料共培养细胞的Ⅰ型胶原和整合素(31 mRNA相对表达水平的测定,检测细胞粘附发生的机制。3、通过溶血试验,热原实验,皮内反应试验来评价复合材料的生物相容性。通过兔下颌骨临界缺损的箱状模型来评价纯硫酸钙(CSH)和纳米晶羟基磷灰石胶原／硫酸钙(nHAC/CSH)复合骨水泥的骨缺损修复性能。采用x光影像学方法观察各组动物的骨缺损修复情况,术后4、8、12周取兔下颌骨,记录骨矿密度(bone mineral density,BMD)数值,骨密度值进行定量分析,以评价骨缺损区的成骨质量。利用脱钙及未脱钙的组织切片及染色技术,观察材料降解、形态改变与组织生长情况,评价其成骨能力。结果1、XRD用来测试含不同比率nHAC和CSH的复合水泥的晶体构成,凝固后的nHAC/CSH复合水泥的主要成分为nHAC和CSD,与文献报道一致,成功合成了复合材料。结果显示,凝固性、可注射性以及骨水泥的机械性能均取决于nHAC和CSH比率。nHAC能使复合水泥的凝固时间延长,nHAC的在复合水泥中所占的比率越高,凝固时间越长。复合水泥抗压强度和压缩模量随着nHAC比率的增加而降低。EDX结果分析证实添加nHAC显著改进了nHAC/CSH复合水泥的生物活性。除了30% nHAC/CSH复合水泥之间在第14天和第21天的结果外,nHAC/CSH复合水泥和纯CSH水泥相比,降解率无显著差异。因此,可以通过调整nHAC和CSH比率,使nHAC/CSH复合水泥的降解度符合各种临床应用；对含不同比例nHAC的复合水泥性能进行测试,筛选出含10% nHAC的复合材料各项性能比较符合临床要求。2、在条件培养基的诱导下,将骨髓间充质干细胞成功诱导为骨髓源成骨细胞,并具有成骨能力。将骨髓源成骨细胞用于MTT试验和流式细胞仪的结果显示,nHAC/CSH有良好的生物相容性。通过将骨髓基质细胞在两种材料表面的培养的扫面电镜结果可见,nHAC/CSH材料更有利于细胞的粘附,Ⅰ型胶原和整合素β1mRNA相对表达水平也表明nHAC/CSH复合水泥有更强的促进细胞粘附的能力。和纯硫酸钙(CSH)相比,纳米晶羟基磷灰石胶原／硫酸钙(nHAC/CSH)复合骨水泥能显著提高材料的生物活性,尤其是在促进细胞粘附方面。3、体内试验的结果也显示,该复合水泥未导致明显的炎症反应,无溶血性、无致热性、无皮肤刺激性,具有良好的生物安全性,符合国家生物医用材料相关标准。将材料植入兔下颌骨临界性箱状缺损修复结果表明,：nHAC/CSH复合骨水泥有良好的骨缺损修复能力,尤其是早期成骨性能优异,与纯CSH水泥相比,更有利于促血管生成和骨改建。结论1、通过调整材料中]nHAC和CSH的比率可控制nHAC/CSH复合水泥的凝固时间、可注射性能、机械性能,使其满足临床需要。与纯CSH水泥相比,复合材料表现出较好的生物活性。并筛选出含10% nHAC的复合材料各项性能比较符合临床要求。2、体外细胞培养实验结果表明,nHAC/CSH复合骨水泥无明显的细胞毒性,有较好的细胞相容性,与纯CSH水泥相比,更有利于细胞增殖和黏附。3、纳米晶羟基磷灰石胶原／硫酸钙(nHAC/CSH)复合骨水泥有较强的骨改建能力,尤其在早期促成骨方面效果显著,具有潜在的临床利用价值。更多还原

【Abstract】 ObjectiveBony defects caused by infection, tumor, trauma, surgery, joint fusion, and fracture nonunion have increased as the population ages. Facing the problems of the irregular bony defects and the increasing popularity of minimally invasive techniques, especially in the application of craniofacial regions, the search for injectable biodegradable scaffold materials with satisfactory compatibility and rapid ossify performance became one of the major hotspots in bone tissue engineering.Injectable bone cements can be classified into four classes:calcium phosphates, acrylic bone cements, calcium sulfates, and composites. Among these materials, calcium sulfate has a long clinical history for use as bone graft substitute in its form known as plaster or gypsum for its self-setting ability "in situ" after filling the defect, the lack of inflammatory response, and the promotion of bone healing. It has been utilized in periodontal disease, endodontic lesions, alveolar bone loss and maxillary sinus augmentation. However, calcium sulfate is inferior to autografts in bone defect repair. However, calcium sulfate cement has some drawbacks that significantly limit its clinical applications, such as lacking the ability to form a chemical bond with bone tissue at the early stage of therapy because of its poor bioactivity. The ideal bone scaffold should promote early mineralization and support new bone formation while at the same time allowing for replacement by new bone. Based on the biomineralization principles, nano-hydroxyapatite/collagen (nHAC) composite, inspired from investigation on natural bone, had been developed by mineralizing typeⅠcollagen in our lab, which has attracted great attention. In this material collagen molecules and nano-hydroxyapatite assembled into mineralized fibril, with high similarity of natural bone both in composition and hierarchical structure. Extensively studies indicated that the nHAC material is bioactive and biodegradable. In order to optimize bioactivity and the performance of bone regeneration of CSH, the previous study suggested that hydroxyapatite (HA) could be incorporated into CSH, creating a composite grout that could be used for bone regeneration as an alternative or adjunct to autogenous bone.In the present study, a novel injectable bone cement was designed as a composite of nHAC and CSH, A series of such injectable composite cements with different ratio of nHAC and CSH were prepared for adjustable self-setting time. Their mechanical properties, in vitro bioactivity and degradability, in vitro and in vivo biocompatibility of the composite cements were determined in this study, which is aimed to have complementary advantages for the non-loading bearing bone defect repair in orthopedics application, such as bone tumor resections and osteoporosis.Methods1. In order to evaluate the biocompatibility and bioactivity of the composite cements, a series of such injectable composite cements with different ratio of nHAC and CSH were prepared for adjustable self-setting time. Their mechanical properties, in vitro bioactivity and degradability, in vitro and in vivo biocompatibility of the composite cements were also determined in this study. Through this research, the suitable proportion of nHAC should be screened for clinical practice.2. Quantitative and qualitative biocompatibility assays with bone marrow stromal cells cultured with materials in vitro were performed for evaluating the biocompatibility. Under the induction of condition culture medium, marrow mesenchyma stem cells were induced to marrow source osteoblast and express the performance of ossification. In vitro, the marrow source osteoblast was detected by MTT assay and Flow cytometric analysis. BMSCs grew on the surface of two samples were detected under scanning electron microscope. Type I collagen and integrinβ1 mRNA relative expression level were also detected to test the mechanism of cell adhesion.3. The the hematolysis experiment, the pyrogen experiment and the intracutaneous response experiment were used to appraise biocompatibility of the composite cement. A critical box-shaped defect model in the mandible of the rabbit was used to evaluate the bone-remodeling ability.4,8,12 weeks after operation, rabbit mandibular in box-shaped defect regions were extracted. X-ray study was used to observe the situation of each group of animal’s bone defect. BMD analysis the of the defect regions were also used to evaluate the level of ossification. Using the decalcify and not the decalcify tissue slice and the dyeing technology to observe the degradation, the shape change and the bone formation of the defect region and also evaluate the ability of bone regeneration of the composite cement.Results1. XRD was used for testing the composition of cement with a serious of different ratio of nHAC and CSH. The main composition of the final setting cement of nHAC/CSH is nHAC and CSD. The diffraction of the nHAC and CSH powders prepared in this paper were the same as that reported previously. The composite cement was synthesized successfully. The results demonstrated that setting behavior, injectability, and mechanical properties of the bone cement were depend on the composition ratio of nHAC and CSH. nHAC has a retarding effect on the setting time of composite cement, a higher setting time was obtained with the increase of the amount of nHAC. The mechanical results indicated that compressive strength and compressive modulus of composite the cement decreased with the increase of the content ratio of nHAC. The results from the EDX analysis indicated that the bioactivity of the nHAC/CSH composite cements has been improved significantly compared to that of the pure CSH cement. It can be seen that there was no significant difference of the degradation rate between the nHAC/CSH composite cements and the pure CSH cement, except that of 30% nHAC/CSH composite cement at 14th day and 21st day. The nHAC/CSH composite cement can be adjusted by modifying the ratio of nHAC and CSH for various clinical applications.10% nHAC was carefully screened for clinical practice.2. Under the induction of condition culture medium, marrow mesenchyma stem cells were induced to marrow source osteoblast. In vitro, favourable biocompatibility was detected by MTT assay and Flow cytometric analysis. BMSCs grew on the surface of two samples were found to be adhered firmly on nHAC/CSH surface compared with pure CSH under scanning electron microscope. Type I collagen and integrinβ1 mRNA relative expression level also suggested nHAC/CSH composite cement have a higher ability to promote cell adhesion than the pure CSH. Results in vitro indicated that the nHAC/CSH significantly improved bioactivity compared with that of CSH, especially in promoting cell adhesion. 3. In vivo experiment indicated that this composite cement has not caused the obvious inflammation, does not have hemolytic, not sends the thermal property, the non-skin irritating quality, has the satisfactory biological security, conforms to the national biology medical material related standard. The in vivo results also show that nHAC/CSH composite cement exhibit favorable bioactivity of bone regeneration than that of CSH, it also could cause an earlier accelerator and better osseointegration for bone repair.Conclusions1. The setting time and injectability and mechanical properties of the composite cement could be controlled by adjusting the content ratio of nHAC and CSH. nHAC/CSH exhibit better bioactivity compared to pure CSH.10% nHAC was carefully screened for clinical practice.2. The in vitro cell culture test showed that the composite cement have no significant cytotoxicity could benefit cell proliferation and adhesion compared with pure CSH cement.3. Furthermore, the in vivo results mean that nHAC/CSH composite have better biocompatibility and a higher bone remodeling activity than that of CSH, especially at the early stage of remodeling. Hence, nHAC/CSH composite cement can be further researched as injectable scaffold for bone regeneration.更多还原