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 harvested from the patient’s hamstring or patellar tendon. Limitations of this approach include donor site morbidity and a limited availability in the quantity of tissue available for harvest. Furthermore, 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 aims to regenerate or replace damaged tissues through a combination of three-dimensional scaffolds, cells and signalling molecules. Currently, ligament tissue engineering strategies that consider the bone-ligament interface have generally focused on the direct osteogenic priming of mesenchymal stem cells (MSCs), or on maintaining an osteoblastic phenotype in the osseous region of a multi-layered scaffold. However, realising the importance of re-establishing a cartilaginous transition tissue between ligament and bone have led to increased interest in the tissue engineering of a cartilaginous interface between the ligament and bone regions of tissue engineered constructs.

This thesis aims to develop a mechanically functional scaffold that provides spatially defined regulatory cues for ligament tissue engineering, 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. To this end aligned and randomly-aligned electrospun microfibrillar scaffolds were seeded with bone marrow derived MSCs and stimulated with transforming growth factor β3 (TGFβ3) or connective tissue growth factor (CTGF), either individually or sequentially. Aligned microfibres were found to facilitate either a ligamentous or a chondrogenic phenotype depending on the specifics of the growth factor stimulation regimen. It was shown that, for the engineering of ligamentous grafts, aligned electrospun microfibres in synergy with CTGF can be used to enhance ligamentous matrix production, while aligned microfibres combined with TGF-β3 can be used to promote cartilaginous matrix production.

A methodology to engineer human scale ligament scaffolds using aligned electrospun PCL fibres was then developed. By high speed collection, electrospun fibres with a higher fraction of unwelded fibres were produced. Increasing the fraction of unwelded fibres during electrospinning reduced the flexural rigidity of the resultant electrospun-sheets, which in turn allowed the bundling of fibres into 3D scaffolds with dimensions comparable to the human ACL. These unwelded fibres allowed for higher interfibrillar spacing, which in turn facilitated the rapid migration of MSCs into the body of the scaffold. The high-speed collection induced higher molecular chain orientation in the PCL fibres, which in turn resulted in the development of scaffolds with 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.

The next stage of the thesis explored controlling the spatial presentation of ECM and growth factors to create contiguous ligament, cartilage and endochondral/osseous regions within an electrospun scaffold. This required developing a strategy to generate a mineralized phase to support an endochondral phenotype within the osseous region of the scaffold. To that end, simulated body fluid was used to deposit hydroxyapatite (HA) onto the fibre scaffolds. The scaffolds functionalised with cartilage ECM and a HA coating were found to support an endochondral phenotype, thereby enabling the production of scaffolds which support spatially defined differentiation of MSCs. Combining growth factors with ECM cues did not further enhance MSC differentiation over that observed with ECM stimulation alone.

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.