BMP异位诱导成骨的Micro-CT评价

【摘要】 借助Micro-CT评价单纯牛骨形态发生蛋白( bovine bone morphogenetic protein， bBMP)异位诱导成骨的长期三维影像学及骨质变化。(20±2) g昆明小鼠21只，麻醉后于双侧股部肌肉中植入bBMP各2 mg，分别于1、2、4、6、8、10、12周各处死3只，切取诱导分化组织，5%戊二醛固定，行Micro-CT扫描和三维重建，运用ABA专用骨骼分析软件测定组织矿含量 (tissue mineral content, TMC)，组织骨密度 (tissue mineral density, TMD)，骨体积分数(bone volume fraction, BVF)，结构模型指数(structure model index, SMI),骨小梁厚度(trabecular thickness, Tb.Th),骨小梁数量(trabecular number, Tb.N)及皮质骨骨密度(bone mineral density, BMD)等参数，运用SPSS10.0统计软件进行统计学分析。bBMP从植入2周开始逐渐形成一椭圆形骨组织块，2～4周，异位生成骨呈疏松的新生骨，4周时组织矿含量达第一个峰值，骨小梁数量最多;随着观察时间的延长(6～12周)，异位诱导生成的椭圆形骨组织内部骨小梁逐渐吸收，数量减少，12周时骨小梁数量最少;而外层骨组织逐渐塑形成为皮质骨，12周时骨矿含量值、骨小梁厚度、组织骨密度和皮质骨骨密度均达最大值。说明bBMP具有强大的异位骨诱导能力，血供不足时，骨质降解吸收;血供充足时，骨质逐渐成熟改建。

【关键词】 骨形态发生蛋白;异位成骨;骨诱导;骨小梁;MicroCT

　The Assessment of Ectopic Osteoinduction by bBMP Using Micro-CTLI Dan, LIU Min, HU Yunyu, BI Long, DUAN Yonghong, JIN Jing, WANG Jun, LIU Yin

　　(Institute of Orthopaedics & Traumatology,Xijing Hospital，The Fourth Military Medical University,Xi'an 710032，China)

　　Abstract：To assess the 3-D image and changes in quality of osseous tissue produced through ectopic osteoinduction by bovine bone morphegenetic protein over an extended period by means of Micro-CT.Twenty-one Kunming rats(20±2)g were used， 2 mg bBMP were implanted into thigh muscles of both sides of the rat under anesthesia. Three animals of each were sacrificed at 1, 2, 4, 6, 8, 10 and 12 weeks after the implantation. After being harvested, the newly formed bone tissue was fixed with 5% glutaraldehyde, then successively subjected to Micro-CT scan and 3-D reconstruction, measurement of tissue mineral consent(TMC) using ABA software and tissue mineral density(TMD), determination of bone volume fraction(BVF), structure model index(SMI), trabecular thickness(Tb.Th), trabecular number(Tb.N), cortical bone mineral density(BMD) and other parameters, 3-D images were created and saved as TIFF files; statistical analyses were carried out by ANOVA (analysis of variance) using Statistics Package for Social Science (SPSS) software.At 2 weeks postimplantation, an oval mass of osseous tissue began to take shape; between 2 to 4 weeks, the ectopic-induced new bone presented as loose cancellous bone tissue, and the mineral content reached the first peak by the 4th week, showing numerous trabeculae; the innermost trabecular of the ectopic-induced lump of osseous tissue were resorbed with their number decreased with time(between 6th and 12th week), approaching the minimum by the 12th week; the outer part remodeled into cortical bone with TMC, Tb.Th, TMD and BMD of the lump as whole reaching the maximum by the 12th week.Bovine BMP possesses strong ectopic osteoinductivity, but the newly formed bone tissue is resorbed when the blood supply is inadequate, whereas the osseous tissue remodels with progression toward a mature tissue when provided with ample blood supply.

　　Key words：Bovine bone morphogenetic protein; Ectopic bone formation; Osteoinduction; Trabecular bone; Microcomputed tomography

　　1 引 言

　　自1965年Urist[1]首次报道骨形态发生蛋白异位成骨能力到1995年中国食品药品监督管理局对重组合异种骨[2](主要成分为bBMP)及美国FDA相继对rhBMP临床应用的批准，BMP的研究已从实验室走向临床。但是，关于BMP仍有许多未解决的问题，例如BMP活性定量，BMP异位成骨的转归等。目前，研究主要集中在基因重组的BMP，如rhBMP-2、rhBMP-4及rhBMP-7等，反而忽视对最早通过生物化学方法提取并纯化的天然BMP的研究。既然BMP具有强大的异位骨诱导成骨能力，这种异位生成骨是否具有应用价值?其转归如何?本实验就小牛骨中生物提取BMP异位成骨过程中组织学的变化进行长期观察，借助MicroCT了解成骨骨质变化情况，探讨这种异位生成骨进一步应用的可能性，并为BMP相关骨修复研究提供参考。

　 2 材料与方法

　　2.1 原材料与设备