Vas, Wollis Jude; (2019) Bioengineering The Fracture Callus: Bone Repair Through Fracture Mimetics. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Fracture non-union is estimated to occur in approximately 5-10% of bone fractures. Tissue engineering strategies that aim to replicate mature tissue may be unresponsive to the cues observed during the initiation of fracture repair, and as such have limited ability to integrate with the host tissues. As such, this thesis aims to develop an implant that mimics the cartilaginous callus, the initial reparative stage of the body’s response to fracture; through the development of decellularised xenogeneic hyaline cartilage in combination with skeletal (stem) cells. This thesis presents data on the optimisation of an osmotic shock based decellularisation methodology (Vac-OS) for costal cartilage. The resultant scaffolds (dcECM) were investigated for the removal of cellular content and maintenance of cartilage-specific proteins such as sulfated glycosaminoglycans. The Vac-OS methodology efficiently lowered DNA content to levels below in vivo immunological response thresholds, while eliminating the highly immunogenic and abundant alpha-gal epitope. Interestingly, immunogenic neutralisation was achieved while retaining over 80% of the sGAG content, surpassing published hyaline cartilage decellularisation methodologies. Extracellular matrix (ECM) integrity was using a novel methodology based on fluorescence lifetime imaging (FLIM) and further assessed using differential scanning calorimetry (DSC). Both FLIM and DSC indicated a lack of significant change in cartilage ECM integrity, post VacOS decellularisation. The bioactivity of the dcECM was subsequently assessed using various skeletal cell populations, indicating an inherent capacity to enhance chondrogenesis. Furthermore, post-implantation in immunocompetent mice, the dcECM promoted a regenerative response, in contrast with native costal cartilage. The dcECM was also fabricated into porous scaffolds capable of being upscale to meet clinical demands and gel coatings that enhance in vitro chondrogenesis. This thesis demonstrates the dcECM’s potential as a fracture callus mimetic. The study, therefore, concludes that these constructs could potentially enhance bone repair in cases of atrophic non-union fracture, where the failure of callus formation is a defining event.

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