MSCs细胞膜片复合EPCs移植修复大鼠放疗区骨缺损的实验研究

After radiotherapy, it is difficult to restore the craniofacial defects. Because the bone used for reconstruction has been subject to insufficient blood supply. In the earlier research, we have found the mesenchymal stem cells (MSCs) sheet could be effective to accelerate bone healing around implant after radiotherapy. But only the MSCs for bone reconstruction was not enough to resolve the blood supply after radiotherapy. The researchs showed endothelial progenitor cells (EPCs) have significantly stimulated angiogenesis effect for the treatment of ischemic diseases, which provides new ideas. Therefore, on the basis of the inbred line F344 rat tibial bone defects model after radiation and of the preliminary work, we intended to study this problem. We cultured mesenchymal stem cells (MSCs) -cell sheet and added the EPCs on it. Then, novel bone tissue was reconstructed with a hydroxyapatite scaffold in vitro. Further the novel tissue engineered bone was put into and repair the bone defect after radiotherapy. The tissue was observed from morphology, cytology and molecular biology. This would provide a novel method for reconstructing the tissue engineered bone tissue.

放疗后颅颌面的骨缺损修复常受到重建区骨组织血供不足、成骨能力弱等问题的限制，修复效果不理想。本课题组在前期研究中发现，骨髓间充质干细胞（MSCs）细胞膜片可有效促进放疗区种植体周围骨组织愈合，但由于干细胞种类单一，放疗区血管再生能力弱的问题尚未解决。研究表明，内皮祖细胞（EPCs）具有明显促血管生成作用，目前已广泛应用于各种缺血性疾病的治疗研究中。本项目拟通过分析不同比例EPCs和MSCs共培养后成血管和成骨相关基因及功能的变化，阐明两种细胞的相互作用机制，通过将EPCs接种于MSCs细胞膜片上，形成新的细胞复合体，再进一步与羟基磷灰石支架整合，体外构建血管化组织工程骨，并将该组织工程骨应用于近交系F344大鼠胫骨骨缺损放疗模型中，借助大体、组织形态学、细胞学以及分子生物学等手段评估新型组织工程骨在放疗后骨缺损修复中的作用，为颅颌面部放疗后的骨缺损重建提供实验依据。

头面部恶性肿瘤患者，通常需要采用手术联合放疗的治疗方案。放疗后颅颌面的骨缺损修复常受到重建区骨组织血供不足、成骨能力弱等问题的限制，修复效果不理想。放疗也会明显降低种植体成功率。本项目拟通过将EPCs 和 MSCs 联合应用于放疗区骨缺损及放疗区种植体周围，为解决放疗区组织缺损修复提供新的解决方案与实验依据。本项目分离培养大鼠骨髓EPCs和BMSCs，通过多种方法检测两种细胞的特性，证明所培养的细胞符合各自特征，将EPCs接种于BMSCs细胞膜片后发现其可提高细胞膜片成骨能力，将构建的复合膜片应用于放疗区骨缺损，发现复合膜片可促进放疗区骨缺损的愈合。将EPCs和BMSCs直接共培养，发现共培养可促进成骨相关基因的表达。EPCs与BMSCs比例为10:1时效果较佳。将EPCs、BMSCs共培养的细胞膜片应用于大鼠放疗区种植体周围可促进新骨形成和改建，促进骨结合。综合本项目研究结果，我们认为EPCs可提高BMSCs膜片的成骨能力，能促进放疗区骨缺损愈合；直接共培养EPCs和BMSCs并诱导形成复合细胞膜片可应用于种植体周围，能促进放疗区种植体骨结合。EPCs与BMSCs的联合应用为改善放疗患者骨再生及提高放疗后种植体固位的修复疗效提供了新思路，为提高颌面部肿瘤患者的生活质量提供了新方法。