Engineering of natural cartilage substitution biomaterials

Cartilage lesions cause pain and a loss of joint motion, and if not repaired will further degenerate into an osteoarthritic state. Currently the only treatment options for end stage osteoarthritis are total joint replacements, which are not preferable for younger or more active patients. Early intervention therapies to repair initial cartilage damage and stem the progression of osteoarthritis are thought to be a more favourable treatment option in appropriate cases. It is hypothesised that an acellular xenogeneic osteochondral scaffold could be used in mosaicplasty-like operations to provide an immunocompatible, off-the-shelf biomaterial for osteochondral lesion repair, retaining the same natural composition, structure and function as native cartilage. Initial characterisation of cartilages from different species (pig, cow and sheep) and joint regions of the hip and knee revealed differences in cartilage biology, biochemistry and biomechanics. Osteochondral tissues from skeletally immature porcine medial condyles were selected along with mature bovine femoral groove tissues as source materials for decellularisation, primarily based on cartilage thickness and glycosaminoglycan (GAG) content. Bovine osteochondral pins were subject to a number of decellularisation protocols and were shown to be successfully decellularised following use of a water pik to remove bone marrow, four cycles of freeze-thaw (two of which were in a hypotonic buffer with protease inhibitors), two cycles of hypotonic buffer followed by incubation in 0.1 % (w/v) SDS with protease inhibitors and treatment with nucleases and sterilisation with 0.1 % (v/v) peracetic acid. The process removed all whole cell nuclei, as visualised by histology and reduced cartilage DNA content per cartilage dry weight to 39 ng.mg-1. However, the process removed 99% of GAGs and resulted in reduced biomechanical properties. Porcine osteochondral pins were fully decellularised following use of the above process with one cycle of incubation in hypotonic buffer and 0.1 % (w/v) SDS in hypotonic buffer. Cartilage DNA content was reduced by 98% and the osteochondral tissues contained no visible cell nuclei. Cartilage GAG content was reduced by 60 %. Further alterations to the protocol to improve GAG retention revealed cartilage damage; histologically appearing highly porous and having greatly increased water content and almost complete GAG loss. A comprehensive investigation was conducted to identify the cause of the damage, however no protocol could be developed which completely eradicated cartilage damage. Further characterisation of acellular bovine osteochondral scaffolds showed that a low concentration of SDS remained in the bone, and that this had cytotoxic effects when incubated in contact with BHK cells. Further washes in PBS were added to the protocol to remove excess SDS, however this increased washing led to damage of the bovine cartilage, as seen previously in porcine osteochondral decellularisation. Finally, the decellularisation process was applied to whole porcine condyles in which the ratio of cut edge to cartilage area was minimised and minimal damage to the decellularised cartilage was seen. In summary, the complex microstructure of cartilage has been shown to be surprisingly fragile ex vivo and a novel approach to the development of clinically relevant acellular osteochondral grafts is required; however significant advances have been made in the current study.