Towards an optimal microenvironment for nucleus pulposus regeneration: a glycobiology approach

Abstract

Neck and low back pain are the two highest causes of job-related disability in the UK and the USA. These pathologies are strongly related to intervertebral disc degeneration (IVD). Disc degeneration diseases (DDD) are characterised by changes in extracellular matrix (ECM) composition which lead to an important disorganisation of IVD tissue. Better knowledge of IVD biology and DDD in the last decades has promoted the development of new tissue engineering approaches to restore the disc function from a biological viewpoint. The ultimate objective of this thesis was to develop an optimal functionalized cell delivery system using an ECMmimicking injectable hydrogel that enhances the production and the deposition of newly synthesised ECM to aid the regeneration of NP tissue. It was hypothesised that the modulation of the glycoenvironment of NP cells will promote the maintenance of their phenotype. In the first phase of this thesis, an injectable type II collagen hydrogel stabilised with poly(ethylene glycol)ether tetrasuccinimidyl glutarate and supplemented with hyaluronic acid was successfully developed. The hydrogel system was stable in culture and had the capability to support cell growth. In addition, NP cells maintained a low type I collagen expression and their cell morphology after culture in the hydrogel. These characteristics, in addition to its injectable properties, make this hydrogel a promising candidate as a carrier of cells for future translation in vivo. The results obtained in this study highlighted the importance of ECM composition on NP cell behaviour. Highly glycosylated, the ECM of IVD tissue plays a crucial role on cell behaviour and IVD biology. Therefore, as a step forward, the glycoenvironment of the IVD was mapped in an effort to understand IVD glycoenvironment and its impact on IVD biology. A subset of specific and selective histological markers to distinguish the cell and ECM phenotypes of NP, AF and cartilage tissue and their stage of maturation was identified. The detailed CS composition and quantity of chondroitin sulfates (CS) revealed a change in sulfation pattern of CS with maturity. The depletion of CS has been shown to greatly affect IVD biology of the intervertebral disc and CS were chosen for the investigations conducted in the last part of this thesis. Therefore, the behaviour of GAGs, specifically of CS, and xylosyltransferase I (XT-I) and glucuronyltransferase I (GTI), two key enzymes involved at crucial points of CS synthesis, was evaluated in a bovine ageing IVD model. Important changes in GAGs composition during disc ageing were highlighted in this study. CS, specifically, were affected at a structural and quantitative levels with important changes in sulfated disaccharide composition upon ageing. A correlation between the expressions of XT-I and GT-I and CS content was shown in this study. The delivery via electroporation restored the expression of both enzymes at a protein level. A trend, although not significant, towards the increase of CS production after delivery of XT-I and GT-I was seen. In accord with the results of this study, the best therapeutic approach to modulate the expression of GAGs might be a dual delivery of XT-I and GT-I or in combination with aggrecan protein core up-regulation. Glycans were shown in this thesis to be essential to IVD biology. A better understanding of their effects on cell behaviour will promote the development of new biological tissue engineering approaches for IVD regeneration