Gonzalez-Fernandez Tomas, Tierney Erica G, Cunniffe Grainne M, O'Brien Fergal J, Kelly Daniel J
1 Trinity Centre for Bioengineering (TCBE), Trinity Biomedical Sciences Institute , Trinity College Dublin, Dublin, Ireland .
2 Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin , Dublin, Ireland .
Tissue Eng Part A. 2016 May;22(9-10):776-87. doi: 10.1089/ten.TEA.2015.0576.
Incorporating therapeutic genes into three-dimensional biomaterials is a promising strategy for enhancing tissue regeneration. Alginate hydrogels have been extensively investigated for cartilage and bone tissue engineering, including as carriers of transfected cells to sites of injury, making them an ideal gene delivery platform for cartilage and osteochondral tissue engineering. The objective of this study was to develop gene-activated alginate hydrogels capable of supporting nanohydroxyapatite (nHA)-mediated nonviral gene transfer to control the phenotype of mesenchymal stem cells (MSCs) for either cartilage or endochondral bone tissue engineering. To produce these gene-activated constructs, MSCs and nHA complexed with plasmid DNA (pDNA) encoding for transforming growth factor-beta 3 (pTGF-β3), bone morphogenetic protein 2 (pBMP2), or a combination of both (pTGF-β3-pBMP2) were encapsulated into alginate hydrogels. Initial analysis using reporter genes showed effective gene delivery and sustained overexpression of the transgenes were achieved. Confocal microscopy demonstrated that complexing the plasmid with nHA before hydrogel encapsulation led to transport of the plasmid into the nucleus of MSCs, which did not happen with naked pDNA. Gene delivery of TGF-β3 and BMP2 and subsequent cell-mediated expression of these therapeutic genes resulted in a significant increase in sulfated glycosaminoglycan and collagen production, particularly in the pTGF-β3-pBMP2 codelivery group in comparison to the delivery of either pTGF-β3 or pBMP2 in isolation. In addition, stronger staining for collagen type II deposition was observed in the pTGF-β3-pBMP2 codelivery group. In contrast, greater levels of calcium deposition were observed in the pTGF-β3- and pBMP2-only groups compared to codelivery, with a strong staining for collagen type X deposition, suggesting these constructs were supporting MSC hypertrophy and progression along an endochondral pathway. Together, these results suggest that the developed gene-activated alginate hydrogels were able to support transfection of encapsulated MSCs and directed their phenotype toward either a chondrogenic or an osteogenic phenotype depending on whether TGF-β3 and BMP2 were delivered in combination or isolation.
将治疗性基因整合到三维生物材料中是增强组织再生的一种有前景的策略。藻酸盐水凝胶已被广泛研究用于软骨和骨组织工程，包括作为转染细胞到损伤部位的载体，使其成为软骨和骨软骨组织工程的理想基因递送平台。本研究的目的是开发能够支持纳米羟基磷灰石（nHA）介导的非病毒基因转移的基因激活藻酸盐水凝胶，以控制间充质干细胞（MSC）的表型，用于软骨或软骨内骨组织工程。为了制备这些基因激活构建体，将与编码转化生长因子-β3（pTGF-β3）、骨形态发生蛋白2（pBMP2）或两者组合（pTGF-β3-pBMP2）的质粒DNA（pDNA）复合的MSC和nHA封装到藻酸盐水凝胶中。使用报告基因的初步分析表明实现了有效的基因递送和转基因的持续过表达。共聚焦显微镜显示，在水凝胶封装前将质粒与nHA复合导致质粒转运到MSC的细胞核中，而裸pDNA则不会发生这种情况。TGF-β3和BMP2的基因递送以及随后这些治疗性基因的细胞介导表达导致硫酸化糖胺聚糖和胶原蛋白产生显著增加，特别是与单独递送pTGF-β3或pBMP2相比，在pTGF-β3-pBMP2共递送组中。此外，在pTGF-β3-pBMP2共递送组中观察到II型胶原蛋白沉积的染色更强。相比之下，与共递送相比，在仅pTGF-β3和pBMP2组中观察到更高水平的钙沉积，伴有X型胶原蛋白沉积的强染色，表明这些构建体支持MSC肥大并沿着软骨内途径进展。总之，这些结果表明，所开发的基因激活藻酸盐水凝胶能够支持封装的MSC的转染，并根据TGF-β3和BMP2是联合递送还是单独递送将其表型导向软骨形成或成骨表型。
