关节腔内骨髓间充质干细胞与同种异体骨的共培养
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摘要
背景:构建组织工程软骨的时候,同种异体骨与骨髓间充质干细胞是常用的支架材料和种子细胞,以往大多采用体外培养的方式。膝关节内游离体可以长期存在于关节腔中,并维持一定的软骨组织学特性。因此,关节腔可能为软骨细胞的生长和发育提供良好的环境条件。目的:探讨在关节腔内骨髓间充质干细胞与同种异体骨复合培养的效果。方法:纳入5只新西兰新生白兔进行骨髓间充质干细胞分离与培养,利用1只成年新西兰白兔制备同种异体骨,进行骨髓间充质干细胞与同种异体骨复合。实验分组:腔内培养组将骨髓间充质干细胞与同种异体骨复合物培养于动物关节腔内,正常对照组设为同腔内正常软骨,体外培养组实施常规体外培养。培养4,8,12周进行组织学苏木精-伊红染色观察及Ⅱ型胶原免疫组织化学染色观察。结果与结论:1培养12周进行苏木精-伊红染色和观察,正常对照组细胞质和软骨基质出现红染,细胞核出现蓝染,软骨细胞按照一定的方向呈紧密状有序排列。腔内培养组细胞质和软骨基质出现红染,细胞核出现蓝染,支架材料基本吸收。软骨细胞长入支架之中,细胞按照一定应力方向排列,且形态变小。体外培养组软骨细胞出现大量增殖,但呈无序状排列;2经免疫组织化学染色和观察,计数可得,随着培养时间的推移,腔内培养组的A值呈现出不断上升的情况,体外培养组和正常对照组均未出现明显的改变。且培养4,8,12周,正常对照组和腔内培养组的A值均显著高于体外培养组(P<0.05)。培养4,8周,腔内培养组的A值均显著低于正常对照组(P<0.05)。但培养12周,腔内培养组与正常对照组A值差异无显著性意义(P>0.05);3实验结果表明,在体外和关节腔内环境下,均可以利用同种异体骨与骨髓间充质干细胞复合培养获得组织工程软骨,且关节腔内培养可以获得更好的培养效果。
BACKGROUND: Bone marrow mesenchymal stem cells and allogenic bones are commonly used as seed cells and scaffolds, respectively, for constructing tissue-engineered cartilage through in vitro co-culture. The loose body of the knee joint can survive in the articular cavity for a long time, and maintain certain characteristics of cartilage tissues. Therefore, the articular cavity can provide a good environment for the growth and development of chondrocytes. OBJECTIVE: To investigate the effect of bone marrow mesenchymal stem cells co-cultured with allogenic bone in the articular cavity. METHODS: Bone marrow mesenchymal stem cells were isolated from five newborn New Zealand white rabbits. One adult New Zealand rabbit was enrolled to prepare allogenic bone for co-culture with bone marrow mesenchymal stem cells. Afterwards, bone marrow mesenchymal stem cells and allogenic bone composites were cultured in the articular cavity(intracavitary culture group) or in vitro as in vitro culture group, respectively; the normal cartilage tissues grew in the articular cavity as control group. Cells were observed by hematoxylin-eosin staining and type II collagen immunohistochemistry staining at 4, 8 and12 weeks of culture. RESULTS AND CONCLUSION: At 12 weeks culture, hematoxylin-eosin staining showed: in the control group, chondrocytes arranged tightly and directionally with red stained cytoplasm and cartilage matrix as well as blue nuclei; in the intracavitary culture group, the scaffold was mostly absorbed and chondrocytes grew into the scaffold in a certain direction with smaller shape, while cytoplasm and cartilage matrix were red stained, blue nuclei appeared; in the in vitro culture group, abundant chondrocytes proliferated in a disordered arrangement. Immunohistochemistry staining showed: the absorbance(A) values in the intracavitary culture group showed a continuous increase, but no obvious change was in the other two groups. Moreover, at 4, 8 and 12 weeks of culture, A values in the control group and intracavitary culture group were significantly higher than that in the in vitro culture group(P < 0.05); at 4 and 8 weeks, A value in the intracavitary culture group was significantly lower than that in the control group(P < 0.05), but at 12 weeks, there was no significant difference in A value between the two groups(P > 0.05). These results suggest that the tissue-engineered cartilage can be constructed by bone marrow mesenchymal stem cells co-cultured with allogenic bone under in vitro and in vivo environment, especially in the articular cavity.
BACKGROUND: Bone marrow mesenchymal stem cells and allogenic bones are commonly used as seed cells and scaffolds, respectively, for constructing tissue-engineered cartilage through in vitro co-culture. The loose body of the knee joint can survive in the articular cavity for a long time, and maintain certain characteristics of cartilage tissues. Therefore, the articular cavity can provide a good environment for the growth and development of chondrocytes. OBJECTIVE: To investigate the effect of bone marrow mesenchymal stem cells co-cultured with allogenic bone in the articular cavity. METHODS: Bone marrow mesenchymal stem cells were isolated from five newborn New Zealand white rabbits. One adult New Zealand rabbit was enrolled to prepare allogenic bone for co-culture with bone marrow mesenchymal stem cells. Afterwards, bone marrow mesenchymal stem cells and allogenic bone composites were cultured in the articular cavity(intracavitary culture group) or in vitro as in vitro culture group, respectively; the normal cartilage tissues grew in the articular cavity as control group. Cells were observed by hematoxylin-eosin staining and type II collagen immunohistochemistry staining at 4, 8 and12 weeks of culture. RESULTS AND CONCLUSION: At 12 weeks culture, hematoxylin-eosin staining showed: in the control group, chondrocytes arranged tightly and directionally with red stained cytoplasm and cartilage matrix as well as blue nuclei; in the intracavitary culture group, the scaffold was mostly absorbed and chondrocytes grew into the scaffold in a certain direction with smaller shape, while cytoplasm and cartilage matrix were red stained, blue nuclei appeared; in the in vitro culture group, abundant chondrocytes proliferated in a disordered arrangement. Immunohistochemistry staining showed: the absorbance(A) values in the intracavitary culture group showed a continuous increase, but no obvious change was in the other two groups. Moreover, at 4, 8 and 12 weeks of culture, A values in the control group and intracavitary culture group were significantly higher than that in the in vitro culture group(P < 0.05); at 4 and 8 weeks, A value in the intracavitary culture group was significantly lower than that in the control group(P < 0.05), but at 12 weeks, there was no significant difference in A value between the two groups(P > 0.05). These results suggest that the tissue-engineered cartilage can be constructed by bone marrow mesenchymal stem cells co-cultured with allogenic bone under in vitro and in vivo environment, especially in the articular cavity.
引文
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[2]王斌,吕浩然,王伟雄,等.异体皮质人工骨复合物治疗骨折不愈合及骨缺损[J].中国组织工程研究,2013,17(51):8821-8826.
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[4]Lee JY,Choi MH,Shin EY,et al.Autologous mesenchymal stem cells loaded in Gelfoam for structural bone allograft healing in rabbits.Cell Tissue Bank.2011;12(4):299-309.
[5]Dong JL,Li LX,Mu WD,et al.Bone Regeneration with BMP-2 Gene-modified Mesenchymal Stem Cells Seeded on Nano-hydroxyapatite/Collagen/Poly(L-Lactic Acid)Scaffolds.J Bioact Compat Polymer.2010;25(6):547-566.
[6]朱肖奇,贺用礼,马雪峰,等.生物衍生骨与骨髓间充质干细胞复合修复免桡骨大段缺损[J].中国组织工程研究与临床康复,2009,13(3):417-422.
[7]Tran CT,Gargiulo C,Thao HD,et al.Culture and differentiation of osteoblasts on coral scaffold from human bone marrow mesenchymal stem cells.Cell Tissue Bank.2011;12(4):247-261.
[8]Reinders ME,de Fijter JW,Roelofs H,et al.Autologous bone marrow-derived mesenchymal stromal cells for the treatment of allograft rejection after renal transplantation:Results of a phase I study.Stem Cells Trans Med.2013;2(2):107-111.
[9]江标,李明.骨组织工程中骨髓间充质干细胞与细胞因子的研究进展[J].重庆医学,2006,35(24):2290-2293.
[10]黄相杰,姜红江,孟鹏,等.自体间充质干细胞移植异体韧带重建治疗膝交叉韧带损伤[J].中国实用医药,2011,6(36):92-95.
[11]Itakura S,Asari S,Rawson J,et al.Mesenchymal stem cells facilitate the induction of mixed hematopoietic chimerism and islet allograft tolerance without GVHD in the rat.Am J Trans.2007;7(2):336-346.
[12]Tang TT,Lu H,Dai KR,et al.Osteogenesis of freeze-dried cancellous bone allograft loaded with autologous marrow-derived mesenchymal cells.Mat Sci Eng.2002;20(1/2):57-61.
[13]刘彩云.骨髓间充质干细胞联合异体骨治疗蒙古羊胫骨缺损的实验研究[D].内蒙古农业大学,2014.
[14]刘阳.富集骨髓间充质干细胞复合n HA/CS材料修复兔骨缺损的实验研究[D].南方医科大学,2011.
[15]王宏彬.同种异体骨髓间充质干细胞的研究现状[J].中国组织工程研究,2012,16(23):4306-4309.
[16]杨楠,何惠宇,胡杨,等.复合骨髓间充质干细胞同种异体支架骨修复羊髂骨极限缺损[J].中国组织工程研究,2013,17(16):2859-2868.
[17]刘晓阳,李广润,刘洪涛,等.硫酸钙人工骨/骨髓间充质干细胞构建组织工程化骨诱导脊柱融合[J].中国组织工程研究,2014,18(21):3281-3286.
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[19]Khojasteh A,Eslaminejad MB,Nazarian H,et al.Vertical bone augmentation with simultaneous implant placement using particulate mineralized bone and mesenchymal stem cells:a preliminary study in rabbit.J Oral Implantol.2013;39(1):3-13.
[20]Wei X,Mao Z,Hou Y,et al.Local administration of TGFbeta-1/VEGF165 gene-transduced bone mesenchymal stem cells for Achilles allograft replacement of the anterior cruciate ligament in rabbits.Biochem Biophys Res Comm.2011;406(2):204-210.
[21]谭新颖.同种异体骨髓间充质干细胞复合同种异体冻干骨修复比格犬半侧下颌骨缺损的实验研究[D].中国人民解放军医学院,2014.
[22]邢志远,张继波,孔令菊,等.深低温冻存羟基磷灰石/骨髓间充质干细胞修复兔桡骨缺损[J].中国组织工程研究,2013,17(25):4629-4636.
[23]刘昌奎,谭新颖,徐娟,等.骨髓间充质干细胞复合同种异体骨支架体内构建组织工程骨修复髁突缺损的实验研究[C].//2013年全国第五届口腔颌面外科修复重建学术会议暨国际研讨会论文集.2013:54-55.
[24]Vulcano E,Murena L,Falvo DA,et al.Bone marrow aspirate and bone allograft to treat acetabular bone defects in revision total hip arthroplasty:Preliminary report.Eur Rev Med Pharmacol Sci.2013;17(16):2240-2249.
[25]Kuo YR,Chen CC,Shih HS,et al.Prolongation of composite tissue allotransplant survival by treatment with bone marrow mesenchymal stem cells is correlated with T-cell regulation in a Swine hind-limb model.Plastic Reconstruct Surg.2011;127(2):569-579.
[26]Nather A,David V,Teng JW,et al.Effect of autologous mesenchymal stem cells on biological healing of allografts in critical-sized tibial defects simulated in adult rabbits.Ann Acad Med.2010;39(8):599-606.
[27]刘昌奎,谭新颖,徐娟,等.骨髓间充质干细胞复合同种异体骨支架体内构建组织工程骨修复髁突缺损的实验研究[C].//第五届全国口腔颅颌面修复重建外科学术会议暨国际研讨会论文集.2013:54-55.
[28]李涛,宋国栋,包崇云,等.骨髓间充质干细胞作为干细胞参与生物材料异位诱导成骨[J].中国组织工程研究与临床康复,2010,14(21):3819-3822.
[29]费志强.BMP-2基因修饰的骨髓间充质干细胞复合PLLA/n-HA人工骨修复大鼠股骨缺损的实验研究[D].中山大学,2013.
[30]李光辉.外周血CD34+细胞与骨髓间充质干细胞共培养修复兔颅骨缺损的实验研究[D].第四军医大学,2013.
[31]王小志.基因转染骨髓间充质干细胞复合同种异体骨修复羊髂骨极限缺损[D].新疆医科大学,2014.
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