Generation of a Novel Multiphasic Bone-Ligament-Bone Construct for Scapholunate Interosseous Ligament Reconstruction

Abstract

Background: Scapholunate interosseous ligament (SLIL) tears are a common wrist injury in young and active patients with suboptimal outcomes after surgical repair. While bone-ligament-bone (BLB) constructs have been successfully developed in orthopaedics for the replacement of damaged anterior cruciate ligaments (ACL), this has not been applied for the reconstruction of small joints. This novel approach could potentially be superior to equivalent autografts due to factors including: (1) Replication of the patient’s SLIL external morphology allowing for identical matching of construct to existing joint architecture (2) Maintenance of equivalent biomechanical properties as the native SLIL (3) Incorporation of biochemical cues to promote vascularisation and tissue regeneration thereby minimising overall healing time and (4) Elimination of donor morbidity when harvesting autografts. Study Aim: The overarching aim of this thesis is to produce an artificial construct with a BLB interface for subsequent implantation into small joints. To this end, additive manufacturing was utilised to create a novel composite bioscaffold suitable for dorsal SLIL replacement in the wrist. Methodology: A series of studies were conducted to achieve the aim. Stage 1 consisted of the design and fabrication of a BLB multiphasic scaffold for dorsal SLIL reconstruction. Two designs with a fibre interdistance of 350μm & 600μm were 3D-printed in a continuous fashion using medical grade polycaprolactone (mPCL). The scaffold was characterised using microCT analysis and mechanical testing (tensile and cyclic loading, compression testing). Stage 2 investigated numerous ways for cell seeding into the compartments using cell sheets and cell spheroids formed from BMSCs as well as augmentation of osteogenic and fibroblastic differentiation using growth factors or drugs. Extensive in vitro characterisation including gene expression, immunofluorescence analysis, cell viability and DNA/collagen quantification were conducted. Finally, Stage 3 involved the characterisation of this BLB scaffold in an ectopic rat model and new rabbit knee model in vivo. Histological and biomechanical analysis of the samples were conducted. Results and Discussion: Stage 1- The scaffold with a 350μm fibre interdistance was mechanically stronger (ultimate tensile force of scaffold= 71±2.66N) and able to withstand normal carpal physiological forces. Stage 2- Firstly, cell sheets treated with ascorbic acid (AA) showed high DNA content and extracellular matrix deposition in vitro prior to implantation. Secondly, ligament regeneration can be enhanced using hypoxic culture conditions. In addition, the BMP-6 or GDF5/BMP6 treatment combo increased protein expression but only at Day 3 in vitro. Thirdly, the AggreWell plates generated spheroids of sufficient quality for seeding into the bone compartment of the BLB scaffold with the addition of hydroxyapatite nanoparticles increasing collagen production. However, it was an inefficient method for this application. Finally, the pretreatment of BMSCs with BMP2/GSK126 synergistically upregulated gene expression for osteogenesis in vitro but this failed to translate into significant bone mineralisation after 8 weeks in vivo. Stage 3- The proof-of-concept study involving the rat subcutaneous model demonstrated that the BLB scaffold can provide sufficient compartmentalisation and fibre guiding properties necessary for the regeneration of the dorsal SLIL. In the rabbit model, tissue integration and vascularisation were noted with increased scaffold mechanical strength at 8 weeks (p<0.05). Bone and ligament tissues were regenerated in their respective compartments with similar structural and mechanical properties of the native ligament. The bone-ligament interface was strong and histological analysis further supported this with the visualisation of aligned transverse collagen fibres. Conclusion: Overall, this research thesis forms the foundation for tissue engineering for application of ligament regeneration in small joints. It has addressed a gap in clinical knowledge and resulted in the design and generation of a novel multiphasic BLB scaffold that can be used for dorsal SLIL reconstruction. While refinement and improvement of this scaffold is still undergoing with several follow up studies planned, it represents an important step towards a future where regenerative medicine can provide personalised solutions to common injuries thereby greatly improving patient outcomes.