同轴电纺法构建双基因活化基质及其在牙周组织再生中的应用

Periodontal disease that involves the deterioration of tooth-supporting structures is one of the most prevalent oral diseases all over the world. It affects 90 percent of human population and is the primary cause of tooth loss among adults. Gene-modified periodontal tissue engineering can take the gene sequence encodings into the periodontal cells.To regenerate the damaged periodontal tissue and supporting bone, a surgical approach is widely performed by placing a space-maintaining barrier membrane between the root surface and the gingival flap. This technique, known as guided tissue regeneration (GTR), is based on the exclusion of gingival epithelial cell from the damaged area by using a barrier membrane, hence allowing the selective repopulation of tissues derived from the periodontal ligament and alveolar bone. The gene-modified cells will synthesize and secrete grow factors, which can facilitate the periodontal tissue regeneration.How to construct a gene activated matrix as a appropriate, safe and efficient scaffold for carrying targeting genes and expressing of all of these genes, is a hard nut to crack to put in the periodontal tissue regeneration.Coaxial electrospinning method is a fast and simple fabrication process which can generate a kind of core-shell nanosized polymer fibers by applying a strong electric field to a liquid polymer droplet.The electrospun fibers have nanoscale diameters, very high surface to volume ratio and a fully interconnected porous network structure which can utmostly mimic the features of natural extracelluar matrix (ECM), which is also very similar to the structure of ECM. In this study, we propose that a new type of nanofibrous membrane can be developed by the combination of gene transfer and electrospinning technique. This new membrane will mimic the chemical composition and porous structure of the core-shell nanofibrous, which contain hOPG plasmid vector in the core layer and hold BMP plasmid vector in the shell layer simultaneously. It can not only physically maintain the space for the tissue regeneration, exclude the invasion of epithelial tissue as well as enhance the attachment, proliferation, and differentiation of periodontal ligament cells, but also empower the scaffolds with the ability of gene transfer.It is expected to greatly enhance the periodontal tissue regeneration as well as physically maintain the space for tissue repair, thus to be a promising and cost-effective GTR membrane candiate.

牙周病及其所导致的牙周组织病损是成人失牙的最主要原因，其发病率在我国高达90%以上。基因修饰的牙周组织工程将相关细胞因子基因编码入种子细胞，使其能持续合成和释放细胞因子，从而更好的促进牙周组织的再生。然而，如何构建基因活化基质来形成种子细胞支架，承载多个目的基因，实现局部持续表达，提高转染效率，是其应用中的一大难题。同轴静电纺丝是一种新兴的芯-壳型纳米纤维支架制备技术，其产品具有纳米级的纤维细度、仿生的三维网络结构和可调控的降解释放速率。本研究拟采用该技术制备芯质含有hOPG质粒载体(阻断/延缓骨质吸收)、壳层含有hBMP2质粒载体(促进骨质重建)的三维纳米纤维状双基因活化基质，使之兼具可调性双基因缓释能力和高度仿生天然细胞外基质的纤维支架形貌特征。研究有望将基因修饰技术和组织工程技术相结合，提高基因转染效率，促进种子细胞的贴附增殖和功能表达，最终实现促进牙周组织修复再生的目的。

牙周病及其所导致的牙周组织病损是成人失牙的最主要原因。基因修饰的牙周组织工程将相关细胞因子基因编码入种子细胞，进而持续合成和释放细胞因子参与牙周组织再生修复。本研究采用同轴静电纺丝技术制备出了芯-壳型结构并载有pEGNP-N1/BMP-2及pDsRed2-N1/OPG质粒的纳米纤维基因活化基质（GAM）。同混纺GAM相比，同轴静电纺丝技术制备GAM的三维网络结构类似，均仿生天然细胞外基质的纤维支架形貌，但其接触角和力学强度略低但降解性能更加平稳。与PDLCs体外培养的结果显示GAM能够承载BMP-2与OPG目的基因于组织再生部位缓慢释放转染PDLCs并成功表达目的基因，同轴静电纺丝技术制备的芯-壳型结构的GAM较混纺制备的GAM细胞毒性小，可以提高hBMP2与hOPG基因的转染效率，促进PDLCs的贴附增殖和功能表达。SD大鼠与beagle犬牙周缺损模型上分别进行的8mm极限骨缺损修复以及牙周缺损组织修复再生研究也表明无论是单基因混纺结构还是双基因同轴芯-壳型结构的GAM均为实验骨组织和牙周组织缺损的重建提供了纳米纤维支架，且在其中释放目的基因BMP-2与OPG，转染周围细胞并表达，最终促进了缺损局部组织的再生修复。其中beagle犬牙周缺损的实验动物模型中，BMP-2、OPG以及BMP-2/OPG组和未转染组相比其的新生牙槽骨面积百分比和新生牙骨质长度百分比均有明显增加；BMP-2/OPG组新生牙槽骨面积百分比和新生牙骨质长度百分比与BMP-2以及OPG组相比也有增高，且差异有显著性。本课题采用的同轴静电纺丝GAM制备方法将可能作为一种新颖、有效的基因转染手段应用于骨组织以及牙周再生的领域。