BIOFABRICATION STRATEGIES TO GUIDE THE SELF-ORGANISATION OF BIOMIMETIC CARTILAGE AND OSTEOCHONDRAL TISSUES

Abstract

The modest clinical impact of musculoskeletal tissue engineering (TE) can be attributed, at least in part, to a failure to recapitulate the structure, composition and functional properties of the target tissue. This has motivated increased interest in developmentally inspired (DI) TE strategies, which seek to recapitulate key events that occur during embryonic and postnatal development, to generate truly biomimetic grafts. Typically, these approaches are scaffold-free and underpinned by processes such as cellular self-assembly and self-organisation. Such TE strategies can be substantially enabled by emerging biofabrication and bioprinting strategies, and in particular the use of cellular aggregates and microtissues as ?biological building blocks? for the development of larger tissues and/or organ precursors. The objective of this thesis was to explore the potential to converge these emerging biofabrication strategies with existing 3D (bio)printing methods to develop scalable methods to engineer highly biomimetic tissues suited to biological joint resurfacing. This thesis first engineered a highly biomimetic articular cartilage (AC) via directed self-organisation. To achieve this, inkjet bioprinting was used to precisely deposit MSCs into a novel fixation device which supported the development of a self-organised cartilage from the high cell density condensations. A microwell array imposed boundary conditions on the developing tissue and served to generate stratification within the collagen network that closely mirrored native AC. Dynamic culture conditions were shown to further enhance the quality of this engineered cartilage, in terms of the matrix richness as well as the hierarchical collagen arrangement. Next, the use of microtissues as biological building blocks for engineering cartilage tissues of scale was explored. To this end a novel microwell method, ideally suited to the biofabrication and cultivation of spheroids, was designed and validated. Principally, this work underpinned future studies that leveraged microtissues as tissue/organ-seeds. However, it also functioned to demonstrate the capacity to form various musculoskeletal spheroid phenotypes using a single platform technology. Cartilage microtissues, capable of fusing to form a homogenous and unified macrotissue, as well as vascular spheroids, which could form pervasive prevascular networks were developed. To generate tissues of scale, DI approaches are inherently dependent on the cell?s capacity to synthesise large amounts of tissue-specific matrix. To improve the feasibility of such approaches, a method which consistently produce high-quality cartilage microtissues was developed. Through a serendipitous discovery, hydrocortisone was found to significantly improve the initial aggregation and chondrogenic potential of heterogeneous MSC populations. Next, it was shown that numerous microtissues can spontaneously fuse and subsequently self-organise into thick, biomimetic and mechanically competent articular cartilage. A 3D printed (3DP) polymer framework was used to further guide the self-organisation of the cartilage macro-tissue, resulting in the formation of a zonal collagen organisation. These microtissues were first used to generate a layer of structurally organised cartilage on top of a novel joint fixation device, for the treatment of focal chondral defects in a caprine preclinical model. While no statistically significant improvements in healing were observed compared to microdrilling, important insights into the regenerative process were gained. This motivated the use of engineered osteochondral (OC) implants as a strategy for biological joint resurfacing. It was also possible to generate hypertrophic cartilage microtissues consisting of a mineralised cartilaginous extracellular matrix (ECM), which were used to form the osseous region of the OC implant. A strategy for the bioassembly of the phenotypically distinct cartilage microtissues within a 3DP polymeric scaffold was also established. Finally, the capacity of this engineered plug to regenerate OC defects was assessed in a preclinical, large animal model. Collectively, the in vivo results indicated that the developmentally inspired OC plug stabilised the defect, restoring a near-normal articular surface. Such biological implants have the capacity to transform the treatment of damaged and diseased synovial joints.