基于iPS细胞的种植体周围骨缺损组织工程化重建研究

Peri-implant defect is the difficult point and hotsport in research on function and aesthetic restoration of dental implant.Based on the conception of guided bone regeneration(GBR) technique from traditional way, the membrane which serves as the physiologic barrier encourages new bone to grow and prevents the growth of fibro tissue and repairs the defect at last. However, this technique is limitted by lack of plenty and activited cells, more complication and less of new bone formation. Recently, the induced pluripotent stem (iPS)cells, typically derived by transfection of stem cell-associated genesinto adult somatic cell,emerged as new strateges and important method in potentially therapeutic use in regenerative medicine and showed great promise in tissue engineering owing to their paticular merits including without the controverial use of embryos and avoiding and immunogenic response. In this project, four stem cell-associated gene of Oct4,Sox2,Nanog and Lin28 with a lentivired system will be used to transform periodontal ligament stem cells(PDLSCs)into iPS. After the biological properties and osteogenic differentiation are explored and identified,these odontogenic iPS cells will be used as seed cells for bone tissue engineering, and a 3D culture model of iPS cells will be established in vitro by these cells in combination with chitosan temperature sensitive gel-a type of injectable scaffold in bone tissue engineering prepared in our previous study.Consequently,these cell-scaffold constructs will be transplanted in vivo to cure peri-implant defect animal models in miniature pig.As a result, histological observation,immunohistochemistry staining,bone histomorphametric analysis were performed to determine the ablility and efficiency of new bone formation in rebuilding peri-implant defects. This study may provide a new way to regenerate peri-implant bone defect by iPS based tissue engineering, and also provide a new platform for the mechanic study of iPS cells in osteogenic differentiation and the formation of new bone formation at cellular levels and at molecular levels.

种植体周围骨缺损是当前种植牙功能和美学修复的难题和研究热点。传统采用骨引导再生术治疗，但因缺乏大量活性细胞，易出现并发症，且再生骨量较为局限，疗效常不稳定。诱导性多能干细胞（iPS）由体细胞重新编程诱导产生，具有类胚胎干细胞无限增殖和多向分化及消除免疫排斥等优点。本课题拟采用干细胞及组织工程技术，应用慢病毒或非病毒介导的Oct4,Sox2,Nanogji Lin28等因子对牙周膜干细胞（PDLSCs）诱导重编程为iPS细胞，在对生物学特性检测分析和成骨方向定向诱导分化的基础上，将其与我们前期研制的可注射温敏凝胶骨组织工程支架材料复合，体外建立iPS细胞三维培养模型，体内移植治疗小型猪种植体周围骨缺损模型，动态观察、对比检测iPS细胞作为种子细胞，用于种植体周围骨缺损重建的效能。为种植体周围骨缺损的组织工程化再生治疗提供了新方法，也为细胞和分子水平上的研究iPS细胞介导的骨形成提供了研究平台

种植体周围骨缺损是长期困扰口腔种植基础研究和临床治疗的重要课题。近年来迅速发展的干细胞、基因工程，尤其诱导性多潜能干细胞（induced pluripotent stem cells, iPS cell）技术等生命科学领域的一系列重要成就，也为种植体周围骨再生重建研究提供了新途径。iPS细胞通过基因重编程，使正常组织的体细胞转变为类胚胎干细胞（ES）样的多潜能干细胞，成为器官和组织再生最有希望的细胞来源，iPS细胞治疗目前正在走向前临床研究。而口腔来源的干细胞较其他组织更易获得组织、更高的iPS细胞形成率独具优势，成为建立iPS细胞库理想、可靠的细胞来源。本项目以牙周膜干细胞（PDLSCs）基础，将Oct4,Sox2，Lin28，Nanog四种基因导入PDLSCs内，诱导其重编程形成iPS细胞，探讨牙周膜来源的iPS细胞用于种植体周围重建的可行性和可靠性。项目组完成了：1）犬PDLSCs原代培养、分离、扩增；2）用载有Oct4,Sox2，Lin28，Nanog的慢病毒载体转染PDLSCs，通过免疫RT-PCR、免疫荧光等方法对其生物学特性及表型进行鉴定；3）诱导其向骨、脂肪、神经细胞方向分化并进行分化潜能检测。4）将PDLSCs来源的iPS细胞与壳聚糖凝胶、羟基磷灰石磷酸三钙复合材料复合后植入犬种植体周骨缺损区，动态观察、评估其用于种植体周骨组织缺损修复情况。结果表明：1）采用免疫磁珠法成功培养、分离了人、犬PDLSCs，发现其具有高度增殖和多向分化潜能。2）采用慢病毒转染法将载有Oct4,Sox2，Lin28，Nanog的基因导入PDLSCs，经免疫组化、免疫荧光、RT-PCR等多种方法检测，表明已成功诱导PDLSCs形成iPS细胞。3）PDLSCs诱导形成的iPS细胞具有分化为功能性成骨细胞、成脂肪细胞、成神经细胞样细胞的潜能。4）成功建立了犬牙列缺损、牙槽嵴缺损、及种植体周围骨缺损模型，将iPS细胞与温敏凝胶复合后局部注射治疗，通过大体观察、x-线观察、组织学切片等结果发现，iPS细胞可促进种植体周骨再生，重建种植体周缺损组织。本研究首次将iPS细胞用于种植体周围骨缺损修复重建治疗，为干细胞基础上的组织工程化再生治疗种植体周围缺损组织提供了新思路、新方法，丰富了iPS细胞前临床应用研究内容，也为细胞和分子水平上研究iPS细胞介导的骨形成提供了研究平台。