Usage of bacterial cellulose-collagen scaffolds incorporating IGF-I and TGFB-1 genes for cartilage regeneration

USAGE OF BACTERIAL CELLULOSE-COLLAGEN SCAFFOLDS INCORPORATED WITH IGF-I AND TGF-β1 GENES FOR CARTILAGE REGENERATION Ultimate goal of tissue engineering and regenerative medicine is to develop substitutes to help the guidance of the growth of new functional tissue using biological, mechanical and structural cues. Scaffolds used in tissue engineering applications have primarily been incorporated with the growth factors in the form of recombinant proteins to boost the therapeutic response. However, using growth factors for that purpose is associated with a number of distinct disadvantages such as the requirement of large doses, the need for repeated applications, poor distribution, expense and especially short half-life. The healing potential of scaffolds for tissue engineering can be enhanced by incorporating them with the genes. The scaffolds can be used as gene delivery vehicles in this technique. Scaffold mediated gene delivery is an advantageous strategy for gene transfer due to localized delivery of a therapeutic gene. DNA delivered from the scaffold is principally taken up by the seeded cells, therefore limiting unwanted exposure in other areas. Bacterial cellulose-collagen composites can be used as a scaffold for genes in cartilage tissue engineering due to their biocompability, high waterholding capacity and biodegradability. Rat bone marrow mesenchymal stem cells are preferable cell source for the tissue regeneration. Rat bone marrow mesenchymal stem cells (RBMSCs) can be easily isolated, expanded and cryo-preserved. Moreover, these cells are able to differentiate into osteo-, adipo- and chondrogenic cells. In this study, rat bone marrow stem cells (RBMSCs) seeded bacterial cellulose-collagen scaffolds that were crosslinked with DHT and/or Genipin were used to investigate the effect of scaffold mediated gene therapy on chondrogenic differentiation process of RBMSCs. The bacterial cellulose-collagen scaffolds were incorporated with phrGFP-II-I plasmids which contain IGF-I and TGF-β1 genes to induce chondrogenic differentiation process of RBMSCs. For this study, MTS cell proliferation assay was used to learn about the cytotoxic effects of untreated and crosslinked scaffolds. For the characterization of RBMSCs, flow cytometry analysis was carried out. Besides, Alcian blue staining was applied to analyze chondrogenic potentials of RBMSCs. In addition, to assess the effect of gene application on protein production of cells, total protein concentration of samples were compared. Confocal microscopy study was carried out to show the presence of cartilage specific proteins. The results of these studies demonstrated that RBMSCs were proliferated and differentiated into chondrogenic cells on crosslinked bacterial cellulose-collagen scaffolds that were incorporated with phrGFP-II-I plasmids which contain IGF-I and TGF-β1 genes better than the others. The combination of an efficient IGF-I and TGF-β1 gene transfer system residing in bacterial cellulose-collagen scaffold that was crosslinked with different crosslinkers provided better chondrogenesis. Ultimately, the application of a DHT/Genipin crosslinked bacterial cellulose-collagen scaffolds containing IGF-I and TGF-β1 genes can be a promising scaffolds for cartilage repair to provide efficient sustained protein expression within a defect without the side effects of the current recombinant protein based technologies.