脱钙骨基质为骨组织工程支架修复骨缺损实验研究

目的：利用骨组织工程原理对节段性骨缺损进行修复,探讨脱钙骨基质（demineralized bone matrix,DBM）在骨组织工程中的应用。方法：从兔股骨分离骨髓间质干细胞（bone mesenchymal stem cells,BMSCs）,经体外诱导分化、条件培养、5-溴-2′-dexyouridine,5-BrdU）标记后,接种到兔海绵状脱钙骨基质（sponge of DBM ,SDBM）支架上,然后分别植入兔背部肌肉内及兔桡骨中段10mm长的骨膜骨缺损内。对照组为缺损内单纯植入SDBM及空白组。术后观察6周。结果：经条件培养的BMSCs可在体外表达Ⅰ型胶原、碱性磷酸酶,并形成钙结节;体内异位植入组织工程骨块可形成软骨及骨组织;组织工程骨块及单纯SDBM在术后6周完全修复骨缺损（6/6）;极限压缩强度测量显示,组织工程骨块植入组新生骨与正常桡骨段差异无显著性（P=0.623）,单纯SDBM植入组新生骨在生物力学方面低于正常桡骨段（P=0.038）。结论：脱钙骨基质在骨组织工程中是一种有效的生物活性支架材料。

An experimental study of demineralized bone matrix to repair bone defects as a scaffold of tissue engineering

OBJECTIVE: To evaluate application of the sponge of demineralized bone matrix (SDBM) in tissue engineering of bone. METHODS: SDBM was prepared from long bone of rabbits. Bone marrow cells were flushed from the bone shaft of femurs of a two-month-old New Zealand white rabbit. After the cells were cultured for 9 days, the flasks were added into dexamethasone (10(-8) mol/L), beta-glycerophosphate sodium (10 mmol/L) and L-ascorbic acid (50 micrograms/ml). After 5 weeks, the cultured cells were collected and marked by 5-Bromo-2'-dexyouridine (BrdU). The grand sum of cells seeded on a piece of SDBM was about (4-6) x 10(6). The composites of cells and SDBM (tissue engineered chip, TEC) were implanted into muscles and bone defects of radius in rabbits. A standard procedure was applied to make a 10 mm long defect bilaterally in the radius of nine skeletally mature male New Zealand white rabbits. All of the 18 defects were randomly divided into three groups: group I, six defects were grafted by TEC; group II, six defects were grafted with SDBM alone; group III, six defects were empty. RESULTS: The results of radiographic and histological evaluation showed that all of the defects were repaired in group I and group II at 6 weeks, none of the defects was repaired in group III. The results of BrdU staining showed that the staining was positive in group I, but negative in group II. Biomechanical test showed that the compressive ultimate strength (CUS) of new bone in TEC implanted group was comparable with normal radius (P = 0.623) and in SDBM implanted group was significant lower than normal radius (P = 0.038). CONCLUSIONS: The TEC can form cartilage and bone tissue in muscles and repair segmental bone defects. SDBM is a kind of effective natural scaffold in tissue engineering of bone.