【作者】 王根林；

【导师】 杨惠林；

【作者基本信息】 苏州大学 ， 骨外科学， 2008， 博士

【摘要】 第一部分可降解多孔型丝素蛋白/羟基磷灰石的制备及其在SD大鼠体内降解的实验观察【目的】制备丝素蛋白/羟基磷灰石（silk fibroin/hydroxyapatite,SF/HA）复合人工骨材料,并将该材料植入生物体内降解,以了解SF/HA在动物体内的降解速率及机体对SF/HA的组织学反应,为后续成骨实验提供可靠的参考数据。【方法】以蚕丝丝素蛋白作为羟基磷灰石沉积的模板,以家蚕丝素短纤维为增强材料,以NaCl为致孔剂,采用等静压的方法制备4种SF/HA复合多孔材料。以羟基磷灰石为对照组,测定4种SF/HA复合材料及羟基磷灰石在0.5h、2h、6h、12h及24h不同时间点的吸水率。将4种SF/HA复合材料及羟基磷灰石植入SD大鼠背部皮下,进行体内降解观察。实验组分别于术后2、6、12、16、20及24周取材,对照组分别于术后2、12及24周取材,进行大体观察及组织学检查。【结果】丝素蛋白、HA、添加剂及致孔剂配比不同,制备的4种SF/HA人工骨材料的孔径、孔隙率及强度等也不同。材料吸水率趋势如下:SF/HA₃、SF/HA₄>SF/HA₂、SF/HA₁ >HA。HA、SF/HA₁及SF/HA₂降解十分缓慢或基本不能降解;SF/HA₃ 20～24周降解完毕;SF/HA₄ 12～16周降解完毕;各种材料周围组织未见明显变性坏死等。【结论】SF/HA₁平均孔径为6.5μm,最大孔径也只有15μm,不适合骨组织工程支架材料的要求。SF/HA₄虽然孔径、孔隙率满足要求,但力学强度较低,压缩强度只有1.59MPa,在水中很快散开,也不适合骨组织工程的要求。故在后续的试验中,我们剔除这两种材料,将对SF/HA₃及SF/HA₂两种材料进行体外细胞相容性研究。第二部分兔骨髓基质细胞的分离培养及其成骨诱导分化的实验研究【目的】体外分离培养兔骨髓基质细胞（bone marrow stromal cells,BMSCs）,并向成骨方向诱导培养,为SF/HA相容性提供检测细胞及为SF/HA组织工程化骨提供适宜的种子细胞。【方法】抽取新西兰白兔的骨髓5ml,通过密度梯度离心法获取BMSCs,体外贴壁培养、扩增、传代,倒置显微镜下观察原代及各代细胞形态、数量生长情况,描绘生长曲线。第2代细胞用含1×10^-8g/l地塞米松、10mmol/lβ-甘油磷酸钠及50μg/l L-抗坏血酸的条件培养液将其定向成骨诱导培养、扩增。采用倒置相差显微镜观察细胞形态变化;采用MTT法测定细胞增殖情况;使用碱性磷酸酶染色及Von Kossa钙结节染色等方法,鉴定其成骨性能。【结果】BMSCs在培养皿中贴壁生长、增殖,条件培养液诱导后表现出明显的成骨活性,细胞增殖良好,体外矿化结节Von Kossa染色阳性、碱性磷酸酶染色阳性,证实其有成骨潜能。【结论】可以通过体外分离纯化兔BMSCs,在一定条件下能够向成骨方向诱导分化,并能使细胞保持较高的成骨活性,适于作为材料相容性的检测细胞及构建组织工程化骨的种子细胞。第三部分可降解多孔型丝素蛋白/羟基磷灰石复合人工骨材料的细胞相容性研究【目的】以成骨诱导的BMSCs作为检测细胞,与两种丝素蛋白/羟基磷灰石（SF/HA）进行体外复合培养,检测SF/HA的细胞相容性。【方法】将兔BMSCs体外成骨诱导至第3代,接种到预湿的SF/HA₂及SF/HA₃材料上复合培养,以单纯BMSCs相同条件下培养作为对照。通过相差显微镜、扫描电镜及HE染色等方法观察细胞在材料中的生长情况。以MTT法检测材料对细胞增殖活性的影响,以碱性磷酸酶活性测定评价其成骨能力,以材料浸提液的细胞毒性试验评估材料是否有细胞毒性。【结果】扫描电镜及组织学观察发现,细胞材料复合后,BMSCs在SF/HA₂及SF/HA₃上能够良好地粘附和增殖。ALP活性测定及MTT法测定OD值均证实,BMSCs细胞活性及成骨性能不受材料影响,与对照组相比,差异无统计学意义（P>0.05）。材料浸提液细胞毒性试验显示,细胞毒性分级均为0～Ⅰ级,两种SF/HA浸提液对细胞生长基本无毒性作用。【结论】SF/HA₂与SF/HA₃对BMSCs的生长和成骨功能无影响,具有良好的细胞相容性,具有构建组织工程化骨的可行性。第四部分可降解多孔型丝素蛋白/羟基磷灰石异位成骨及其组织相容性的实验研究【目的】探讨SF/HA₂及SF/HA₃体内异位成骨的能力,并通过肌肉植入实验进一步观察两种材料的组织相容性,为SF/HA修复节段性骨缺损提供实验依据。【方法】将体外诱导培养的兔骨髓基质细胞,以5×10⁷/ml的密度接种到SF/HA₂,作为实验组1;接种到SF/HA3,作为实验组2,并于3～5d后植入兔背部肌肉中。植入单纯SF/HA₂与SF/HA₃,作为对照组。对照组亦作为材料的肌肉植入实验模型,观察组织相容性。于术后第4、8、12周,每次随机选取5只兔处死后取材,大体观察及HE染色组织学观察,并进行骨组织形态计量学分析,定量比较各组新骨形成情况。【结果】实验组术后4、8、12周均有新骨形成,随着时间延长,新骨生成量增多;骨组织形态计量学分析显示,实验组1优于实验组2（P<0.05）;对照组则均无新骨形成。肌肉植入实验未见肌肉变性坏死等。【结论】肌肉植入实验、第一部分的皮下植入实验及第三部分的细胞相容性研究均显示,两种SF/HA具有良好的组织相容性。SF/HA₃与SF/HA₂复合细胞后构建的组织工程化骨,具有良好的异位成骨能力,可望应用于临床修复骨缺损。但从生物降解性及异位成骨的效果看,SF/HA₃更适合于作为人工骨材料,将进一步研究该材料修复节段性骨缺损的能力。第五部分可降解多孔型丝素蛋白/羟基磷灰石组织工程化骨修复节段性骨缺损的实验研究【目的】探索SF/HA₃组织工程化骨的成骨作用及作为骨替代材料的可行性,期望为临床治疗骨缺损提供新的人工骨材料。【方法】在本实验中将SF/HA₃更名为SF/HA,与诱导的兔BMSCs复合,构建组织工程化骨。麻醉生效后,充分显露兔左侧桡骨中上段,造成15mm节段骨缺损。实验组植入SF/HA+BMSCs复合物,实验对照组植入SF/HA材料,空白对照组骨缺损区不植入任何材料。于术后第4、8、12及16周行X线摄片及16周时行螺旋CT扫描重建,观察骨缺损修复及骨塑形情况。参照Lane-Sandhu X线评分标准对各组桡骨缺损的骨修复程度评分。标本行HE染色及Masson染色组织学观察,按照Lane-Sandhu组织学评分法比较12周及16周时各组动物的骨修复情况。【结果】实验组、实验对照组及空白对照组的放射学及组织学检查评分显示,新骨生成有统计学差异（P<0.05）,实验组优于实验对照组优于空白对照组;大体观察及放射学检查证实:实验组兔桡骨缺损完全愈合;实验组对照组缺损处少部分骨愈合;空白对照组缺损基本无骨修复作用,由纤维组织充填。【结论】SF/HA与BMSCs复合,降解速率适宜,能较快被骨组织取代,具有相当于自体骨的骨缺损修复能力,基本达到现代骨组织工程学的要求。但材料本身缺乏骨诱导作用,单独用于节段性骨缺损修复作用有限。更多还原

【Abstract】 PartⅠ: Preparation of Degradable Porus Silk Fibroin/Hydroxyapatite （SF/HA） Composite and Degradation of the Composite in SD rats in Vivo【Objective】To prepare a novel bone substitute of silk fibroin/hydroxyapatite （SF/HA） composite for bone tissue engineering, and to explore the composite degradation rate and tissue reaction on it in SD rats in vivo.【Methods】Silk fibroin （SF） and Hydroxyapatite （HA） composite powders were synthesized. 4 types of porous silk fibroin/hydroxyapatite （SF/HA） composite could be prepared when adding silk short fibers and using NaCl as porogen by isostatic pressing. Using HA as a control group, water absorption rate of 4 composites and HA were measured at 0.5, 2, 6, 12 and 24 h after these materials soaked in deionized water. These materials were implanted into SD rats’back subcutaneously to observe their degradation rate and tissue reaction on them. Specimens in experiment group were observed in general and from the aspect of histology at post-operative 2, 6, 12, 16, 20 and 24 weeks respectively, while control group at post-operative 2, 12 and 24 weeks.【Results】Pore size, porosity and strength of these 4 type of SF /HA were different when proportion of SF, HA, affix and porogen was different. Absorption rate was as follows: SF/HA₃ and SF/HA4>SF/HA₂ and SF/HA₁>HA. HA, SF/HA₁ and SF/HA₂ had little degradation. SF/HA₃ had complete degradation during post-operative 20～24 weeks, while SF/HA4 had complete degradation during post-operative 12～16 weeks. Histology showed that tissue surrounding SF/HA had no apomorphosis and necrosis.【Conclusions】SF/HA₁ is unsuitable for bone tissue engineering scaffold because its average pore size was 6.5μm, and its maximum pore size was only 15μm. SF/HA4 is also unsuitable for bone tissue engineering scaffold because its strength is only 1.59MPa and it quickly spread in water. In the future experiment, we will exclude these two composites, and only carry out study on SF/HA₃ and SF/HA₂. PartⅡ: Experimental Studies on Cultivation of Bone Marrow Stromal Cells （BMSCs） from Rabbits and BMSCs Osteogenic Differentiation【Objective】To differentiate and proliferate bone marrow stromal cells （BMSCs） from rabbit’s marrow into osteoblasts in vitro to prepare seed cells for bone tissue engineering and compatibility test.【Methods】Primarily cultured BMSCs isolated from a rabbit’s bone marrow by density gradient centrifugation were subcultured in mineralization medium to induce their differentiation into osteoblasts, whose morphological characteristics and proliferation status were observed by phase-contrast microscope. Von Kossa staining and alkaline phosphatase （ALP） activity test were employed to assess BMSCs’osteoblastic differentiation and the generation of calcified extracellular matrix. MTT assay detected the osteoblast proliferation.【Results】BMSCs cultured in vitro showed obvious osteogenic capacity in mineralization DMEM. Von Kossa staining of the mineralized nodules and alkaline phosphatase detection of the passaged cells both yielded positive results. MTT assay showed the osteoblast proliferation was normal.【Conclusions】BMSCs could be cultured, differentiated and proliferated with active osteogenic function by differentiation culture medium in vitro. So it could be suitable for seed cell for bone tissue engineering.PartⅢ: Study on Cellular Compatibility of the degradable porus composite of SF/HA【Objective】To evaluate SF/HA cellular compatibility as well as the feasibility of the composite to serve as a scaffold in tissue engineering by using co-culturing of osteogenic BMSCs and SF/HA.【Methods】The third passage BMSCs were transplanted into SF/HA₂ and SF/HA₃ after being induced to differentiate into osteoblasts and then seeded into the materials for 3 to 5 days. BMSCs alone were cultured at the same condition to act as controls. The cellular morphology and function （attachment, proliferation and differentiation） were assessed separately by means of phase contrast microscope, HE, SEM and MTT assay, ALP activity. The material leaching liquor were used to test cell toxicity. 【Results】BMSCs could adhere to SF/HA₂ and SF/HA₃, and proliferate and grow on the surface of the composites normally. The cellular activity and function were not affected by the materials, and no statistical difference was found between the two groups and the control group （P>0.05）. Cell toxicity test discovered these materials had no toxic effect on BMSCs.【Conclusion】SF/HA₂ and SF/HA₃ have a good biocompatibility and can be used as a tissue engineering scaffold.PartⅣ: Studies on ectopic bone formation of SF/HA with rabbit BMSCs and the composite tissue compatibility【Objective】To explore the ectopic osteogenesised ability of the tissue engineered bone fabricated by osteoblasts which derived from rabbit BMSCs co-cultured with SF/HA, and to observe the material tissue compatibility by placing it into rabbits’muscle.【Methods】The differentiated osteoblasts which were derived from BMSCs, with 5×107/ml density, were seeded onto SF/HA （SF/HA₂+BMSCs as Group1; SF/HA₃+BMSCs as Group2）. Then, co-cultured for 5 days, the compounds with cells and SF/HA were implanted into muscular pouch of back of rabbits and SF/HA alone was implanted as a control group. The effectiveness of bone formation was assessed separately by means of gross observation and histology after implantation for 4, 8, 12 weeks.【Results】New osteogenesis was detected at the end of the 4th, 8th and 12th week after implantation respectively in experimental group. The quantity of new osteogenesis in experimental group 1 was better than in experimental group 2 by histology morphometry （P<0.05）. There was no new bone formation in the control group. Muscle surrounding all materials had no apomorphosis and necrosis from the aspect of histology.【Conclusion】Muscle implantation test, Subcutanea implantation test in PartⅠand cell compatibility in PartⅢshow SF/HA₂ and SF/HA₃ have a good biocompatibility. The tissue engineered bone fabricated by osteoblasts which derived from BMSCs cultured onto SF/HA have a good ability of ectopia osteogenesis in vivo. It is supposed to be a good way to repair clinical bone defect. However, SF/HA₃ is more suitable than SF/HA₂ because SF/HA₃ has a better bio-degradation rate and ectopia osteogenesis effect. PartⅤ: Experimental study on rabbit segmental radial defects repaired by SF/HA co-cultured with rabbit BMSCs【Objective】To discuss the bone formation of SF/HA₃ tissue engineered bone co-cultured with rabbit’s BMSCs and the feasibility of SF/HA₃ as a bone substitute.【Methods】SF/HA₃ is named SF/HA in the current study. The purified, culture-expanded, and osteogenic BMSCs were combined with SF/HA in vitro according to condition of cell culture. A segmental bone defect （15 mm in length） was created at left radial in each rabbit. The composite grafts were implanted into the bone defects in rabbits through open operation. The curative effect was evaluated by radiographic examination, histology analysis and eyes observation in experimental groups, experimental control group and blank control group at post-operative 4, 8, 12, 16 week, respectively.【Results】The bone defects that had been treated with grafts exhibited new bone formation increased with time by radiography, histology and eye observation. The rate and quality of new bone formation were significantly different in the experimental groups, experimental control groups and blank control groups （P<0.05）. The segmental bone defects were complete union in the experimental groups, while the defects were partial union in the experimental control groups. In the defects in blank groups, there were no formation of new bone after operation and bone defects were finally repaired only by fibrous tissue.【Conclusion】SF/HA has a suitable degradation rate and can be replaced by normal bone tissue appropriately. The bone formation ability of SF/HA combined with BMSCs is equivalent to the ability of autogenous bone. It is supposed to be a good way to repair clinical bone defect. However, the composite has no bone induction, and using the material alone has little bone formation in the segmental bone defect.更多还原