Development of Direct Cartilage Repair with a Layer-by-Layer 3D Bioscaffold

Abstract

The avascular articular cartilage composed of low density chondrocytes embedded within a dense extracellular matrix so its injury has limited self-repairing ability Several clinical approaches have been proposed; however none of them provide outstanding long term healing outcome so far Tissue engineering provide the possibility to regenerate the damaged cartilage or osteochondral; nevertheless there are still some challenges in the engineering cartilage In this study for better delivery of scaffold with cells to fill the irregular cartilage defect the layer-by-layer poly (ethylene glycol) diacrylate (PEGDA) scaffold seeded with rabbit adipose-derived stem cells (ADSC) were evaluated in vitro and ex vivo To investigate cytotoxic response to photopolymerized PEGDA scaffold which was combined with rabbit ADSC was fabricated in the layer-by-layer (LBL) fashion rabbit endothelial progenitor cells (EPC) that were co-cultured with rabbit ADSC were incubated in vitro in the presence of differentiating and proliferating required growth factors for principle of stem cell simulation The completed scaffold assembly was generated beyond a modified mold with multiple holes (Diameter 3mm; height 3mm) In PEGDA scaffold the life-supporting function for most ADSC was proved through LIVE/DEAD staining after 6 days The cell viability to photopolymerization was analyzed by MTS assay following the recommended procedures (ISO 10993-5 and ISO 10993-12) and the result has suggested that photocrosslinking process was not the risk factor for cytotoxicity in LBL assembly for 3 days Taken together these results showed that such scaffold assembly could have potential of forming repaired cartilage tissue with porcine osteochondral plug (OC plug) For ex vivo study the different cell distributions (ADSC EPC/ADSC and 3T3) were applied to LBL PEGDA scaffold to help deal with the ADSC chondrogenesis associated with EPC then each scaffolds developed into 3-mm-diameter cylindrical cartilage defect (Depth 3mm) in porcine middle of 6-mm-diameter OC plug and culture ex vivo for 2 weeks In EPC/ADSC group the underlying scaffold was especially encapsulated with EPC in order to incorporate former findings into the introduction of bioactive factors from the bone marrow of OC plug in whole culture system The rest layers combined with ADSC were fabricated by LBL assembly and the other two groups (ADSC and 3T3 groups) of scaffolds were completely produced as well According to results of histology the fibrous-like tissue was appeared to cover the native cartilage in ADSC groups after a week as well as in EPC/ADSC group after two weeks Under such ex vivo culture system in EPC/ADSC group the LBL assembly could have the potential of fabricating repaired tissue with enhanced extracellular matrix deposition and structure to lesion site of OC plug for 2 weeks The shear strength between scaffold and cartilage was examined though push-out test Interestingly the findings of histology and mechanical analyze were corroborated the concept that EPC could assist ADSC chondrogenesis found in the interface of scaffold-cartilage during the period of 1-2 week Although the scaffold integrity to cartilage was decreased within each groups during 2 weeks the statistic results showed that there was no statistical significance of difference It was demonstrated that the shear strength within each groups was stable during 2 weeks of culture In summary layer-by-layer PEGDA bioscaffold with ADSC can be a promising candidate for tissue engineering application with evenly distributed cell distribution and future cartilage tissue printing by advanced rapid prototyping

Date of Award	2014 Aug 22
Original language	English
Supervisor	Ming-Long Yeh (Supervisor)