The potential of SRY (sex determining region Y) - Box 9 and telomerase reverse transcriptase genes transfection for articular cartilage tissue engineering

Abstract

This study incorporates gene transfer with tissue engineering to evaluate the feasibility of cartilaginous tissue formed using SRY(Sex Determining Region Y)-Box 9 (SOX9) and Telomerase Reverse Transcriptase (TERT) genes transfected chondrocytes. The aim of this research is to improve on the current cartilage treatment strategies with undue limitations and to work toward an alternative treatment for cartilage damage. The experimental settings involve monolayer cell culture, in vitro three-dimensional (3D) culture and in vivo ectopic implantation. The cells were isolated from six rabbit’s articular cartilage and cultured until passage-1 (P1). The P1 cells were transfected with SOX9/TERT-, SOX9-, and TERT-gene. The non-transfected chondrocytes serve as the control group. For monolayer study, the cells were sub-cultured until P3 and evaluated in each serial passage. The in vitro 3D construct was formed by seeding the P1 cells in poly(lactic-co-glycolic acid) (PLGA) and PLGA/fibrin hybrid scaffolds with cells density of 1×105 per scaffold. The resulted cell-scaffold constructs were evaluated at week-1, -2 and -3 of culture. For in vivo study, the week-3 in vitro constructs formed by SOX9/TERT-transfected chondrocytes were subcutaneously implanted at the dorsum of the athymic mice. The constructs were evaluated at week-2 and -4 post-implantation. The analyses include growth kinetics profile, cell proliferation analysis, compression-stress analysis, macroscopic, microscopic visualisation, histological stains, quantitative sulphated glycosaminoglycan (sGAG) content analysis and gene expression study using real-time polymerase chain reaction (RT-PCR) of cartilaginous markers (SOX9, COL2A1, ACAN), COL1A2, TERT and collagenolytic marker (MMP13). A total of 60.4% transfection efficiency can be achieved using Lipofectamine® 3000 reagent. The upregulation of the transferred genes was noted in the cell groups indicating the effectiveness of the procedure. The monolayer cultured cells were unable to retain their cartilaginous phenotype. However, the in vitro 3D culture successfully exhibited the cartilaginous tissue formation. The cells and extracellular matrix (ECM) were densely distributed in the constructs at week-3. The cell number was increased in the constructs. The ECM components (sGAG, proteoglycan and collagen type-II) were visualised in the constructs. The cartilaginous genes expression was upregulated in the SOX9/TERTtransfected chondrocytes constructs group. Hence, this group was selected for the in vivo study. The in vivo constructs have the appearance which resembles cartilage. In terms of the construct’s rigidity, there are no changes in the groups from week-2 to week-4 post-implantation. The cells and ECM distribution were homogenous in the in vivo constructs, which is better than the one observed in the in vitro constructs. The presence of ECM components was noted in the constructs indicates the cartilaginous tissue development. The cartilaginous genes expression was particularly upregulated in SOX9/TERT-PLGA/fibrin construct. The SOX9/TERT-PLGA/fibrin construct has the potential to be developed into a functional cartilaginous tissue and translated into clinical application. Since the end goal of this present study is to benefit the humankind, proper research guidelines to ensure safety and efficacy of the engineered tissue must be followed with good intention and values. The approach is in-line with the teaching of Islam – there should be neither harming nor reciprocating harm.