The Development of Cell-Mediated Tissue Engineered Collagen-Based Strategies for Osteochondral Defect Repair

Smooth articular cartilage is vital for the pain free movement of the joint, therefore the formation of osteochondral defects remains a challenge for orthopaedic surgeons. The hyaline cartilage, however is avascular and is poorly supplied with cells roughly 1.7% cell volume density in the medial femoral condyle (Hunziker, 1999). Therefore, any injury to the joint surface does not heal easily. The latest focus is on tissue engineering techniques to design scaffolds that can be implanted to stimulate the body’s own healing. In the design of these scaffolds the gradient nature of the tissue at the joint surface must be considered. One such scaffold has been designed in the Tissue Engineering Research Group of the Royal College of Surgeons in Ireland. This collagen based scaffold has been developed using a novel iterative freeze-drying technique with three seamlessly integrated layers representing the bone layer, the intermediate, or tidemark layer, and the cartilage layers which include the gliding, transitional and radial zones (Levingstone et al., 2014). The overall objective for this thesis is to develop and optimise a tissue engineered biomaterial scaffold based method of osteochondral defect repair that facilitates cell-seeding onto the scaffold as a single-stage procedure, with the possibility of implantation via arthroscopic technique. In order to achieve this, the specific aims of the thesis were to assess the modalities through which the scaffold directs cell differentiation in the different layers, to identify the optimal cell seeding regimen that can be used for the scaffold on the defect to produce best outcome of hyaline cartilage generation, to perform a large animal in vivo trial to assess the outcomes at 3, 6 and 12 months, and finally to develop an arthroscopic delivery device that can allow the scaffold be implanted through a minimally invasive procedure that will further decrease patient morbidity.

In Chapter 2 of this thesis the intrinsic properties of an RCSI developed, collagen-based tri-layered scaffold, to direct stem cell differentiation as required within each layer were examined. The bone layer was found to have osteoinductive as well as osteoconductive abilities when seeded with rat mesenchymal stem cells. It was shown that the intermediate layer was chondroconductive and would not allow the encroachment of mineralised tissue into the cartilage layer protecting the tidemark, even when the scaffold is cultured all together in osteochondrogenic media. The cartilage layer proved itself to be chondroconductive as well as mildly chondroinductive. It was also shown that there was not a significant amount of contraction in the tri-layered scaffold in vitro. These results taken together show that the scaffold is suitable for use in press-fit technique into osteochondral defects and should ensure good outcomes of bone and cartilage formation in the relevant layers.

Chapter 3 of this thesis focused on establishing an optimal cell-seeding regime for this multi-layered collagen-based scaffold. Currently cell based approaches require an in vitro expansion phase of roughly six weeks (Brittberg et al., 1994), however it has been shown here that the use of a co-culture of rapidly isolated primary chondrocytes (CC) and fat pad derived mesenchymal stem cells (FPMSCs) in a ratio of 1:3 is a valid alternative, and the co-culture of FPMSCs:CC was also shown to have a synergistic effect that augmented the results of using merely chondrocytes alone. These FPMSCs and CCs can be harvested on the morning of surgery, seeded back onto the scaffold after isolation and re-implanted without the expensive in vitro expansion phase. This eliminates a large cost element of the procedure and simplifies the procedure for both patient and surgeon.

In Chapter 4 a large caprine in vivo study was undertaken to assess the cell-seeded and cell free version of the scaffold implanted in a critically sized defect of a caprine stifle joint and compare it to a commercially available biomaterial for osteochondral repair. Results demonstrated that the cartilage was satisfactorily repaired both macroscopically and microscopically, and that the tri-layered scaffold was able to generate cartilage and subchondral bone that was of a similar standard to the commercial product both as a cell-free, off-the-shelf-product and as a cell-seeded scaffold. There was no significant difference between the cell-seeded scaffold group and the cell-free scaffold group in this study.

Chapter 5 focusses on the development and testing of an arthroscopic delivery device for implanting the multi-layered scaffold into a human knee via a minimally invasive arthroscopic technique. A device was developed that involved a reusable trigger handle and a single-use windowed cassette in various sizes that would allow safe delivery of the scaffold to the osteochondral defect inside the joint. Benchtop testing of the arthroscopic delivery device showed that the delivery device successfully implanted the scaffolds in a consistent manner without overly compressing them and causing the cell suspension to be extruded.

Collectively the results from this thesis have shown that the tri-layered scaffold has intrinsic properties to optimally repair osteochondral defects by directing cell differentiation appropriately for the individual layers. Cartilage repair can be augmented by pre-seeding the scaffold with cells prior to implantation, and best results for this are obtained using a co-culture of FPMSCs:CC, which can be harvested and isolated using a rapid isolation technique. These tri-layered scaffolds have been proven in in vivo trials to generate hyaline-like cartilage in a large animal model, and a device has been designed to enable the implantation of this device by arthroscopic technique.