Spatial presentation of tissue specific extracellular matrix components and growth factors on porous electrospun fibre scaffolds for bone-ligament interface tissue engineering

In many clinical situations ligament or tendon replacements are required, such as for the surgical replacement of a torn anterior cruciate ligament (ACL). The current ?gold standard? treatment is resection of the torn ligament and replacement by an autologous tendon graft. Limitations of this approach include that the interface between the tendon graft and the bone heals as a loose fibrovascular tissue instead of a resilient fibrocartilage enthesis, which leads to long term mechanical instability of the replacement graft. This has motivated the search for alternative strategies to regenerate damaged ligaments. The field of tissue engineering (TE) aims to regenerate or replace damaged tissues through a combination of three-dimensional (3D) scaffolds, cells and signalling molecules. This thesis aims to develop a mechanically functional scaffold that provides spatially defined regulatory cues for ligament TE, with the specific goal of regenerating the stratified interface between ligament and bone. This thesis began by investigating how fibre alignment and growth factor stimulation interact to regulate the chondrogenic and ligamentous differentiation of MSCs. It was shown that, for the engineering of ligamentous grafts, aligned electrospun microfibres in synergy with connective tissue growth factor (CTGF) can be used to enhance ligamentous matrix production, while aligned microfibres combined with transforming growth factor ?3 (TGF-?3) can be used to promote cartilaginous matrix production. A methodology to engineer human scale ligament scaffolds using aligned electrospun PCL fibres was then developed. Electrospun fibres with a higher fraction of unwelded fibres were produced by high speed collection. Increasing the fraction of unwelded fibres allowed the bundling of fibres into 3D scaffolds with dimensions comparable to the human ACL and allowed for higher interfibrillar spacing that facilitated the rapid migration of MSCs into the body of the scaffold. Furthermore, these scaffolds showed a Young?s modulus approaching that of the native human ACL. Next, the tissue-specific bioactivity of cartilage and ligament extracellular matrix (ECM) to direct MSC fate was examined after immobilization onto electrospun scaffolds. It was shown that functionalising electrospun scaffolds with the solubilized ligament ECM promotes homologous bioactivity over and above that observed with commercially available type 1 collagen. It was also found that the immobilisation method (physical adsorption or covalent conjugation) played a key role in the bioactivity of the solubilized ECM. Functionalising electrospun scaffolds with the solubilised cartilage ECM provided a substrate to support the development of a more cartilaginous tissue characteristic of the enthesis. Finally, this thesis explored controlling the spatial presentation of ECM and growth factors to create contiguous ligament, cartilage and endochondral/osseous regions within an electrospun scaffold. Hydroxyapatite (HA) deposited via simulated body fluid was used to generate a mineralized phase to support an endochondral phenotype within the osseous region of the scaffold. The scaffolds functionalised with cartilage ECM and a HA coating were found to support an endochondral phenotype. To conclude, this thesis describes a novel methodology to develop a human sized, mechanically functional scaffold for ligament tissue engineering with spatially defined regions with the potential to regenerate the stratified interface between ligament and bone. This work provides insights into the appropriate combinations of biophysical and biochemical factors that can be used to engineer the interface between ligament/tendon and bone, the application of which will be significant as tissue engineering strategies move towards enthesis regeneration