胎牛松质骨来源组织工程骨的构建及其理化、生物学特性研究

发布时间：2018-05-22 16:08

本文选题：无机小牛骨 + 原子力显微镜　；参考：《南京大学》2015年博士论文

【摘要】：背景与目的骨量不足是口腔颌面外科手术中面临的主要问题之一,其原因包括牙周病、外伤、肿瘤等。目前在临床上主要通过自体骨、异体骨、异种骨、人工合成材料等材料进行植入,其中自体骨移植长期以来都被认为是植骨材料的“金标准”但自体骨也存在着明显的缺点,如增加取骨术区、取骨区并发症等。因此,研发接近自体骨植骨效果的组织工程骨一直是生物材料领域的热点。目前国外组织工程骨领域的研究已经使大量的材料在临床使用和商品化,而国内在相关科学研究和成果转化之间的衔接上仍然存在一些不足。本研究立足于骨粉材料基础研究,着眼于组织工程骨的构建和后期转化的可行性操作,希望可以实现组织工程骨材料的自主研发。之前我们通过大量临床经验积累,总结了目前使用的各种商品化骨粉,发现这些骨粉仍然存在一些不足。化学合成的骨替代材料由于与天然骨理化性质的差异以及后期代谢成份的影响,其在临床的使用并不广泛,目前仍需要进一步完善研究。天然来源的骨替代材料如各种动物松质骨来源的骨粉,由于具有和骨组织相近的多孔结构和近似的矿物质组成,目前是临床使用的主要材料,如小牛松质骨来源的BioOss骨粉。该类型的煅烧骨主要成份为骨无机基质,并没有骨诱导性,所以在临床上存在体内滞留时间过长,新生骨骨质不理想等缺点。近年开发的组织工程骨如PepGen/P-15,其过于简单的多肽表面修饰方法使所添加多肽P-15的活性和稳定性不明确,也一定程度上限制了在临床的应用。本课题将全面地对组织工程骨材料的研发和相关生物学评价进行系统性研究,旨在(1)研发具有可靠生物安全性和成骨性的胎牛松质骨无机骨基质；(2)对生产的无机骨基质进行表面修饰,提高其成骨效率； (3)明确多肽P-15对于细胞粘附的作用。材料与方法选取胎牛松质骨为原料,利用化学及煅烧法制备胎牛骨无机骨基质(Fetal Anorganic Bovine-derived Matrix, FABM)。使用MTT法、SEM扫描电镜、染色体畸变实验等方法,检测所制得FABM的生物安全性。使用球磨仪粉碎FABM,过筛制备纳米级FABM后压制成片。在制得的FABM片上培养人颌骨间充质干细胞(Human jaw mesenchymal stem cells, HJMSC),观察多肽P-15对细胞粘附形态的影响。通过常温共沉积法在FABM片表面通过双矿化法沉积磷酸钙结晶,对FABM进行表面改性,提高FABM对成骨细胞的粘附及其成骨性。利用原子力显微镜(Atomic Force Microscope, AFM)的单细胞检测技术,研究P-15对HJMSC弹性模量的影响,和P-15对于FABM颗粒与HJMSC细胞之间粘附力的影响。制备兔颅骨临界骨缺损,植入所制得的骨粉,制备组织切片,观察其远期成骨性和骨材料的体内代谢时间。结果1.所制备骨粉的物理化学性质制得骨粉外观为白色或类白色多孔颗粒,颗粒尺寸为0.5-2mm。其中超过粒度上限的颗粒重量不超过3%,小于粒度上限的颗粒重量不超过5%。X射线衍射谱中无明显的其他磷酸钙和结晶物质峰,羟基磷灰石含量为96.9%。检测pH值为：7.42,骨粉钙磷比：1.76±0.27。2.所制备骨粉的生物安全性测试细胞毒试验中,不同浓度的材料浸提液与细胞共培后,MTT检测结果均未显示明显细胞毒性。热源反应实验中,三只动物(兔)体温升高均小于0.6℃,判断骨粉无热源反应。急毒试验中,试验组和对照组动物活动无异常,未观察到毒性反应,试验组体重变化与对照组动物相比无明显差异。皮肤刺激试验中,所选用的三只家兔,注射浸提液后,试验样品与溶剂对照平均记分差为0.49,小于1.0,表明无明显皮内反应出现。染色体畸变试验中显微镜下观察染色体畸变,选取分散良好的中期分裂相(染色体数为2n±2)进行染色体畸变分析。结果得出骨粉材料未致染色体畸变率升高。3.多肽P-15对HJMSC的影响在20min和12h共培养时间条件下,通过SEM电镜结果可见,有无P-15存在的FABM片表面附着的HJMSC,其粘附后的形态有差异。主要表现为细胞伸展状态及丝状板状伪足的多少等形态学表现。AFM实验中,有无P-15吸附的FABM颗粒,其对HJMSC的粘附力基本在30pN左右,无显著差异,但在拉脱FABM颗粒与细胞时,有P-15参与的组别所消耗的能量为无P-15组的四倍。提示P-15能更有效地促进细胞和FABM之间的粘附。在P-15对于HJMSC的弹性模量研究中,细胞经P-15共培养后,AFM试验发现P-15改变了细胞膜弹性模量,提示P-15对细胞骨架有一定影响,并和P-15浓度有关(P0.05)4.骨粉植入动物实验在兔颅骨制备1.8cm × 1.8cm的临界骨缺损,植入制得的FABM,在不同时间点处死动物,得到组织切片。切片结果提示FABM生物相容性较好,未发现较明显异物反应,在体内的代谢时间为26周左右。结论(1)所制得FABM具有符合标注的理化性质和良好的生物安全性,符合三类医疗器械的临床前生物监测标准。(2)多肽P-15对于HJMSC具有促粘附作用。P-15对细胞的弹性模量有影响,提示其可能改变细胞的细胞膜结构,从而影响细胞的粘附。多肽P-15可以作为有效的促成骨生长因子用于骨粉表面修饰。(3)共沉积法可以在FABM表面形成稳定有序的结晶,通过沉积固定的生长因子可以起到缓释长效的作用。(4)动物体内实验提示FABM有良好的生物安全性和成骨特性,其体内代谢周期符合临床需要。
[Abstract]:Background and objective bone shortage is one of the main problems in oral and maxillofacial surgery, which includes periodontitis, trauma, tumor and so on. It is now mainly implanted in the clinical materials such as autogenous bone, allograft bone, xenogeneic bone, artificial synthetic material and so on. Autogenous bone transplantation has long been considered as the "gold mark" of bone graft materials. However, there are obvious defects in the autogenous bone, such as increasing the area of osteotomy and the complication of bone taking. Therefore, the research and development of tissue engineering bone, which is close to the effect of autogenous bone graft, has always been a hot spot in the field of biological materials. There are still some shortcomings in the connection between scientific research and the transformation of results. This study is based on the basic research of bone powder materials, focusing on the construction of tissue engineering bone and the feasibility of the later transformation. We hope to realize the independent research and development of tissue engineering bone materials. We have accumulated a lot of clinical experience and summed up the current use. A variety of commercialized bone powder has been found that there are still some deficiencies in these bone powders. The chemical synthetic bone substitute is not widely used in clinical use because of its differences in physical and chemical properties of natural bone and the effect of later metabolic components. The natural source of bone substitute, such as various animal cancellous bones, is still needed. Bone meal is a major clinical material, such as BioOss bone powder derived from calf cancellous bone. The main component of this type of calcined bone is bone inorganic matrix and no bone inducibility, so there is a long time in the body and new bone bone in the body. The tissue engineering bone, such as PepGen/P-15, has been developed in recent years, such as the simple method of polypeptides surface modification, which makes the activity and stability of the added polypeptide P-15 not clear, and to some extent restricts the clinical application. This topic will make a comprehensive study on the research and development of tissue engineering bone material and the related biological evaluation system. The purpose of the study is (1) to develop an inorganic bone matrix with reliable biological safety and osteogenesis in fetal bovine cancellous bone; (2) surface modification of the inorganic bone matrix produced to improve its osteogenesis efficiency; (3) the effect of polypeptide P-15 on cell adhesion. Materials and methods were used to prepare fetal bovine cancellous bone as raw material and prepared by chemical and calcining methods. Fetal bovine bone mineral matrix (Fetal Anorganic Bovine-derived Matrix, FABM). Using MTT method, SEM scanning electron microscopy, chromosome aberration test and other methods to detect the biological safety of FABM. W mesenchymal stem cells, HJMSC), observe the effect of polypeptide P-15 on the cell adhesion morphology. By depositing calcium phosphate crystallization on the surface of FABM slices through the normal temperature co deposition method, the surface modification of FABM to improve the adhesion and osteogenesis of FABM to osteoblasts is improved. The single detail of the primary subforce microscope (Atomic Force Microscope) is used. The effect of P-15 on the modulus of elasticity of HJMSC and the effect of P-15 on the adhesion between FABM particles and HJMSC cells. The critical bone defect of the rabbit skull was prepared and the bone powder was implanted, and the tissue sections were prepared, and the long-term osteogenesis and the metabolism time in the body were observed. Results the physical and chemical properties of the 1. preparation of bone powder were made. The appearance of bone powder is white or white like white porous particles, the particle size of which is 0.5-2mm. above the upper limit of particle size is not more than 3%. The weight of particles less than the upper grain size is not more than other calcium phosphate and crystalline material peak in the 5%.X ray diffraction spectrum, and the content of hydroxyapatite is 96.9%. detection pH value: 7.42, bone meal calcium and phosphorus. In the biological safety test of bone powder prepared by 1.76 + 0.27.2., in the cytotoxicity test of bone powder, the results of MTT detection did not show obvious cytotoxicity. In the heat source reaction experiment, the body temperature of three animals (rabbits) was less than 0.6 degrees C, and the bone powder had no heat source reaction. There was no significant difference in animal activity and no observation of toxic reaction. There was no significant difference in weight change between the experimental group and the control group. In the skin irritation test, the average score of the test samples and the solvent was 0.49, less than 1 after the injection of the extract, which showed no apparent intradermal reaction appeared. In the chromosome aberration test, there was no obvious intradermal reaction in the three rabbits. Chromosome aberration was observed under the microscope, and the chromosomal aberration was analyzed with a well dispersed medium division phase (2n + 2). The results showed that the effect of.3. polypeptide P-15 on HJMSC was not caused by the increase of the chromosome aberration rate of bone powder material. The results of SEM electron microscopy showed that there was a FABM slice without P-15 under the co culture time of 20min and 12h. The morphology of the adherent HJMSC is different. The main expression is the morphology of the cell extension state and the number of filamentous pads. In the.AFM experiment, there is no P-15 adsorbed FABM particles, and the adhesion force to HJMSC is basically around 30pN, and there is no significant difference, but when the FABM particles and the cells are removed, there are P-15 participating groups. The energy consumption was four times that of the non P-15 group. Suggesting that P-15 could promote the adhesion between cells and FABM more effectively. In the study of the modulus of elasticity of HJMSC by P-15, after P-15 co culture, the AFM test found that P-15 changed the elastic modulus of the cell membrane, suggesting that P-15 has a certain effect on the cytoskeleton, and that the concentration of P-15 is related to the P-15 concentration (P0.05) 4. bone powder implantation In the animal experiment, the critical bone defect of 1.8cm x 1.8cm was prepared in the rabbit skull, and the FABM was implanted. The animals were killed at different time points and the tissue sections were sliced. The results showed that the biocompatibility of FABM was better, and the metabolic time in the body was 26 weeks in the body. Conclusion (1) the FABM has the physical and chemical properties. Quality and good biological safety conforms to the pre clinical biological monitoring standards of three kinds of medical instruments. (2) polypeptide P-15 has an effect on the elastic modulus of cells with the adhesion action of.P-15 to HJMSC, suggesting that it may change the cell membrane structure of the cell, thus affecting the cell adhesion. Polypeptide P-15 can be used as an effective factor to promote bone growth factor. The surface modification of bone powder. (3) co deposition can form a stable and orderly crystallization on the surface of FABM. By deposition of fixed growth factors, it can play a sustained and long-term effect. (4) in vivo experiments in animals suggest that FABM has good biological safety and osteogenic characteristics, and its metabolic cycle in vivo meets the clinical needs.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318.08;R687

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