CAJ | 학술논문

背景:诱导因子及软骨微环境是影响骨髓基质干细胞成软骨分化及软骨形成的主要因素.目的:利用少量软骨细胞以共培养方式诱导骨髓基质干细胞体内成软骨.设计、时间及地点:2004-09/2005-03在解放军第四军医大学口腔医院病理教研室完成的随机对照动物实验.材料:选择清洁级新西兰兔15只用于细胞支架复合物的种植,随机分为混合细胞组、软骨细胞组、骨髓基质干细胞组,5只/组.取新生1～3 d龄新西兰兔5只用于骨髓基质干细胞、软骨细胞的分离培养.聚羟基乙酸无纺网支架为上海易括公司产品,材料直径15 μm,平均间距150～200 μm,孔隙率97%,厚2 mm.方法:混合细胞组将骨髓基质干细胞与软骨细胞按3:1混匀,调整细胞密度为6.0×1010 L-1,接种于经培养液预湿的塑形聚羟基乙酸 5 mm×5 mm的支架上,然后在复合物周围滴加含胎牛血清的DMEM液培养1周.软骨细胞组、骨髓基质干细胞组的细胞密度调整为6.0×1010 L-1后同法接种于支架上.各组兔麻醉后于背部一侧皮下组织植入对应的细胞-支架复合物.主要观察指标:植入后第8周行新生软骨大体观察和苏木精-伊红、Masson三色染色.结果:各组细胞与聚羟基乙酸支架黏附情况良好.植入8周后,混和培养组和软骨细胞组均形成了成熟的软骨样组织,并基本保持复合物初始的大小和形状,两组新生软骨外观及组织学特征较接近.骨髓基质干细胞组在体内培养过程中未形成软骨组织,而是形成了纤维结缔组织.结论:骨髓基质干细胞与软骨细胞按3:1混合形成的微环境,能有效诱导骨髓基质干细胞向软骨细胞分化,并促进骨髓基质干细胞的体内成软骨.
배경:유도인자급연골미배경시영향골수기질간세포성연골분화급연골형성적주요인소.목적:이용소량연골세포이공배양방식유도골수기질간세포체내성연골.설계、시간급지점:2004-09/2005-03재해방군제사군의대학구강의원병리교연실완성적수궤대조동물실험.재료:선택청길급신서란토15지용우세포지가복합물적충식,수궤분위혼합세포조、연골세포조、골수기질간세포조,5지/조.취신생1～3 d령신서란토5지용우골수기질간세포、연골세포적분리배양.취간기을산무방망지가위상해역괄공사산품,재료직경15 μm,평균간거150～200 μm,공극솔97%,후2 mm.방법:혼합세포조장골수기질간세포여연골세포안3:1혼균,조정세포밀도위6.0×1010 L-1,접충우경배양액예습적소형취간기을산 5 mm×5 mm적지가상,연후재복합물주위적가함태우혈청적DMEM액배양1주.연골세포조、골수기질간세포조적세포밀도조정위6.0×1010 L-1후동법접충우지가상.각조토마취후우배부일측피하조직식입대응적세포-지가복합물.주요관찰지표:식입후제8주행신생연골대체관찰화소목정-이홍、Masson삼색염색.결과:각조세포여취간기을산지가점부정황량호.식입8주후,혼화배양조화연골세포조균형성료성숙적연골양조직,병기본보지복합물초시적대소화형상,량조신생연골외관급조직학특정교접근.골수기질간세포조재체내배양과정중미형성연골조직,이시형성료섬유결체조직.결론:골수기질간세포여연골세포안3:1혼합형성적미배경,능유효유도골수기질간세포향연골세포분화,병촉진골수기질간세포적체내성연골.
BACKGROUND:Inducing factor and chondrogenic microenvironment is a primary factor, which influences chondrogenic differentiation and chondrogenesis of bone marrow-derived mesenchymal stem cells (MSCs). OBJECTIVE:To explore the feasibility of in vivo chondrogenesis by co-culture of bone marrow-derived MSCs and chondrocytes. DESIGN, TIME AND SETTING:A randomized controlled animal experiment was performed at Department of Pathology, Stomatological Hospital, Fourth Military Medical University of Chinese PLA between September 2004 and March 2005. MATERIALS:Fifteen New Zealand rabbits of clean grade were used for cell-scaffold construct transplantation. The rabbits were randomly divided into co-culture, chondrocyte, and bone marrow-derived MSC groups, with 5 rabbits in each group. Five neonatal New Zealand rabbits, aged 1-3 days, were used for isolation and culture of bone marrow-derived MSCs and chondrocytes. Polyglycolic acid (PGA) scaffold material (Shanghai Yikuo Company, China) has a fiber diameter of 15 μm, with an average interval of 150-200 μm, an interval porosity of 97% and 2-mm thickness. METHODS:In the co-culture group, bone marrow-derived MSCs and chondrocytes were mixed at a ratio of 3:1. The mixed cells were seeded onto a pre-wetted PGA scaffold (5 mm×5 mm )at the ultimate concentration of 6.0×1010 L-1. Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine serum was dropwise added to peripheral compound for 1 week of culture. In the chondrocyte, and bone marrow-derived MSC groups, chondrocytes and bone marrow-derived MSCs of the same ultimate concentration were seeded respectively onto the PGA scaffold. Then, the cell-scaffold constructs were transplanted into subcutaneous tissue of adult rabbits. MAIN OUTCOME MEASURES:Gross observation and hematoxylin-eosin & Masson staining of neo-cartilage were performed after in vivo culture for 8 weeks. RESULTS:Cell in all groups had a fine adhesion to the scaffold. In both co-culture and chondrocyte groups, the cell-scaffold constructs could maintain the original size and shape during in vivo culture and formed homogenous mature cartilage after 8 weeks of in vivo culture. Furthermore, the neo-cartilages in both groups were similar to each other in gross appearance and histological features. In the bone marrow-derived MSCs group, connective tissue rather than cartilage was found during in vivo culture. CONCLUSION:Chondrocytes can provide a chondrogenic microenvironment to induce a chondrogenic differentiation of bone marrow-derived MSCs and thus promote the chondrogenesis of bone marrow-derived MSCs in vivo.