In vitro chondrogenesis by BMP6 gene therapy

In this study, the promotion of in vitro chondrogenesis was investigated by using chitosan scaffolds and rat bone marrow-derived mesenchymal stem cells (rBMSCs) which are transfected by BMP6 (bone morphogenetic protein-6) encoding gene. For this purpose, plasmid DNA (pShuttle-rBMP6), the expression vector consisting of the coding sequence of the BMP6 was obtained, and then, it was entrapped in chitosan scaffolds to obtain a gene-activated matrix (GAM). The chitosan scaffolds performed the controlled and sustained release of plasmid DNA, thus they continuously provided the modification of rBMSCs to induce chondrogenic differentiation. In addition, the cells were transfected by lipid-based agent (Lipofectamine) and then, these modified cells were inoculated into the chitosan scaffolds. Furthermore, a group of chitosan scaffolds with nontransfected rBMSCs with recombinant BMP6 free in culture medium was used as control. Comparative results showed that, mitochondrial activities of modified rBMSCs by Lipofectamine and chitosan GAM were significantly higher than those of nontransfected rBMSCs. The observations from scanning electron microscopy analysis confirmed that BMP6 gene-modified rBMSCs differentiated to the chondrogenic phenotype. Highest amount of glycosaminoglycan contents of rBMSCs on GAM concluded that BMP6 gene-activated chitosan scaffold has a potential in the application of cartilage regeneration. © 2012 Wiley Periodicals, Inc

Platelet-rich plasma-loaded chitosan scaffolds: Preparation and growth factor release kinetics

The aim of this study is to compare the effects of different platelet-rich plasma (PRP) preparation methods on platelet activity and to investigate the growth factor (GF) release kinetics from PRP-loaded chitosan scaffolds for tissue engineering applications. Flow cytometry analysis showed that centrifugation processes used for PRP preparation did not cause significant effect on platelet activation levels by means of markers investigated. Two different methods were used to prepare PRP-loaded chitosan scaffolds: (i) PRP was added to chitosan gel before freeze-drying to prepare scaffolds called as "GEL" and (ii) PRP was embedded to freeze-dried chitosan scaffolds to prepare scaffolds called as "SPONGE." In addition, nonactivated PRP and PRP activated with type-I collagen were used as control groups. Scanning electron microscopy images demonstrated that, in GEL group, there is no deterioration on the scaffolds porous, 3D, and interconnected structure. GF release kinetics was determined by enzyme-linked immunosorbent assay for platelet-derived GF-BB, transforming GF-ß1, and insulin-like GF-1. A sustained release of GFs was achieved in GEL group while a sharp burst release was observed for all the GFs from the SPONGE groups. Moreover, platelet-derived GF-BB, insulin-like GF-1, and transforming GF-ß1 releases were prolonged to 20 days in GEL groups, and the biological activities of all GFs released from GEL and SPONGE scaffolds were preserved. This study demonstrated that chitosan scaffold that was called GEL could be an appropriate carrier for PRP applications by providing sustained release of GFs. © 2012 Wiley Periodicals, Inc