【作者】 何君仁；

【导师】 卫小春；

【作者基本信息】 山西医科大学 ， 骨科学， 2008， 硕士

【摘要】 目的1探讨体外提取分离培养兔骨髓间充质干细胞（BMSC）的方法。2寻求更好的关节软骨生物支架材料,使其在孔隙率,比表面积,弹性模量等物理特性和细胞毒性、溶血试验、生物降解性等生物相容性方面更接近天然软骨支架材料的要求。3探讨异种异体脱细胞软骨支架材料（acellular cartilage material, ACM）复合同种异体兔骨髓间充质干细胞（rabbit bone marrow-derived mesenchymal stem cells, rBMSC）修复兔股骨内髁关节软骨缺损的效果。方法1采用密度梯度离心和差速贴壁法获得兔骨髓间充质干细胞.镜下观察细胞生物学形态及生长状态,细胞计数板下对第1、3、5代细胞计数,绘制细胞生长曲线,细胞表面抗原标记对其进行纯度分析。2①将猪膝关节软骨冻干加工为粉末,胰酶消化,曲拉通洗脱,蒸馏水洗净冻干,紫外线照射（UVI）后成型。电镜观察孔径分布,压汞仪测定孔隙率等参数;②细胞毒性测定:材料浸提液培养细胞进行细胞形态大体观察,MTT法观察细胞活性;③急性全身毒性反应:材料浸提液注射入SD大鼠腹腔观察材料对动物表现及体重变化的影响;④溶血试验:材料浸提液与稀释动物鲜血混合观察红细胞溶解情况,492nm下检测OD值计算相对溶血率;⑤观察细胞复合材料共培养情况并将材料埋植于动物皮下检测材料的生物降解性。3①密度梯度离心和差速贴壁法获得原代兔BMSC。选择第三代BMSC作为种子细胞;②利用冷冻干燥、胰酶消化和化学去垢剂等方法制备脱细胞软骨支架材料;③3月龄新西兰兔内髁制备直径4mm,深3mm动物关节软骨缺损模型,24只新西兰兔以2个时间段随机分为3组,ⅠACM-BMSC组:第三代BMSC 1×10⁶个/mL与ACM于37℃、5%CO₂饱和湿度复合48h;ⅡACM组;Ⅲ空白对照组;④移植6,12周后大体及组织学观察,免疫组化染色观察修复组织Ⅱ型胶原,Wakitani评分评估修复效果。结果1原代培养的BMSC呈圆形、梭形、多角形等,48h可见贴壁细胞有伸展现象,呈梭形,多角形,成纤维细胞样展开,细胞核清晰,14d左右可达90%融合;1、3、5代骨髓间充质干细胞的生长曲线:细胞贴壁48h增殖缓慢,处于潜伏期;对数增殖期为3～4d,第6d后进入平台期;③第2代BMSC CD44表达阳性,标记率为93.0%。2①脱细胞生物支架材料为白色,略微发黄,外表呈多孔疏松网状结构。脆性大,有一定的弹性。孔隙率为68.54%,平均孔径为47.13μm;②细胞毒性试验:24h、48h、72h各时间段内三组细胞OD值两两比较（P>0.05）,无显著差异,ACM细胞毒性为0级;③动物急性毒性实验:Ⅰ生物材料处理组和Ⅱ生理盐水处理组对动物体重影响没有差异（P>0.05）,Ⅰ生物材料处理组和Ⅲ苯酚处理组对动物体重有显著差异（P<0.05）;④溶血试验:材料的相对溶血率为2.92%,低于5%的标准,没有明显溶血现象;⑤材料直接接触试验:细胞嵌入软骨支架材料中,细胞成圆形和椭圆形,部分细胞可见细胞核,材料成颗粒状,染色均一;动物皮下埋植:组织学观察可见试样周围存在少量淋巴细胞和嗜中性粒细胞,致密纤维囊壁将复合细胞的材料包裹,材料被降解为细小颗粒,细胞均匀散在材料中成椭圆形,可见分裂相。3①大体观察及组织学观察:6和12周ⅠACM-BMSC组再生组织与正常关节软骨面平齐,修复部位表面较平整,界限模糊,接近正常软骨。ⅡACM组修复组织表面不平整并有明显下陷,修复组织全层可见成纤维样细胞,深层可见极少数透明软骨样细胞。Ⅲ空白旷置组未见明显修复,肉芽组织形成伴成纤维样细胞增生;②Wakitani组织学评分可见在不同的时间段内Ⅰ和Ⅱ组均低于Ⅲ组,差异有统计学意义（P<0.05）,Ⅰ和Ⅱ组间组织学评分统计学无显著差异（P>0.05）;③免疫组织化学: ACM-BMSC组修复组织的细胞为软骨样细胞,可见柱状排列,周围软骨基质Ⅱ型胶原染色阳性。结论1依据实验方法,BMSC可以较快良好的生长,且纯度较高,为BMSC的研究提供方便的提取和培养方法。2 ACM在孔隙率、比表面积、细胞毒性、溶血实验、动物急性毒性反应、生物降解性方面符合软骨组织工程中对于支架材料的要求,提示支架材料有良好的生物相容性和安全性。3以ACM为支架材料,同种异体BMSC为种子细胞制备的组织工程化软骨对兔股骨内髁关节软骨缺损有修复作用,形成的新生软骨为透明软骨样组织。更多还原

【Abstract】 Objectives1 To investigate the more effective methods of isolation, culture of bone marrow mesenchymal stem cells (BMSC) in vitro.2 To explore the more conformable cartilage biomaterial that conforms to the natural biomaterial in total porosity, total specific surface area, average pore radius, elastic modulus and biocompatibility, such as test for in vitro cytotoxicity, test of hemolysis and biodegradation.3 To investigate the feasibility of repairing the whole layer cartilage defects of medial femoral condyles of adult rabbits with the BMSC/ACM materials.Methods1 Bone marrow stem cells of New Zealand Rabbit were obtained and purified by gradient centrifuge an adhesion culture in vitro. The morphology of BMSC were observed with phase contrast microscope; Drawing the growth curve of BMSC:The cells(P1,P3,P5)were assimilated by trypsin and cultivated in 24一well plate.Three bores were assimilated every day for 8 days, and then growth curve with average was drawn; Identifying the cel1-surface marker:The percentage of the wel1 growth P2 cells were identified by CD44 staining by flow cytometry.2①The articular cartilage of pigs were processed into powder. The components of cells were eliminated by Trypsin, TritonX-100 and distilled water. Finally the powder was freezed out by freezedryer and irradiated by ultraviolet. The microcosmic images of ACM were showed in electron microscope and the parameters of pore radius distribution were measured by sorptomtic Instrument;②Test for in vitro cytotoxicity: The BMSC were cultured in the special L-DMEM that ACM were dipped in and then observed through microscope and MTT;③Test for acute systemic toxicity: The changes of the behavior and the avoirdupois of the SD rats were evaluated after the special L-DMEM was injected into the SD rats by intraperitoneal injection;④Test of hemolysis: The grade of hemolysis was made in the macroscopical phenomenon and the measures of optical density at 492nm;⑤The growth state of the BMSC that immerged into ACM was described. Then the biodegradation of the ACM was estimated after the ACM was hypodermically embedded.3①Bone marrow stem cells of New Zealand Rabbit were obtained and purified by gradient centrifuge and adhesion culture in vitro. The passage 3 BMSC were used for the seeding cells of cartilage tissue engineering;②The acellular pig articular cartilage was prepared by lyophilization, trypsinization and chemical subtraction.③Full thickness empty defects measuring 4 mm in diameter by 3 mm deep were prepared in the medial femoral condyles of 24 3-month-old New Zealand White rabbits, which were randomized to select two groups. Each group was randomized to receive transplantations with ACM-BMSC (8 knees), ACM (8 knees), or no grafts (8 knees) into the cartilage defects.④The repairing effects of the condyles were macroscopically observed and the morphological changes of repaired defects were evaluated 6 and 12 weeks after the operation. The histological scores and the type-Ⅱcollagen immunohistochemistrical stains were carried.Results1①Observing the morphology of BMSC with inverted microscope:Cells showed fiber figure and swirl growth.Primary cultured BMSC were oval,spindle-shaped or polygonal,and adhered to plastic surface within 48h and reached 90% confluence within 14 days.After purification and proliferation,they were uniformly long spindle-shaped form;②Cells growth cure of BMSC;The BMSC were still in latent phase after being adherent to the bottom 2 days;3-4 days later, cells were in log phase;6 days later,and cells came into platform phase;③The result of the cell-surface markers:Cell membranes were colored up evidently after CD44 staining and CD44 of the passage 2 BMSC positive rate were 93%.2①The surface of the pale yellow ACM was porous and netty and the bouncy ACM was fragile. The results showed that the Average pore radius was 47.13μm and the Total porosity came to 68.54%;②Test for in vitro cytotoxicity: At every time interval, the OD scores of three groups were not significant differences (P>0.05).The grade of the cytotoxicity of the ACM was 0;③Test for acute systemic toxicity: The avoirdupois scores of both groupⅠandⅡwere not statistically significant differences (P>0.05), but the differences between groupⅠandⅢwere statistically significant(P<0.05);④Test of hemolysis: Hemolysis Percentage was 2.92%;⑤Observing the morphology of BMSC in the ACM with inverted microscope:BMSC were oval and nucleoli appeared in a few cells. And the ACM were decomposed into particulates; the histological observation after the ACM was hypodermically embedded: There were few lymphocytes and polymorphonuclear leucocyte around the ACM. And the ACM were closed in by compact fibrosis wall and were decomposed into small parts.3①In the 6th and 12th week after the operation, the morphology, distribution and arrangement of the regenerated tissues were similar to normal cartilage in the knees with ACM-BMSC transplantation, and the regenerated tissues grew to be integrated with the surrounding normal cartilage with obscure boundary between them. In the ACM group, the rough surface of regenerated tissue sunk obviously and the fibroblasts in all layer and the few chondrocytes in the deep layer were found. While the thin reddish grey layer of soft granulation tissue formed in the defect and the fibroblasts increased in non–transplantation group;②At every time interval, the histological scores of groupⅢbased on Wakitani scoring excelled both groupⅠandⅡwith statistically significant differences(P<0.05),but the differences betweenⅠandⅡwere not significant(P>0.05);③Immunohistochemistrical stains showed that cells in the zones of repaired tissues were abundant, arranged columnedly and the typeⅡcollagen staining was positive.Conclusions1 BMSC were easily isolated and cultured in vitro and proliferate prosperously, and more purified. The protocol should make it possible to undertake.2 ACM accords with the biomaterial standards of cartilage tissue engineering in Total porosity, Total specific surface area, test for in vitro cytotoxicity, test of hemolysis, tests for acute systemic toxicity and biodegradation.3 Tissue-engineering cartilage based on BMSC seeded into ACM can repair the defects of the whole layer cartilage defects of medial femoral condyles of rabbits. The repair tissue was confirmed to be hyaline cartilage.更多还原