## 3 An Overview of Selected Chemical Crosslinkers
### Ultraviolet Radiation  
The use of UV to crosslink collagen is a relatively novel technique that relies on the formation of highly reactive radicals to facilitate the formation of crosslinks within the structure. There is no strict chemical mechanism due to the non-specificity of radical based reactions, but amino acid analyses give evidence of crosslinks through aromatic residues, like phenylalanine and tyrosine [24; 25] that are able to stabilise the radicals within their de-localised \(\pi\)-systems.  
An investigation by [PERSON] on UV-irradiated films made from acid-soluble rat tail tendon collagen revealed a reduction in thermal stability and the creation of surface flaws such as wrinkles and micro-cracks after UV crosslinking at \(\lambda=254\) nm, 0.196 J cm\({}^{-2}\)min\({}^{-1}\), for 2, 4 and 8 h [26]. The UV radiation applied was suggested to disrupt the hydrogen bonds within and across the collagen in these samples, thus initiating the release of water and the production of collagen fragments [26]. This fragmentation of collagen has been shown to be limited through the use of glucose by inhibiting the unwinding of the triple helices in bovine insoluble dermal and Achilles tendon collagen constructs [24; 27].  
[PERSON] et al. conducted an assessment of the physical properties of UV-irradiated collagen films and scaffolds both with and without the presence of glucose. Type I bovine dermal scaffolds and bovine Achilles tendon collagen scaffolds and films were produced, and subsequently treated at a range of UV conditions (\(\lambda=254\) nm for 30 min). Scaffolds were irradiated at a range of intensities (0.06-0.96 J cm\({}^{-2}\)), whereas films were only irradiated at either 0.42 or 0.96 J cm\({}^{-2}\). The crosslink densities achieved were very low despite long exposure times, giving rise to a maximum Young's modulus under 2 kPa for tendon collagen treated with glucose, and 0.5 kPa without any additives [24]. Moreover, the influence of UV-crosslinking on degradation stability in water was found to be dependent on the collagen source. UV crosslinking improved the stability of tendon collagen, displaying no dependence on irradiation intensities whereas the stability of dermal collagen was improved at the lowest intensity and decreased at higher values. This was reconciled by the competing effects of collagen crosslinking and collagen denaturation that occur during UV-treatment [24].  
In the same study, the authors also investigated the in vitro cellular integrin-mediated cellular response to UV crosslinking. \(\alpha_{2}\beta_{1}\) integrin mediated binding of HT1080s and platelets, as well as spreading and proliferation of HT1080s was found to be unaffected by UV crosslinking, suggesting that the GXOGER sequences were unaffected by the UV-treatment [24]. Building upon these results, the synergistic effects of UV crosslinking (\(\lambda=254\) nm, 0.42 or 0.96 J cm\({}^{-2}\) for 30 min) and EDC-NHS crosslinking (0-200% of the standard condition as defined in Section 3.2) was investigated by [PERSON] et al. on Type I insoluble bovine Achilles tendon collagen. UV-irradiation was found to inhibit the adhesion of \(\alpha_{2}\) I-domains above the expected effect from EDC-NHS alone, with a EDC-NHS concentration-dependent inhibition of HT1080 cell adhesion and cell coverage). This was proposed to occur due to the involvement of phenylalanine (F) in UV crosslinking, resulting in the loss of GFOGER crosslinking motifs, the most strongly binding of the GXOGER motifs. As a result, binding is  
Figure 4: Dehydrothermal mechanism of crosslinking. Illustration redrawn from mechanisms by [PERSON] et al. [13].  
compensated through the GLOGER motifs, where L represents Leucine, which is compensatory for \(\alpha_{1}\)-I domain binding, but a weak association for \(\alpha_{2}\)-I domains [28].