组织工程材料大鼠颅骨缺损修复过程中的磨片三色染色观察

Trichrome staining observation of grinded section in rat critical-sized calvarial defect repairment with tissue engineered bone

目的:用自体细胞复合含孔磷酸钙陶瓷修复颅骨极量骨缺损的SD大鼠骨组织工程模型标本,制作不脱钙磨片并通过改良Masson氏三色染色以观察新骨形成及其与支架材料的关系。方法:成年雄性SD大鼠 14只,随机分成实验组和对照组,每组 7只。全麻及无菌条件下取其左侧胫、股骨骨髓,体外诱导培养大鼠自体骨髓基质细胞。实验组将已分化的自体成骨细胞与含孔磷酸钙陶瓷复合用于修复同体大鼠颅骨极量骨缺损;对照组颅骨缺损处仅置入无细胞的单纯陶瓷。分别于术后 4、8、12、16、20、24及 28周每组各处死1只动物并取材,制成不脱钙磨片并进行改良Masson氏三色染色,光学显微镜下观察比较颅骨修复情况及其矿化程度。结果:实验组 4周时不脱钙磨片,即可见骨缺损修复中央区陶瓷孔隙内蓝色的新骨形成;随着时间的推移,形成新骨逐渐矿化成为红色的成熟骨组织, 28周时骨缺损修复区可见大片新骨呈岛状或条索状排列,其间的陶瓷支架部分已降解,同时加以墨汁灌注的磨片显示骨组织中已形成大量新生血管网。结论:以含孔磷酸钙陶瓷为支架的组织工程骨缺损修复明显优于单纯生物陶瓷修复法,磨片三色染色方法既可了解新骨形成及其矿化程度,又可观察新骨形成与陶瓷支架之间的关系。

Objective: To observe osteogenesis and mineralization with trichrome staining of grinded sections in reconstruction of rat critical-sized calvarial defects using tissue engineered bone.Methods:Fourteen male adult Sprague-Dawley rats were assigned randomly into experimental and control groups. Bone marrow was obtained from left femurs and tibias of all rats under general anesthesia. In experimental group, respective autogenous osteoblasts obtained from bone marrow stromal cells were differentiated and proliferated in vitro and then seeded and subcultured on porous calcium phosphate ceramics. The cell-ceramic composites were used to repair critical-sized (8 mm in diameter) calvarial defects in the corresponding rats. In control group, the solitary ceramic without cell was used. One rat in each group was sacrificed postoperatively at four-week interval till the 28th week. All involved samples were used to make undecalcified grinded sections and stained with modified Masson's trichrome staining and observed under light microscope. Results:In experimental group, blue colored new bone was observed in the center of defect areas four week after reconstructive surgery. With the time went by, newly formed bone among ceramic pores gradually mineralized and matured characterized by its red color staining. Widespread bone islands and abundant blood vessel bundles permeated into ceramic pores were observed in grinded sections with trichrome staining and India ink intravascular injection at 28th week postoperatively. These phenomenons only appeared on bone incisal edges in control group. Conclusions:Tissue engineered bone graft with porous calcium phosphate ceramic may be used for calvarial defects repair.