富血小板血浆诱导人骨髓基质干细胞的体内异位成骨

Ectopic osteogenesis of platelet-rich plasma induced human bone marrow stromal stem cells in vivo

背景：近年来的研究发现,结合组织工程技术利用经诱导转化的骨髓基质干细胞能成功地在动物体内再生出骨组织,并在大型哺乳动物负重骨缺损的修复实验中取得了较好的修复效果。目的：观察富血小板血浆诱导培养人骨髓基质干细胞在体内的成骨特性,探讨采用富血小板血浆诱导培养的人骨髓基质干细胞与珊瑚羟基磷灰石材料体内异位成骨的可行性。设计、时间及地点：配对样本观察,于2007-10/2008-04在中山大学组织工程实验室完成。材料：4周龄BALB/C裸鼠14只,体质量22-24g,麻醉后将裸鼠两侧股部切开,于股部肌间隙内制成肌袋模型。方法：14只裸鼠左侧肌袋内植入珊瑚羟基磷灰石复合富血小板血诱导培养的人骨髓基质干细胞,作为实验组;右侧背部肌袋内植入单纯珊瑚羟基磷灰石材料为对照组。主要观察指标：分别于植入后4,8,12周对比观察两组裸鼠活动及进食情况;X射线平片观察阻射率;苏木精-伊红染色观察骨形成情况。结果：14只裸鼠均进入结果分析。①植入材料后裸鼠活动及进食均正常,伤口愈合良好。材料随植入时间的延长无明显吸收,而材料周围的肌肉组织等软组织由外向内逐渐长入材料孔隙内有所增多。②随时间延长两组X射线平片阻射影像密度逐步增加。植入材料后4,8,12周实验组与对照组相比,差异有非常显著性意义（P〈0.01）。③植入材料后4周,实验组可见珊瑚羟基磷灰石表面有细胞生长,孔隙内有结缔组织长入;对照组仅见珊瑚羟基磷灰石表面有细胞生长。8周时珊瑚羟基磷灰石表面有新生骨形成,孔隙内和孔隙边缘可见骨样组织沉积和少量软骨样组织形成;对照组仅见少量纤维结缔组织长入。12周时珊瑚羟基磷灰石材料表面有较多成熟编织骨形成,部分区域可见髓腔样结构形成,并有血管长入;对照组仍未见新骨及骨样组织形成。结论：富血小板血浆诱导培养人骨髓基质干细胞与珊瑚羟基磷灰石材料在裸鼠体内能够良好的成骨,随时间的延长,成骨特性越明显;体内采用肌袋包裹的方法能有效增加血运及促进组织工程骨血管化生成,促进成骨。

BACKGROUND： Recent studies have confirmed that bone marrow stromal stem cells （BMSCs） can successfully regenerate in animals using tissue engineering technique following induction and transformation, and have great repair outcome in studies of large mammalian loading bone defects. OBJECTIVE： To observe the osteogenic property of platelet-rich plasma induced human BMSCs in vivo and to explore the feasibility of ectopic osteogenesis of platelet-rich plasma induced human BMSCs and combined coralline hydroxyapatite. DESIGN, TIME AND SETTING： The pairing sample study was performed at the Laboratory of Tissue Engineering of Sun Yat-sen University from October 2007 to April 2008. MATERIALS： A total of 14 4-week BALB/C nude mice, weighing 22-24 g were selected for this study. Following anesthesia, rat bilateral femur was incised to create thigh muscle pouch models. METHODS： Platelet-rich plasma induced human BMSCs and combined coraUine hydroxyapatite were implanted into the left thigh muscle pouch （experimental group）. Coralline hydroxyapatite was implanted into the right thigh muscle pouch （control group）. MAIN OUTCOME MEASURES： At 4, 8 and 12 weeks following implantation, nude mouse activity and eating were observed in both groups. Radiopacity was observed using radiographs. Bone formation was observed utilizing hematoxylin-eosin staining. RESULTS： A total of 14 nude mice were included in the final analysis. Activity and eating of rats were normal following implantation, and the wound was well healed. Material was not significantly absorbed with prolonged implanted time. Soft tissues surrounding the materials became more and grew into the materials. Radiopacity was gradually increased in both groups over time. At 4, 8 and 12 weeks following implantation, significant differences were detected between the experimental and control groups （P 〈 0.01）. At 4 weeks following implantation, cells on the surface of coraUine hydroxyapatite were found and connective tissues were seen in the material pores in the experimental group. Cells on the coralline hydroxyapatite were observed only in the control group. At 8 weeks, new bone formation was detected on the surface of coralline hydroxyapatite; bony tissue deposition and a few chondroid tissues were found in the pores or surrounding the pores in the experimental group. A few fibrous connective tissues were observed in the control group. At 12 weeks, abundant mature woven bone was detected on the surface of coralline hydroxyapatite; medullary cavity-like structure and vessels were found in some regions in the experimental group. No new bone or bony tissues were found in the control group. CONCLUSION： Platelet-rich plasma-induced human BMSCs and coralline hydroxyapatite have a good osteogenetic effect in the nude mice. With prolonged time, the osteogenetic property is significant. Muscle pouch package method in vivo can effectively increase blood transportation, promote vascularization formation of tissue engineered bone and accelerate osteogenesis.