## 3 An Overview of Selected Chemical Crosslinkers
### Riboflavin  
Riboflavin, or vitamin B2, has also arisen as a biocompatible means to achieve command-set crosslinking of collagen constructs with blue light. Riboflavin crosslinking of collagen is of particular interest due to the short processing times (15 min) needed to observe significant improvements in mechanical properties (2.5-fold increase in elastic modulus) [35]. [PERSON] et al. established that riboflavin can create crosslinks within a collagen construct (Type I insoluble, bovine Achilles tendon membranes) specifically through the arginine, histidine and lysine amino acids. Although the loss of arginines can result hinder integrin-mediated adhesion, [PERSON] et al. hypothesised that arginines are not as critical in stabilising a divalent cation in the GFOGER motifs as the glutamines which are lost during EDC-NHS crosslinking [36]. This was confirmed with cell attachment assays where only the \(\alpha_{2}\beta_{1}\) integrin-mediated binding was affected, as evaluated with HT1080 fibrosarcoma, whereas the integrin binding in human dermal fibroblasts expressing a range of integrins was unaffected. Unlike EDC-NHS crosslinked films however, riboflavin-crosslinked films did not result in an increase in non-specific binding, whilst possessing comparable ultimate tensile strengths to EDC-NHS crosslinked collagen membranes [36]. Additionally, an investigation of plastically compressed collagen scaffolds by [PERSON] et al. observed a decrease in oxygen diffusivity and in human dermal neonatal fibroblast viability after crosslinking a compressed collagen (Type I, rat tail, single molecule) scaffold [37]. However, this cytotoxicity may arise from an interaction between the compression and crosslinking, since greater degrees of plastic compression resulted in a more significant decrease in cell viability (67%) than the riboflavin crosslinking process (79%).  
Transglutaminase  
Transglutaminases refer to a family of transferase enzymes that crosslink proteins through the formation of a bond between an \(\varepsilon\)-amine (lysine) and \(\gamma\)-carboxyl in glutamines, as shown in Figure 6. There are many types of transglutaminases, including microbial transglutaminase, factor XII, epidermal, keratinocyte and tissue transglutaminases, which are commonly found as crosslinking agents in skin, hair and blood clots in vivo. Of these enzymes, tissue transglutaminase 2 (TG2), is a calcium-dependent enzyme that has shown an exceptionally high cellular response, including an increase in the number of osteoblast adhesion after TG2-crosslinking of freeze-dried Type I calf-skin-derived collagen scaffolds [38].  
Transglutaminases bind to glutamines in the polypeptide chain, activating them for further reactions, as depicted in Figure 6[39]. In the presence of water, this results in the conversion of the glutamine to a glutamate residue, whereas, in the presence of a suitable amine, an amide bond is formed at the site of the activated glutamine [39]. Thus, TG2 can either serve to act as an amide crosslinker that does not utilise pre-existing aspartic or glutamic acids (E and D) in the formation of crosslinks, or, conversely, could help to re-introduce glutamates (E) in the substrate, which may increase the number of MIDAS motifs available on the substrate for integrin recognition.  
In an investigation of the mechanical properties of TG2 by [PERSON] et al., the global mechanical properties such as tensile modulus and failure strength of TG2-treated films were lower than non-crosslinked Type I bovine dermal insoluble collagen films, whereas the strain to failure and plasticity was observed to increase [14]. Regardless, the human dermal fibroblasts seeded on these substrates were well-spread and attached to the substrate, with low cytotoxicity and high cell proliferation.