Abstract：
Despite various clinical options, human anterior cruciate ligament (ACL) lesions do not fully heal. Biomaterial-guided gene therapy using recombinant adeno-associated virus (rAAV) vectors may improve the intrinsic mechanisms of ACL repair. Here, we examined whether poly(sodium styrene sulfonate)-grafted poly(ε-caprolactone) (pNaSS-grafted PCL) films can deliver rAAV vectors coding for the reparative basic fibroblast growth factor (FGF-2) and transforming growth factor beta (TGF-β) in human mesenchymal stromal cells (hMSCs) as a source of implantable cells in ACL lesions. Efficient and sustained rAAV-mediated reporter (red fluorescent protein) and therapeutic (FGF-2 and TGF-β) gene overexpression was achieved in the cells for at least 21 days in particular with pNaSS-grafted PCL films relative to all other conditions (up to 5.2-fold difference). Expression of FGF-2 and TGF-β mediated by rAAV using PCL films increased the levels of cell proliferation, the DNA contents, and the deposition of proteoglycans and of type-I and -III collagen (up to 2.9-fold difference) over time in the cells with higher levels of transcription factor expression (Mohawk, Scleraxis) (up to 1.9-fold difference), without activation of inflammatory tumor necrosis alpha especially when using pNaSS-grafted PCL films compared with the controls. Overall, the effects mediated by TGF-β were higher than those promoted by FGF-2, possibly due to higher levels of gene expression achieved upon rAAV gene transfer. This study shows the potential of using functionalized PCL films to apply rAAV vectors for ACL repair.
摘要：
尽管有各种临床选择，人类前交叉韧带(ACL)损伤不能完全愈合。使用重组腺相关病毒（rAAV）载体的生物材料指导的基因治疗可能会改善ACL修复的内在机制。这里,我们研究了聚(苯乙烯磺酸钠)接枝的聚(ε-己内酯)(pNaSS接枝的PCL)膜是否可以在人骨髓间充质基质细胞(hMSCs)中递送编码修复性碱性成纤维细胞生长因子(FGF-2)和转化生长因子β(TGF-β)的rAAV载体,作为ACL病变中可植入细胞的来源.相对于所有其他条件,在细胞中实现有效和持续的rAAV介导的报道分子(红色荧光蛋白)和治疗性(FGF-2和TGF-β)基因过表达至少21天,特别是用pNaSS移植的PCL膜(差异高达5.2倍)。应用PCL膜对rAAV介导的FGF-2和TGF-β的表达增加了细胞增殖水平,DNA含量，随着时间的推移，在转录因子表达水平较高的细胞中，蛋白聚糖和I型和III型胶原蛋白的沉积（差异高达2.9倍）（Mohawk，巩膜轴)(差异高达1.9倍)，与对照组相比，尤其是使用pNaSS移植的PCL膜时，不会激活炎性肿瘤坏死α。总的来说,TGF-β介导的作用高于FGF-2促进的作用，这可能是由于rAAV基因转移后基因表达水平较高。该研究显示了使用官能化PCL膜应用rAAV载体进行ACL修复的潜力。
