Surgery (St Vincent's) - Theses

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Subject: tissue engineering × Subject: arthritis ×

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    Osteochondral repair using structured biological scaffold and stem cell technologies
    Ye, Kenneth ( 2015)
    Introduction: Articular cartilage damage can result in pain and loss of function for many patients. The traditional management of moderate to severe defects has been difficult due to the lack of intrinsic capacity for cartilage to regenerate. Current methods of cartilage repair include microfracture, osteochondral grafting and autologous chondrocyte implantation. Whilst some individual studies comparing these techniques have shown improvements in long-term clinical outcomes in some patient groups compared to microfracture, major randomised control trials have failed to show consistent long-term differences in clinical outcomes between microfracture, osteochondral grafting, and autologous chondrocyte implantation. Hence, there is a clinical need to explore novel methods of cartilage repair and regeneration using biological techniques such as tissue engineering, stem cells, biomaterials, and growth/differentiation factors to improve cartilage regeneration. The aim of this project was to develop a technique using human infrapatellar fat pad adipose stem cells (IPFP-ASCs) in 3D cultures for chondrogenesis; this required extensive characterisation of the cartilage formed in the 3D cultures/scaffolds (3D pellet culture, chitosan and acellular dermal matrix) for in vitro chondrogenesis. In vivo testing and characterisation of osteochondral defect repair was achieved using a small animal rabbit model for preliminary testing of the ADM-engineered structures. This preliminary testing in the small animal model may then lead to pre-clinical trials in larger animals and human pilot studies in the future. Materials and methods: In vitro IPFPs were harvested from total knee replacements and digested to release adipose stem cells (IPFP-ASCs) which were expanded in vitro. Pellet cultures were developed using TGF-β3 and BMP-6 for chondrogenesis. IPFP-ASCs were seeded onto 3D printed chitosan scaffolds and acellular dermal matrix (ADM) material under the same chondrogenic conditions as the pellet cultures. Four-week cultures were analysed using histology, immunohistochemistry, and gene expression analysis using qPCR. In vivo Osteochondral defects were drilled into distal femoral condyles of adult New Zealand White rabbits. The defects were repaired using either (1) ADM alone (2) autologous IPFP-ASC (3) ADM with autologous IPFP-ASC) or (4) left empty as control. The animals were euthanised at 12 weeks. Repairs were analysed using histology and immunohistochemistry for collagen Type II and Type I. The modified O’Driscoll score was for histological scoring. Further image analysis was conducted to assess quality and quantity of repair. Results: IPFP-ASCs were capable of undergoing chondrogenesis in vitro using pellet cultures and when cultured directly on 3D chitosan and ADM scaffolds using the growth factor combination of TGF-β3 and BMP-6. The method of chondrogenesis was robust and was replicated across both human and rabbit IPFP-ASCs. A one-step single site surgical process was developed for the in vivo modelling of osteochondral defect repair and autologous IPFP-ASC implantation. In rabbits, the rabbit ADM only group achieved the highest ratio of collagen Type II to Type I (77.3%) on image analysis using area measures based from protein expression by immunohistochemistry. This indicated a higher quality of cartilage repair resembling hyaline or hyaline-like cartilage (p<0.05). Conclusion: IPFP-ASCs exhibited robust chondrogenesis under in vitro conditions used in this study; the combination of TGF-β3 and BMP-6 for generation of hyaline cartilage has demonstrated the potential for improving cartilage repair in vitro. ADM, as a support matrix, promoted ASC ingrowth in vitro and proved to be an excellent substrate to promote formation of hyaline-like cartilage in vitro. In the small animal in vivo experiments, it was clear that ADM exhibited positive outcomes when used as a substrate for osteochondral defect repair. These experiments need to be performed in a larger animal model to consolidate these findings prior to consideration of translational to pre-clinical studies.