## 4 Translational Preclinical Models Used to Investigate the Intervertebral Disc Degeneration  
Studying IVD degeneration using human subjects is challenging because of the ethical restrictions hindering the access of the clinical data as well as differentiating the different factors influencing the disc degeneration which include ageing, genetic predisposition, mechanical loading, and inflammation [86]. Therefore, developing reliable and representative animal models is necessary. As a result, different animal models emerged comprising rodents, canines, porcine, rabbit, sheep, and goat even though these animals are different in their anatomy, development, and tissue mechanical characteristics compared to human discs [87; 88]. Such models were utilized to evaluate different hyaluron-based therapeutic approaches for the IVD repair (Table 2). Several disc degeneration replicas are present to study disc degeneration in in vivo models: spontaneous (genetic aberration induces early disease onset) [89] puncture (physical trauma activating inflammatory response) [90; 91] mechanical loading (non-traumatic induction) [92; 93] and biochemical (Chondroitinase ABC treatment) [94]. Preclinical models were also used to introduce novel surgical approaches, especially in large animals [95]. Rodent tails have been extensively used to study the pathophysiology of the IVD degeneration and assess the effect of different therapeutics because they are easily accessible with minimal interference with the surrounding tissues and their normal physiological functions [92; 96; 97; 98; 99; 100; 101; 102; 103; 104]. The typical age of the rats used in such studies is three-months-old and above because they can only reach skeletal maturity at that age [105]. Notochondrial cells can be found in the IVD of rats throughout adulthood while they are lost in humans [93]; however, these cells are lost in many degenerative rat-tail models due to factors such as mechanical loading [106]. [PERSON] et al. developed a model of IVD degeneration in a rat-tail in vivo model by using Ilizarov apparatus to load the tail discs, which was first used by [PERSON] et al. in 1999 [107]. The Ilizarov-type apparatus was set on the cocygeal 8-9 (C8/9) discs to induce loading on C9/10 while C10/11 remained unloaded as a control. As a result, it was found that after seven day, the notochordal cells were lost in the rat-tail discs upon static compression up to day 56. Moreover, on day seven, it was noted that the extrinsic apoptotic pathway was activated in the AF and NP that progressively decreased. In contrast, the intrinsic mitochondrial pathway remained continuously active until day 56 [108]. Under disc compression, ECM turnover was explained through tissue inhibitors of metalloproteinase one (TIMP-1), MMP-2 and MMP-13 up-regulation [109; 110]. In parallel, it was reported that disc loading leads to imbalances in the manifestation of MMPs, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), and TIMPs in rat-tail discs [92]. Small and large animal models were utilized over the decades to target dysregulated biomarkers and signalling pathways (Table 1) to mitigate the IVD degeneration using HA-based biomaterials summarised in Table 2. Different HMW HA and HA-based therapeutic platforms were tested in vitro, in vivo and human trials to repair the disc (Figure 2). \begin{table}
\begin{tabular}{p{113.8 pt} p{113.8 pt} p{113.8 pt} p{113.8 pt}} \hline
**HA Composition and Cross-linking Method** & **Species/Platform** & **Objectives** & **Outcome** & **Reference** \\ \hline
**Collagen-I and HA Ultraviolet (UV) cross-linking** & Bovine In vitro excluding GF. & To assess the efficacy of collagen-I and HA injection in IVD repair, both including and excluding GF. & In terms of ECM components, there were no evident changes between AF and NP in synthesizing collagen, aggreen and leucine-rich proteoglycans. In both types of cells, TGF-B1 induced ECM synthesis.