Osteoarthritis (OA) is a pervasive disease worldwide which affects the articular cartilage and the underlying bone in synovial joints such as the knee. Currently, the only treatment for a severely degenerated knee joint is a total or partial joint replacement with a metal and polymer prosthesis. Whilst these procedures are well established, failures are not uncommon resulting in a more complicated revision surgery. The aging worldwide population and the increase in the instances of younger patients being diagnosed with OA are primary motivations behind the pursuit of new treatment options. Tissue engineering approaches have been gaining traction in recent years, having being successfully translated to the clinic to treat small focal defects. These therapies combine cells, scaffolds and signalling molecules to drive tissue formation and maturation to regenerate damaged tissues. 3D printing technology can be used in tandem with tissue engineering strategies to fabricate constructs that mimic the shape and function of a joint and to accurately position cells and biological cues within such biological implants.
The overall objective of this thesis is to tissue engineer an anatomically accurate, mechanically reinforced biological implant for total joint regeneration. To this end, this thesis set out to investigate the following; 1) whether a developmentally inspired cartilage template formed through in vitro priming of bone marrow derived stem/stromal cells (BMSCs) in an alginate hydrogel can be used to regenerate a critically sized osteochondral defect; 2) to identify suitable cell sources derived from OA joints for hyaline cartilage tissue engineering; 3) to identify a suitable thermopolymer that can be 3D printed to mechanically reinforce cartilage templates and subsequently engineer bi-layered cartilage templates with a top chondral layer containing a co-culture of mesenchymal stromal/stem cells (MSCs) and chondrocytes and a bottom endochondral layer of BMSCs, and to then evaluate these constructs in vivo subcutaneously in nude mice and within caprine osteochondral defetcs; 4) to develop a technique to biofabricate an anatomically accurate, mechanically reinforced, bi-layered cartilage template for whole joint resurfacing.
The thesis began by investigating if a tissue engineered cartilage template could undergo spatially defined differentiation to form bone and cartilage in a critical sized osteochondral defect in a rabbit model. The osteochondral unit develops postnatally from a cartilaginous precursor that undergoes endochondral ossification during skeletal maturation. Evaluation after 3 months demonstrated that the engineered cartilage template can enhance osteochondral repair, although consistent hyaline cartilage regeneration was not observed, suggesting room for further improvement.
Co-cultures of chondrocytes and MSCs are commonly used to enhance chondrogenesis. Recognising that the proposed biological implant is targeted toward patients with OA, the next part of the thesis examined the potential to use co-cultures of cells derived from osteoarthritic joints for cartilage tissue engineering. A co-culture of freshly isolated or culture expanded chondrocytes and infrapatellar fat pad derived MSCs underwent robust chondrogenesis when maintain in low oxygen.
The next stage of the thesis examined mechanically reinforcing the cartilage templates with 3D printed thermopolymers, specifically either PCL, PLA, PLGA (85:15 and 65:35). Whilst all of the polymers supported the chondrogenic phenotype, only PCL maintained its mechanical properties over the 4 weeks of in vitro culture and therefore was chosen as the reinforcing network for the remainder of this thesis. PCL reinforced bi-layered engineered cartilage templates, consisting of a top layer of MSCs and CC co-culture in either alginate or agarose, and bottom layer of BMSCs in alginate, were capable of supporting hyaline cartilage and vascularised bone respectively. The top cartilage layer formed with agarose best suppressed mineralisation and therefore, was chosen as the material to support cartilage formation in a goat model.
Prior to evaluating the reinforced bi-layered cartilage template in a goat model, expansion and differentiation conditions of goat BMSC were optimised. 5% oxygen tension and high glucose availability were identified as the optimal conditions for expanding and differentiating goat BMSCs. Using these culture conditions the bi-layered template was primed in vitro preceding implantation in a critical sized defect in both the medial femoral condyle and the trochlear ridge. After 6 months the bi-layered scaffold supported hyaline-like repair, however, there was some animal to animal variability in the quality of bone repair pointing to areas for improvement in the design of future implants.
Finally a strategy using 3D printing was implemented to develop a tissue engineered, anatomically accurate biological implant for knee joint regeneration. An implant mimicking the geometry of a rabbit medial femoral condyle was printed using the geometrical information obtained from a CT scan of a rabbit knee. BMSC encapsulated in a GelMA bioink were used to engineer a hypertrophic cartilage tissue within the osseous layer. To cover the curved surface of the scaled-up implant, GelMA containing a co-culture of BMSCs and CCs was deposited in droplets on the uneven surface of the osseous region of the implant. The reinforcement frame had mechanical properties exceeding the compressive loads assumed to exist in a rabbit joint. However, three months after implantation of the scaled-up implant, it was observed that the scaffolds had failed mechanically. In most cases it appeared that this was due to delamination of the body of the implant from the stem.
To conclude this thesis describes a novel approach for the regeneration of whole joints by utilising developmental processes to direct repair of both articular cartilage and bone. Additionally, a suitable cell source for engineering articular cartilage using cell derived from OA joints has been identified. This work demonstrates how a combination of 3D printing and tissue engineering strategies can be used to engineer biological implants for synovial joint regeneration.
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