TGF-β3、BMP-2双基因转染的骨髓基质干细胞联合DBM修复关节软骨缺损的实验研究
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摘要
[目的]探讨同种异体脱钙骨基质(DBM)作为软骨组织工程支架材料的可行性；探索DBM与TGF-β3和BMP-2双基因转染的骨髓基质干细胞(BMSCs)体外复合培养后的最佳移植时机并观察DBM复合双基因转染的BMSCs修复滇南小耳猪膝关节软骨缺损的效果,以明确基因增强组织工程技术修复关节软骨缺损的可行性。
[方法] 1.根据Urist描述的方法制得同种异体DBM,扫描电镜观察DBM的超微结构,在体外将TGF-β3、BMP-2双基因转染的滇南小耳猪BMSCs,与制备好的DBM复合培养,MTT法测定BMSCs在DBM上生长增殖曲线,并以扫描电镜观察BMSCs在DBM上的增殖情况。
2.制备滇南小耳猪膝关节软骨全层缺损的动物模型,将BMSCs与DBM复合培养后植入小耳猪膝关节软骨缺损模型中。选用健康成年滇南小耳猪8头,制造双侧膝关节软骨缺损,同时设四组：右膝股骨外髁关节负重面为实验组A组(DBM／双基因转染的BMSCs);右膝股骨内髁关节负重面为对照组B组(DBM／未经转染的BMSCs);左膝股骨内髁关节负重面为空白支架组C组,(单纯植入DBM,不含BMSCs);左膝股骨外髁关节负重面为空白组D组(单纯空白缺损)。于术后2周、4周、8周及12周各处死2只小耳猪,取材对修复组织进行大体、组织学及免疫组化染色观察,根据O'driscoll法对修复组织进行评分,数据输入SPSS11.5软件进行统计学分析,比较各组的评分差异是否具有统计学意义。
[结果] 1.DBM支架材料外观均呈白色海绵状,SEM观察DBM呈天然的高密度孔隙网架结构；双基因转染的BMSCs能在DBM上附着生长并增殖,且复合培养第6天时达到峰值。
2.术后12周A组修复组织呈透明样软骨,表面光滑平坦,与周围软骨及软骨下骨结合良好；B组呈透明软骨样,表面光滑,界限仔细可辨；C组有少部分类软骨样修复；D组纤维性修复,凹陷明显。实验组A组术后12周Ⅱ型胶原免疫组化染色呈阳性,术后8周、12周检测TGF-β3和BMP-2蛋白表达呈阳性。修复组织O'driscoll评分结果显示：2周时,各组无明显统计学差异,从4周起,各时间点实验组评分与实验对照组和空白组评分间均具有统计学差异,即实验组修复效果优于实验对照组和空白对照组,实验对照组优于空白对照组；实验组A组四个时间点评分间均有统计学差异,即12周时修复效果优于8周及4周,8周时修复效果优于4周,4周时修复效果优于2周。
[结论] 1.DBM具有三维天然网状结构和良好的细胞相容性,DBM与双基因转染的BMSCs复合培养后最佳移植时间为第6天。
2.应用基因增强组织工程学技术,将双基因转染的BMSCs复合DBM支架材料修复全层关节软骨缺损是可行的,为临床运用基因治疗软骨缺损提供实验依据。
[Objective] To observe the possibility of using Demineralized bone matrix as a scaffold for cartilage tissue engineering, determine the optimal co-culture time for implantation, and the feasibility of articular cartilage repair of Diannan small-ear pigs with bone marrow stromal cells transfected by adenovirus vector mediated TGF-β3 and BMP-2 seeded on demineralized bone matrix.
[Methods] 1.Demineralized bone matrix was made according to Urist's method. The Ultra structure of DBM was observed with scanning electron microscope (SEM). Bone marrow stromal cells transfected TGF-β3 and BMP-2 were cultured with DBM, and MTT assay was used to analyze the growth curve of Bone marrow stromal cells on DBM. The growth of Bone marrow stromal cells on DBM was also observed with SEM.
2. Prepared full thickness defects both knee joints of Diannan small-ear pig. The DBM/BMSCs transfected TGF-β3 and BMP-2 was co-cultured and transplanted into full thickness defects on femoral condyle of both knee joints. Eight adult Diannan small-ear pigs were used and divided into four groups.Group A is the weight-bearing articular surface of lateral femoral condyle of the right knee (DBM/BMSCs transfected by two genes). Group B is the weight-bearing articular surface of medial femoral condyle of the right knee (DBM/BMSCs). Group C is the weight-bearing articular surface of medial femoral condyle of the left knee (only DBM).Group D is the weight-bearing articular surface of lateral femoral condyle of the left knee(empty defects). Two Diannan small-ear pigs were killed at 2th,4th,8th and 12th week after the operation, and the reparative tissue samples were evaluated grossly, histologically, immunohistochemically and evaluate the repairing effect by O'driscoll's score system. SPSS 11.5 software was applied to proceed the statistic analysis.
[Results] 1.The DBM scaffold was white three-dimensional cancellous feature which had great biocompatibility, could promote cell adhesion and proliferation. So it should be a good scaffold for tissue-engineered cartilage. SEM showed the holes were connected each other, BMSCs transfected by two genes co-cultured with the DBM ampilified,which had the peak until the sixth day.
2. Defective regions of group A were repaired by hyaline cartilage at 4 weeks after transplantation and were barely recognizable with natural articular cartilage at 12 weeks.TypeⅡcollagenase immunohistochemistry stain expression was positive after 12 weeks.The positive reaction was showed in TGF-β3,BMP-2 immunohistochemistry stain after 8、12 weeks.
[Conclusion] 1. The DBM scaffold had a three-dimensional structure of tissue engineering scaffold. DBM could promote the proliferation of target cells. So it should be a good scaffold for tissue-engineered cartilage.
2. DBM/BMSCs transfected by two genes could be an efficient alternative for articular cartilage defects repair.
[方法] 1.根据Urist描述的方法制得同种异体DBM,扫描电镜观察DBM的超微结构,在体外将TGF-β3、BMP-2双基因转染的滇南小耳猪BMSCs,与制备好的DBM复合培养,MTT法测定BMSCs在DBM上生长增殖曲线,并以扫描电镜观察BMSCs在DBM上的增殖情况。
2.制备滇南小耳猪膝关节软骨全层缺损的动物模型,将BMSCs与DBM复合培养后植入小耳猪膝关节软骨缺损模型中。选用健康成年滇南小耳猪8头,制造双侧膝关节软骨缺损,同时设四组：右膝股骨外髁关节负重面为实验组A组(DBM／双基因转染的BMSCs);右膝股骨内髁关节负重面为对照组B组(DBM／未经转染的BMSCs);左膝股骨内髁关节负重面为空白支架组C组,(单纯植入DBM,不含BMSCs);左膝股骨外髁关节负重面为空白组D组(单纯空白缺损)。于术后2周、4周、8周及12周各处死2只小耳猪,取材对修复组织进行大体、组织学及免疫组化染色观察,根据O'driscoll法对修复组织进行评分,数据输入SPSS11.5软件进行统计学分析,比较各组的评分差异是否具有统计学意义。
[结果] 1.DBM支架材料外观均呈白色海绵状,SEM观察DBM呈天然的高密度孔隙网架结构；双基因转染的BMSCs能在DBM上附着生长并增殖,且复合培养第6天时达到峰值。
2.术后12周A组修复组织呈透明样软骨,表面光滑平坦,与周围软骨及软骨下骨结合良好；B组呈透明软骨样,表面光滑,界限仔细可辨；C组有少部分类软骨样修复；D组纤维性修复,凹陷明显。实验组A组术后12周Ⅱ型胶原免疫组化染色呈阳性,术后8周、12周检测TGF-β3和BMP-2蛋白表达呈阳性。修复组织O'driscoll评分结果显示：2周时,各组无明显统计学差异,从4周起,各时间点实验组评分与实验对照组和空白组评分间均具有统计学差异,即实验组修复效果优于实验对照组和空白对照组,实验对照组优于空白对照组；实验组A组四个时间点评分间均有统计学差异,即12周时修复效果优于8周及4周,8周时修复效果优于4周,4周时修复效果优于2周。
[结论] 1.DBM具有三维天然网状结构和良好的细胞相容性,DBM与双基因转染的BMSCs复合培养后最佳移植时间为第6天。
2.应用基因增强组织工程学技术,将双基因转染的BMSCs复合DBM支架材料修复全层关节软骨缺损是可行的,为临床运用基因治疗软骨缺损提供实验依据。
[Objective] To observe the possibility of using Demineralized bone matrix as a scaffold for cartilage tissue engineering, determine the optimal co-culture time for implantation, and the feasibility of articular cartilage repair of Diannan small-ear pigs with bone marrow stromal cells transfected by adenovirus vector mediated TGF-β3 and BMP-2 seeded on demineralized bone matrix.
[Methods] 1.Demineralized bone matrix was made according to Urist's method. The Ultra structure of DBM was observed with scanning electron microscope (SEM). Bone marrow stromal cells transfected TGF-β3 and BMP-2 were cultured with DBM, and MTT assay was used to analyze the growth curve of Bone marrow stromal cells on DBM. The growth of Bone marrow stromal cells on DBM was also observed with SEM.
2. Prepared full thickness defects both knee joints of Diannan small-ear pig. The DBM/BMSCs transfected TGF-β3 and BMP-2 was co-cultured and transplanted into full thickness defects on femoral condyle of both knee joints. Eight adult Diannan small-ear pigs were used and divided into four groups.Group A is the weight-bearing articular surface of lateral femoral condyle of the right knee (DBM/BMSCs transfected by two genes). Group B is the weight-bearing articular surface of medial femoral condyle of the right knee (DBM/BMSCs). Group C is the weight-bearing articular surface of medial femoral condyle of the left knee (only DBM).Group D is the weight-bearing articular surface of lateral femoral condyle of the left knee(empty defects). Two Diannan small-ear pigs were killed at 2th,4th,8th and 12th week after the operation, and the reparative tissue samples were evaluated grossly, histologically, immunohistochemically and evaluate the repairing effect by O'driscoll's score system. SPSS 11.5 software was applied to proceed the statistic analysis.
[Results] 1.The DBM scaffold was white three-dimensional cancellous feature which had great biocompatibility, could promote cell adhesion and proliferation. So it should be a good scaffold for tissue-engineered cartilage. SEM showed the holes were connected each other, BMSCs transfected by two genes co-cultured with the DBM ampilified,which had the peak until the sixth day.
2. Defective regions of group A were repaired by hyaline cartilage at 4 weeks after transplantation and were barely recognizable with natural articular cartilage at 12 weeks.TypeⅡcollagenase immunohistochemistry stain expression was positive after 12 weeks.The positive reaction was showed in TGF-β3,BMP-2 immunohistochemistry stain after 8、12 weeks.
[Conclusion] 1. The DBM scaffold had a three-dimensional structure of tissue engineering scaffold. DBM could promote the proliferation of target cells. So it should be a good scaffold for tissue-engineered cartilage.
2. DBM/BMSCs transfected by two genes could be an efficient alternative for articular cartilage defects repair.
引文
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1 Jadlowiec JA,Celil AB,Hollinger JO.Bone tissue engineering advances and promising therapeutic agents.Expert Opin Biol Ther,2003,3(3):409-423.
2 Martin J Stoddart,Sibylle Grad,David Eglin Mauro Alini. Cells and biomaterials in cartilage tissue engineering.Regen Med,2009,4(1):81-98.
3 Joanne Raghuriatch,John Rollo,Kevin M.Sales,Biomaterials and scaffold design:Key to tissue-engineering cartilage.Blotechnol.Appl.Blochem.2007,(46):73-78.
4韩雪峰,杨大平,郭铁芳,等.纤维蛋白胶塑形、异体微粒软骨脱细胞基质支架的人工软骨研究.中国临床康复,2005,9(2)：56-58.
5 Fox DB,Cook JL,Arnoczky SP,et al.Fibrochondrogenesis of free intra articular small intestinal submucosa scaffolds.Tissue Eng 2004,10(1-2):129-137.
6鄂征,刘流.医学组织工种技术与临床应用[M].北京出版社,2003.
7 Cima L QVacanti J P,Vacanti C, et al. Tissue engineering by cell transplantation using degradable polymer substrates. J Biomech Eng,1991 (5):123-143.
8 颜文龙,孙恩杰,郭海英.组织工程支架材料.上海生物医学工程,2004,25(1)：51.
9 Wefer J,Schlote N,Sekido N,et al.Tunica albuginea acellular matrix graft for penile reconstruction in the rabbit:a model for treating Peyronie's disease.BJU Int,2002,90(3):326-331.
10 Kajbafzadeh AM,Payabvash S,Salmasi AH,et al.Time-dependent neovasculogenesis and regeneration of different bladder wall components in the bladder acellular matrix graft in rats.J Surg Res,2007,139(2):189-202.
11 郑锦标,梁杰,张培华.脱细胞真皮基质的制备、作用机理、免疫原性及其在整形外科中的应用现状.广东医学院学报,2008,26(2)：208-210.
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