【作者】 陈刚；

【导师】 廖前德；

【作者基本信息】 中南大学 ， 外科学， 2011， 博士

【摘要】 因外伤、感染、肿瘤等因素造成骨缺损是骨科临床常见疾病。较小范围的骨缺损可以由机体自身的再生得到修复,大范围骨缺损由于成骨细胞难以越过而不能正常愈合,若不采用适当治疗则最终仅由纤维组织充填,形成骨不连。而大范围骨缺损的修复目前仍是临床上难以完全解决的问题。目前常用的有自体骨移植、异体／异种骨移植、人工合成骨替代品等方法修复骨缺损,都有其优缺点。骨组织工程学是利用组织工程原理对骨缺损进行修复和重建的一门学科,近年来,将种子细胞与特定支架材料复合,在体外构建具有生命的活性组织工程化复合材料是骨组织工程学研究的热点。本实验根据骨组织工程学基本原理,培养人脐带间充质干细胞,成骨诱导后复合纳米羟基磷灰石／聚酰胺66支架材料,植入裸鼠体内观察其成骨的情况,为进一步建立骨组织工程的动物模型及满足治疗骨缺损的临床需要提供理论基础。第一章脐带间充质干细胞的提取、分离、纯化增殖及生物学特性目的研究探讨人脐带间充质干细胞的分离、培养、增殖方法,并研究其生物学特性,以期将其作为骨组织工程种子细胞。方法取足月妊娠剖宫产健康胎儿的脐带,D-Hank’s液充分洗涤,将脐带剪碎至1mm3大小组织块,运用胶原酶消化法从人脐带中分离获取间充质干细胞。加入含胎牛血清体积分数为0.1的DMEM/F12培养基中进行原代培养；待细胞生长至80-90%融合时,1：3传代培养,倒置显微镜观察细胞形态。MTT法测定第3、5、7代细胞生长曲线。流式细胞学检测第3代细胞的表面抗原分子。结果原代培养后24小时可见少量散在贴壁细胞,2-3天后可见梭形和多角形贴壁细胞；5-6天后可见梭形类贴壁细胞明显增多,增长速度增快,10-12天左右细胞融合接近80-90%。传2代以后细胞形态均一,呈长梭形,接近融合状态时可呈漩涡样生长。流式细胞仪检测第3代细胞有97%表达CD105,95%表达CD29,96%表达CD44,97%表达CD13,而只有0.8%表达CD45,1.8%表达CD31,1.2%表达HLA-DR。生长曲线显示传3、5、7代脐带间充质干细胞均有较强的增殖能力。结论人脐带间充质干细胞分离提取操作简便易行,且具有一般间充质干细胞生物学特性,可作为骨组织工程的种子细胞。第二章人脐带间充质干细胞的体外成骨诱导分化目的探讨人脐带间充质干细胞经体外诱导向成骨细胞定向分化的条件及能力,为进一步体内成骨研究奠定基础。方法(1)如第一章方法,分离培养人脐带间充质干细胞。(2)以含10%FBS、地塞米松、抗坏血酸、维生素C、β-甘油磷酸钠的高糖DMEM作为成骨诱导培养基,诱导第3代脐带间充质干细胞向成骨细胞分化。倒置显微镜、HE染色观察细胞形态变化。进行碱性磷酸酶染色及活性定量检测,茜素红S标记钙结节形成情况。(3)RT-PCR检测骨桥蛋白、骨钙素、碱性磷酸酶的mRNA的表达。(4)免疫细胞化学染色及Western blotting检测Ⅰ型胶原蛋白的表达。结果(1)人脐带间充质干细胞经成骨诱导后,细胞体积增大,呈短梭形、多角形；细胞呈集落样层叠生长。(2)成骨诱导14天,碱性磷酸酶染色呈阳性表达；成骨诱导14天内,可见碱性磷酸酶含量随着培养时间的延长逐渐增高。茜素红S染色呈红色。(3)诱导后7天、14天后RT-PCR鉴定骨桥蛋白、骨钙素、碱性磷酸酶mRNA的表达,在151bp、155bp及182bp有单一的目的条带,诱导后14天的细胞骨桥蛋白、骨钙素、碱性磷酸酶mRNA的表达水平均强于诱导后7天的细胞。(4)成骨诱导7天,Ⅰ型胶原蛋白免疫组化染色及Western blotting检测可见阳性表达,诱导组Ⅰ型胶原蛋白表达强度明显高于对照组(P<0.05)。结论人脐带间充质干细胞在体外成骨诱导培养下可定向成骨分化,并能够稳定表达成骨细胞特异性表型,具有成骨能力。第三章人脐带间充质干细胞与nHA/PA66复合支架生物相容性的体外实验研究目的研究人脐带间充质干细胞与nHA/PA66复合支架的生物相容性,探讨nHA/PA66复合支架作为人脐带间充质干细胞载体的可能性,为骨组织工程进一步实验提供理论依据。方法(1)如前述方法制备人脐带间充质干细胞。(2)取成骨诱导后的第3代人脐带间充质干细胞接种于备好的nHA/PA66支架材料上,观察hUCMSCs在nHA/PA66支架材料的生长情况,MTT法测定nHA/PA66对成骨诱导后第3代hUCMSCs生长增殖的影响。(3)检测碱性磷酸酶染色、活性定量检测及Ⅰ型胶原蛋白的表达。(4)检测成骨诱导后第3代hUCMSCs在nHA/PA66支架上的细胞粘附率,分别于2、4、8天取样并计算材料细胞毒性。(5) RT-PCR检测第3代hUCMSCs接种nHA/PA66支架7天OPN、OCN、ALP的mRNA的表达。(6)取材行扫描电镜观察细胞在材料的生长增殖情况。结果(1)成骨诱导后的第3代hUCMSCs在nHA/PA66支架材料上生长分化良好。MTT法测定OD值证实,hUCMSCs生长增殖活性不受材料影响,与对照组相比,差异无统计学意义(P>0.05)。(2)hUCMSCs复合nHA/PA66多孔材料培养,ALP和Ⅰ型胶原免疫组化染色为阳性,.成骨诱导14天内,可见碱性磷酸酶含量随着培养时间延长而逐渐增高。(3)细胞4h、8h、12h、24h粘附率分别为69.1±2%、78.4±2%、82%±2%及91%±2%。(4)第2天、第4天时间点支架材料细胞毒性评级均为0级,第8天毒性评级为1级,均评价为无毒。(5)在细胞复合支架后7天,RT-PCR鉴定骨桥蛋白、骨钙素、碱性磷酸酶mRNA的表达结果显示,实验组在151bp、155bp及182bp有单一的目的条带,其表达强度明显高于对照组(P<0.05)。(6)1天后扫描电镜观察,可见细胞在支架材料表面附着生长；7天后可见细胞在材料上生长良好,细胞可往空隙生长,材料空隙大量充填。结论纳米羟基磷灰石／聚酰胺66多孔支架材料可作为骨组织工程中人脐带间充质干细胞的细胞载体,无毒,生物相容性良好,不影响该细胞的生长增殖及成骨分化,满足骨组织工程的需要。第四章人脐带间充质干细胞复合nHA/PA66支架异位成骨作用的实验研究目的探讨了成骨诱导人脐带间充质干细胞复合nHA/PA66支架材料在裸鼠体内异位成骨的情况,为进一步骨缺损动物实验提供理论依据。方法(1)如第三章所述方法,将成骨诱导人脐带间充质干细胞接种nHA/PA66支架材料复合培养；(2)分别将成骨诱导后hUCMSCs悬液、单纯nHA/PA66支架材料、成骨诱导hUCMSCs与nHA/PA66支架复合体植入裸鼠背部肌袋内,观察其异位体内成骨情况。(3)术后4、8、12周取材行HE染色及骨钙素蛋白免疫组织化学染色观察表达情况。(4)扫描电镜观察其成骨能力。结果成骨诱导后hUCMSCs组、单纯nHA/PA66支架材料组在裸鼠背部肌袋中无骨质形成,成骨诱导hUCMSCs与nHA/PA66支架复合体组有骨质形成,同时材料部分降解。结论成骨诱导的hUCMSCs与nHA/PA66支架材料复合后制备的生物复合材料在裸鼠体内具有异位成骨能力。更多还原

【Abstract】 The bone defect caused by trauma,infection,tumor and so on,are one of the common diseases.Damaged bone will spontaneously heal provided the defect is not over a critical limit,but when the defect is critical-sized,it will be filled by fibrous tissue because the osteoblast cells can not pass the gap between two broken bones.The clinic treatment of wide-boud bone defects is still hard to tackle at present.Autogenous bones,allogenic/allogenous bones,and artificial bone substitutes are now usually used as obturation materials for bone defects,which have their respective strong and weak points.Bone tissue engineering is a subject which applying engineering and medicine principles to repair and reconstruct bone defects.Recently,researchers are mainly focusing on constructing bioactive compounds with seed cells and biological scaffolds.According to the basic principle of bone tissue engineering, we would isolate and culture the human umbilical cord mesenchymal stem cells (hUCMSCs) and then induce cells ossification, Latterly we seed osteoblastic-induced hUCMSCs in nano-hydroxyapatite crystals and pnolyamide 66 (nHA/PA66) material to construct cells-scaffold composites, and then observe its ossification circumstances after raising this compound in nude mouse’s body. Establish large animal model of bone tissue engineering and further satisfy treatment bone defection and damage of clinical demand in order to provide theories foundation.Chapter 1 Experimental study of the isolation、cultivation、proliferation and biological characteristics of human umbilical cord mesenchymal stem cellsObjective To investigate methods for the isolation, cultivation, proliferation and biological characteristics of human umbilical cord mesenchymal stem cells(hUCMSCs) in order to provide seed cells for bone tissue engineering.Methods The full-term umbilical cords from healthy neonate were collected and washed in D-Hank’s balanced salt solution.The mesenchymal tissue was then diced into cubes of approximately 1mm3.We isolated and cultured MSCs derived from human umbilical cord in vitro by utilizing collagenase digestion method.We cultivated origin cells by the DMEM/F12 which contain the fetus cow blood-serum physical volume a score to 0.1. While needing a cell growth to 80-90% fusion,1:3 spread generation culture. We observed the characteristics of the cells by the inverted microscope, and measured the P3、P5 and P7 cells growth curve by MTT method. Flow cytometry examined the surface antigen of the P3 cell.Results In 24 hours a few adherent cells could be observed after cultivation. The primary hUCMSCs indicated spindle-shape or multangular shape after the second-3rd day while it could be observed cell division. Fibroblast-like cells manifold progressively and proliferate rapidly after the 5th-6th day.Cells fused more than 80%～90% after the 10th～12th day. The shapes of cells are homogeneous long spindle-shape after the second passage, and then it could be observed circinate-like in fusion time.Flow cytometry shows that 97%,95%,96% and 97% of the 3rd passage cells expressed CD105,CD29,CD44 and CD 13 respectively, while only 0.8%,1.8% and 1.2% of the cells expressed CD45,CD31 and HLA-DR respectively.The growth curve showed that proliferation ability of the 3rd,5th and 7th passage cells was powerful.Conclusion The method of isolation and culture of hUCMSCs was simple and feasible. The characteristics of cells obtained from human umbilical cords were similar to the mesenchymal stem cells and could be accorded as a kind of seed cells for tissue engineering.Chapter 2 Osteogenic differentiation of human umbilical cord mesenchymal stem cellsObjective To explore the osteogenic differentiation potential of hUCMSCs cultured with a specific medium in vitro and make preparations for the further research about bone tissue construction in vivo.Methods (1)Human umbilical cord mesenchymal stem cells were isolated and cultured according to chapter I. (2) The 3rd passage hUCMSCs were cultured in the high sugar DMEM which contained 10% fetus cow blood-serum, dexamethasone, antiscorbic acid,vitamin C andβ-sodium glycerophosphate to be induces into osteoblast. Be observed the metamorphosis of cells by the inverted microscope and HE staining.The Alkaline Phophatase(ALP) staining was verified and the levels of ALP were measured.The formation of calcium nodules were confirmed by Alizarin red S staining. (3) The expression of osteopontin、osteocalcin and alkaline phosphates mRNA were examined by RT-PCR in 7 and 14 days. (4) The secretion of collagen type I were verified by the immunocytochemistry staining and Western blotting in 7 days.Results (1)After osteogenic induction,the cells became larger and polygonal, and could assemble themselves together.(2)The ALP staining appeared positive after osteogenic induction for 14 days.The levels of Alkaline Phophatase gradually became higher in 14 days.The Alizarin red S staining appeared positive,which verified the formation of calcium nodules,when osteoinducted 14 days. (3)The expression of OPN、OCN and ALP mRNA showed positive at 151bp、155bp and 182bp after osteogenic induction for 7 and 14 days. There were more expression for 14 days.(4)The secretion of collagen type I were positive with immunocytochemistry staining and the Western blotting after osteoinduction for 7 days.The Western blotting verified the expression of collagen type I in the experiment group were obviously higher than that of the control group(P<0.05)Conclusion The hUCMSCs could be osteoinduced to osteoblastic and stably expressed osteoblastic phenotype in vitro. The hUCMSCs could be used to construct bone tissue.Chapter 3 The study on biocompatibility of nano-hydroxyapatite crystals and polyamide 66 (nHA/PA66) material composite with human umbilical cord mesenchymal stem cells in vitroObjective To study the biocompatibility of nHA/PA66 material composite with hUCMSCs and explore the possibility of nHA/PA66 material as scaffold used in the carrier for hUCMSCs.To provide theoretical foundation for further experiments in bone tissue engineering.Methods (1) hUCMSCs were isolated and cultured according to chapterⅠ.(2)The 3rd passage osteoblastic-induced hUCMSCs were seeded in nHA/PA66 material in vitro to construct cells-scaffold composites. We observed the compound situation of cells and the scaffold by the inverted microscope;tested the influence that the scaffold has upon cells multiplication differentiation by MTT.(3) The staining of Alkaline Phophatase(ALP) and collagen type I were verified. The levels of ALP were measured by MTT. (4) The adhesion ratio of nHA/PA66 scaffold composite with the 3rd passage osteoblastic-induced hUCMSCs were detected;The RGR in this scaffold were measured by MTT and counted the cytotoxicity of this material in 2、4、8d.(5) The expression of osteopontin、osteocalcin and alkaline phosphates mRNA were examined by RT-PCR in 7 days.(6) The growth and proliferation of hUCMSCs in this scaffold were observed by scanning electron microscope.Results (1)The 3rd passage osteoblastic-induced hUCMSCs had a good quality of growth, multiplication and differentiation on the surface of nHA/PA66 scaffold.The cellular activity and function were not affected by the materials,and no statistical difference were found between two groups(P>0.05).(2)After compound cultivation,the secretion of ALP and collagen type I were positive with immunocytochemistry staining. The levels of Alkaline Phophatase gradually became higher in 14 days. (3) The adhesion ratio of the 3rd passage osteoblastic-induced hUCMSCs were 69.1±2%、78.4±2%、82%±2% and 91%±2% in the nHA/PA66 scaffold at 4、8、12、24h. (4) The cytotoxicity of this scaffold to cells ranged from zero to one degree and the scaffold were all considered innocuity.(5)The expression of OPN、OCN and ALP mRNA in the experiment group showed positive at 151bp、155bp and 182bp after compound cultivation for 7 days. The RT-PCR verified the expression of OPN、OCN and ALP mRNA in the experiment group were obviously higher than that of the control group(P<0.05).(6)The osteoblastic-induced hUCMSCs adhered and grew on the surface of nHA/PA66 scaffold after one day, The cells extened and proliferation in the nHA/PA66 scaffold were observed by scanning electron microscope after 7 days.Conclusion The nHA/PA66 material could be a good scaffold for hUCMSCs in bone tissue engineering. It was an innocuity、better histocompatibility material and hadn’t a negative impact on the growth、proliferation and osteoinduction of cells.It could satisfy the demand of bone tissue engineering.Chapter 4 The study on ectopic bone formation of nHA/PA66 material composite with human umbilical cord mesenchymal stem cellsObjective To explore the ability of ectopic bone formation of nHA/PA66 material composite with osteoblastic-induced hUCMSCs in nude mouse back and provide theories foundation for further large animal experiments of bone tissue engineering.Methods (1) The 3rd passage osteoblastic-induced hUCMSCs were seeded in nHA/PA66 material in vitro to construct cells-scaffold composites according to chapterⅢ.(2)The osteoblastic-induced hUCMSCs, nHA/PA66 material, nHA/PA66+ osteoblastic-induced hUCMSCs were implanted into the muscular pouch of nude mouse back respectively.All of implant were performed examination of HE staining and immunocytochemical staining of osteocalcin protein to observed the expression after operation 4、8、12 weeks.(3) The composites and products were observed by the scanning electron microscope.Results Neither osteoblastic-induced hUCMSCs nor nHA/PA66 material were sclerotized in the muscular pouch of nude mouse back.The nHA/PA66+ osteoblastic-induced hUCMSCs were maturely sclerotized in large amount,the material partly absorbed.Conclusion The nHA/PA66 material composite with osteoblastic-induced hUCMSCs have a good ability of ectopic bone formation.更多还原