## 3 An Overview of Selected Chemical Crosslinkers
### Genipin  
Genipin, a compound extracted from the fruits of the _Gardenia jasminoides Ellis_, emerged as an alternative crosslinker for crosslinking biomaterials in light of the cytotoxicity of crosslinkers such as glutaraldehyde and formaldehyde [29]. Due to the established food safety of genipin, the crosslinking method has been suggested for use in collagen, gelatin and chitosan-based scaffolds and drug-delivery systems [29]. Genipin has been used as a crosslinker in chitosan-based scaffolds and is proposed to follow the same two-step mechanistic pathway in both chitosan and collagen. One of the crosslinking steps has been reported in earlier studies to involve a secondary amide linkage of a free amine to the genipin via an \(S_{\text{N}}2\) nucleophilic substitution [30; 31], and, more recently, the second crosslink has been described to form via two further pathways to complete the crosslinking to collagen, through either two imide crosslinks, or two amide crosslinks, as shown in Figure 5[31]. Genipin crosslinks gelatin through lysine and arginine groups and is expected to follow a similar mechanistic pathway in collagen [32]. The crosslinks formed with genipin offer collagen scaffolds increased elastic modulus by nearly an order of magnitude, as shown by [PERSON] et al. The authors found that treating Type I rat tail tendon derived collagen scaffolds of 92% porosity was most effective at high crosslinking concentrations (0.7812 _w/w_%) and temperatures (20-37 \({}^{\circ}\)C), producing compressive elastic modulus of 30 kPa when compared against a non-crosslinked control at 5 kPa [33].  
Figure 5: Crosslinking mechanism of genipin. First mechanism proposed in [PERSON] et al. [30] Second proposed in [PERSON] et al. [31]. Image modified from [PERSON] et al. [14] licensed under CC-BY 4.0.  
There is some evidence to support the cytotoxicity of genipin at very large concentrations (\(>\)5 mM), according to a study by [PERSON] et al. which evaluated the effect of genipin-crosslinked Type I collagen gels without washing post-treatment with genipin [32]. However, the effect on washed scaffolds was found to be minimal. In addition, following neurocompatibility and long-term large animal studies, [PERSON] et al. investigated the use of genipin which was directly injected into the spine of five patients to alleviate their chronic lower back pain by improving the mechanical properties of the annulus [34]. At the end of their six month study, the authors observed an improvement in the flexion-extension instability reduction and patient satisfaction in 75% of their patients. Patients only experienced temporary central pain associated with the procedure, and the leg pain experienced by a patient was treated and successfully resolved by the ninth week of the experiment, supporting the safety of genipin for use in vivo.  
Furthermore, an investigation of the mechanical properties of genipin by [PERSON] et al. using Type I bovine dermal insoluble collagen, revealed that genipin at the highest crosslinking concentrations (1.5624 _w/w_ % at room temperature) can act as an alternative for the intermediate crosslinking conditions of EDC-NHS, improving both the Young's modulus and the stress to failure [14]. Integrin-specific binding was also observed to be unaffected by the genipin crosslinking, resulting in high human dermal fibroblast proliferation rates and low cellular toxicity [14].