摘要
背景:骨髓间充质干细胞具有自我更新及多向分化的能力,经诱导后能够向成骨细胞分化,与基质材料结合可以具备形态功能良好的骨缺损修复效应。目的:观察骨髓间充质干细胞与基质材料复合后同体移植修复节段性骨缺损的成骨性能。设计:开放性实验。单位:无锡市第三人民医院细胞实验室。材料:实验于2004-05/2005-03在无锡市第三人民医院细胞实验室完成。选取清洁级6月龄新西兰兔18只,随机数字表法分为细胞支架复合组、单纯支架组、空白对照组,6只/组。支架材料由清华大学材料系生物组提供纳米晶羟基磷灰石胶原材料,具备天然骨微结构特征(组成:羟基磷灰石约57%,胶原约30%,聚乳酸约13%)。方法:①分离培养兔骨髓间充质干细胞,取生长良好的第2代细胞应用EPICS-ALTRA流式细胞仪检测细胞表面抗原表达。②支架材料按实验需求切割成6mm×6mm×4mm大小,60Co辐照灭菌,使用前DMEM-LG浸润。收集培养两三代的细胞,调整浓度为109L-1,与纳米晶羟基磷灰石胶原材料于体外联合培养。③各组均建立兔桡骨节段性缺损模型。细胞支架复合组将已备好的复合物采用同体移植的原则嵌入骨缺损中,单纯支架组将空白支架材料嵌入骨缺损中,空白对照组不植入任何材料。各组动物均不作内外固定,严密缝合软组织、皮肤。④扫描电镜下观察各组材料表面结构及细胞附着情况。⑤各组分别于术后4,8,16周行大体观察、组织学分析及X光摄片,了解成骨情况。主要观察指标:①骨髓间充质干细胞分离培养及鉴定情况。②各组扫描电镜观察结果。③术后各组放射学检测结果。④术后各组大体形态观察结果。⑤术后各组组织学检测结果。结果:实验选取清洁级新西兰兔18只,全部进入结果分析。①原代培养4h后可见有贴壁细胞,静置培养48h后贴壁细胞呈克隆样生长,细胞多为不规则鳞片状,胞体大,核居中。流式细胞仪检测可见CD29,CD44,CD105呈阳性,3种阳性细胞比率分别为97.4%,98.1%,86.2%。②单纯支架组表面不规则,孔隙较多。细胞支架复合组材料表面及孔隙内均有细胞附着。③术后4,8,16周各时间点细胞支架复合组X线片吸光度均高于单纯支架组、空白对照组,且16周时细胞支架复合组可见骨性愈合。④术后16周细胞支架复合组骨痂已能环包植入物,单纯支架组植入物中段仍有部分未被骨组织覆盖,空白对照组没有成型骨痂形成。⑤细胞支架复合组术后各时间点的成骨能力均强于单纯支架组,且16周时有骨小梁形成,成骨细胞排列有序。单纯支架组骨组织大多集中于材料表面,材料吸收少。空白对照组缺损近端有少许新生骨样组织形成,中段则未见。结论:兔骨髓间充质干细胞在体外可以大量增殖,与纳米晶羟基磷灰石胶原材料复合培养具有很强的成骨作用,是一种较好的组织工程种子细胞。
BACKGROUND： Bone bone bone marrow derived mesenchymal stem cells （MSCs） has the capacities of self-renewal and multi-directional differentiation, which can differentiate into osteoblasts after induction. Combined with basal hydroxyapatite materials, MSCs can repair the bone defeet with satisfactory, shape and function.
OBJECTIVE： To observe the bone formation performance of segmental bone defect repaired by autologous transplantation after combination of MSCs and basal hydroxyapatite materials.
DESIGN： Open experiment.
SETTING： Department of Cells, Third People＇s Hospital of Wuxi.
MATERIALS： The experiment was conducted in the Department of Cells, Third People＇s Hospital of Wuxi between May 2004 and March 2005. Eighteen New Zealand rabbits of clean grade, aged 6 months were selected and randomly divided into cytoskcleton group, simple scaffold group and blank control group with 6 rabbits in each group. Nano-collagen basal hydroxyapatite bone were provided by the Department of Material, Tsinghua University, which is characteristic of natural-bone microstrueture. Composition： about 57% were hydroxyapatite,and 30 %. were collagen and 13% were polylactic acid.
METHODS： （1）bone bone marrow derived MSCs of rabbits were isolated and cultured. Well-grown cells of the second generation were inspected of the expression of cell surface antigen （CSA） with EPICS-ALTRA flow cytometry. （2）Scaffold materials were cut into blocks with the size of 6 mm ×6 mm×4 mm, sterilized by ^60Co irradiance and infihrated with DMEM-LG before using. Cells of the 2^ndl and 3^rd generations were collected, cultured, the concentration of which was regulated to 10^9 L^-1. Cells were co-cultured with nano-eollagen basal hydroxyapatite in vitro. （3）Model of segmental defects in radial of rabbits were established in all groups. The prepared compounds in the eytoskeleton compound group were embedded in the bone defect according to autograft principle, and blank scaffold materials were embedded in the bone defects of simple scaffold group. Nothing was transplanted in the blank control group. Rabbits in all groups received no internal or external fixation, whose soft tissues and skins were closely sutured. （4）The surface structure of materials and cell adherence in all groups were observed under scanning electron microscope （SEM）.（5）Bone formation was studied by general observation, histological analysis and X-rays at 4, 8 and 16 weeks after operation respectively.
MAIN OUTCOME MEASURES： （1）The isolated culture and identification of MSCs.（2）Observation of all groups under SEM.（3）Results of radioiogical inspection of all groups. （4）General morpbous of cells in each group after operation.（5）Results of histological inspection of all groups after operation.
RESULTS： A total of 18 enrolled New Zealand rabbits were involved in the analysis of results.（1）There were adherent cells at 4 hours after primary. culture, which were like cloned at 48 hours after static culture. Cells were in irregular scale-shape with large cell body and the nucleus was in middle. Inspection with flow cytometer showed that CD29,CD44,CD-105 were positive, and the ratios of three positive cells were 97.4% ,98.1% and 86.2% respectively. （2）The surface of simple scaffold group was irregular with many ventages. There were cells adherent to the surface of materials as well as the inside of ventage in the eytoskeleton compound group.（3）The absorbance of X-rays at 4. 8 and 16 weeks after operation were higher in the cytoskeleton compound group than blank control group and simple scaffold group, moreover, bone union eould be Seen at the 16^th week in the eytoskeleton compound group. （4）The implants was embedded in the osteotylus at 16 weeks after operation in the cytoskeleton compound group, while parts of the middle segment of implants in the simple scaffold group was not covered by bone tissues. No porosis was found in the blank control group.（5）The ossifying capacity at each time point after operation was better in the cytoskeleton compound group than simple scaffold group, moreover, there was bone trabecula formed at the 16^th week, and the osteoblasts were arranged in order. The bone tissues in the simple scaffold group mainly concentrated on the surface of materials, which sucked little. There were some bony tissues formed in the proximal end of defects in the blank control group, while none was seen in the middle parts.
CONCLUSION： Rabbit MSCs can greatly proliferate in vitro, which has strong osteogenesis by being co-cultured with nano-collagen basal hydroxyapatite. It is a good kind of seed cells in tissue engineering,
出处
《中国临床康复》
CSCD
北大核心
2006年第41期195-197,F0003,共4页
Chinese Journal of Clinical Rehabilitation
基金
无锡市社会发展项目资助(CS20020004)~~
