A traumatic osteochondral lesion is a severe type of articulating tissue injury which disrupts and causes damage to both surface cartilage and underlying sub-chondral bone layers. Afflicted patients generally present with early symptoms such as joint discomfort and severe pain which if left untreated can ultimately progress to the pathological disease known as post-traumatic osteoarthritis. At this late stage, the recommended treatment option is total joint replacement surgery; a procedure which is highly invasive and only has a finite functional in-situ lifespan. In physically active younger patients, the latter property is sub-optimal and thus more effective early stage interventions are needed to fully repair the osteochondral tissue unit. Gene activated matrices (GAMs) are considered an exciting tissue engineering treatment solution, whereby single or multiple reservoirs of therapeutic DNA payloads are incorporated specifically within a biomaterial matrix to facilitate high quality bone and cartilage tissue formation. However, a limitation of current gene activated matrices is their limited efficacy in gene delivery and lack of spatial patterning during fabrication and incorporation. As osteochondral tissue is complex with gradient-like features, a GAM containing a gradient payload distribution is hypothesised to be more effective than current unpatterned approaches. To achieve this, a novel agarose gel electrophoresis platform was utilised to fabricate porous agarose scaffolds which contained spatially controlled in-situ deposited DNA payloads.
Experimentally, this project for the first time sought to comprehensively evaluate and characterise the above platform in terms of its inherent physical biomaterial properties, DNA payload patterning capabilities (DNA-CaP, DNA-PEI), biomaterial adhesion, cytotoxicity profile and 3D transfection performance. To achieve an acceptable osteochondral-like porous biomaterial GAM, it was found that a combination of freeze-drying and higher inherent agarose hydrogel concentrations (>3wt%) were required. Upon conversion into suitably porous scaffolds, the agarose GAM systems were then shown to be cytocompatible but crucially appeared to be lacking inherent 3D cellular adhesion characteristics. Following this major finding, various surface functionalisation methods (fibronectin, LAP-PEO, polydopamine) were tested for scaffold-wide cell adhesion enhancement. The polydopamine coating method appeared remarkably effective in this regard, enhancing C2C12 and Y201 3D cell-scaffold attachment and proliferation specifically after 7 days of seeding. Histological analysis further showed effective differentiation towards osteogenic and chondrogenic lineages when in the presence of respective differentiation media conditions for 4 weeks. 3D transfection analysis revealed, however, that all agarose-polydopamine GAM scaffolds, developed via the electrophoresis patterning platform, failed to induce any tangible gene expression over a 7 day seeding period. Future experimentation is therefore needed to elucidate the exact failure mechanisms shown in this transfection study, with the aim that corrective measures can be generated which can ultimately produce a transfection capable agarose GAM product.
In an additional explorative study, it was also shown that an alternative transfection-capable vector type (CCHLV) could be synthesised from the gene expression cassette region of a plasmid vector. This was significantly aided by the novel use of an ELAN type IIS restriction enzyme synthesis strategy, which could more efficiently produce the final nucleic acid vector product in comparison to current gold standard strategies (ELAN type II). Future experimentation is therefore required to fully characterise these newly developed vectors for its 2D and 3D transfection capabilities.
Overall, huge strides were made in the advancement of a completely novel early stage continuous gradient GAM concept, of which ultimately serves to expand not only the knowledge and understanding of applying agarose DNA electrophoretic patterning in the field of GAM system development and manufacture, but furthermore elucidates the substantial limitations this specific concept inherently possesses which may restrict its adoption as a osteochondral defect regeneration device.
