Investigating the effect of anti-inflammatory hyaluronic acid towards regenerating the annulus fibrosus

Neck and low-back pain are major causes of disability leading to a serious socio-economic burden worldwide. The pathology is strongly correlated with the intervertebral disc degeneration (IVD). Disc degenerative disease (DDD) is described as lack of cellularity and transformation in the extracellular matrix (ECM) composition, which result in adverse changes in the IVD tissue. The lack of a full understanding of the aetiology underlying the DDD in the last decades has promoted the exploration of new tissue engineering approaches to restore the disc function by identifying new inflammatory molecular markers and signalling pathways to target the disc degenerative disease. Therefore, the ultimate goal of this thesis was to identify molecular markers and signaling pathways to develop an optimal anti-inflammatory platform using an ECM- mimicking injectable hydrogel that enhances the cell survival and boosts the synthesis and the deposition of the ECM to regenerate the annulus fibrosus (AF) tissue. It was hypothesised that high molecular weight HA (HMW HA) will act as an anti-inflammatory molecule by down-regulating IFNα signalling, enhancing cell survival by decreasing Interferon-induced Protein with tetratricopeptide repeat 3 (IFIT3) and IGF-binding Protein 3 (IGFBP3) as well as Caspase 3 and as a matrix modulatory by up-regulating the production of the matrix proteins such as aggrecan, collagen I (COLI) and hyaluronan and proteoglycan link protein 1 (HAPLN1). In the first phase of this thesis, Interferon α2β (IFNα2b) was identified as the most dysregulated inflammatory signalling pathway analysing a range of downstream molecular markers comparing a microarray data from degenerative and non-degenerative human annulus fibrosus tissue. Consequently, an injectable HMW HA hydrogel was developed and evaluated as an anti-inflammatory biomolecule in the second phase. The hyaluronan hydrogel was evaluated in bovine organ culture system to investigate its capability to act as anti- inflammatory and to support extracellular matrix synthesis. In addition, this hydrogel has been further tested in an in vivo rat tail disc injury model to identify its anti-inflammatory and matrix modulatory properties in addition to its role in regulating the glyco-environment. The results obtained in this study highlighted the significance of hyaluronan in reducing inflammation by down-regulating IFNα and its signalling molecules, interferon α receptor 1 (IFNAR1) and interferon α receptor 2 (IFNAR2), signal transducer and activator of transcription 1 (STAT1), Signal transducer and activator of transcription 2 (STAT2) and janus kinase 1 (JAK1). In addition to down-regulating the anti-proliferative IGFBP3 and anti-apoptotic IFIT3 which resulted in lower caspase 3 expression. On the other hand, results have shown that annulus fibrosus ECM key components were up-regulated such as aggrecan, COLI and HAPLN1 which are major proteins contributing in the normal organization of the matrix. Hence, the glyco-environment of the IVD was evaluated as an attempt to recognise the influence of HA on the IVD glyco- environment comparing to the healthy and injured tissue. A set of specific and selective lectins were used to distinguish the switch in glycans of AF in healthy tissue, inflammation and hyaluronan treatment. The major finding in this phase of the study was that all sialylation highlighting motifs were down-regulated in healthy and hyaluronan treated tissue in contrast to injured tissue. CS composition and quantity of chondroitin sulfates (CS) revealed a change in sulfation pattern of CS on day 7 and 28 where C0S was significantly up- regulated on day 28 and similarly C4S on day 7. The results obtained from this thesis show that hyaluronan acts as an anti-inflammatory and matrix modulatory molecule in an animal model which is closer in disc dimensions and degenerative process to that of humans.2019-05-1