## 3 An Overview of Selected Chemical Crosslinkers
### Edc-Nhs  
EDC(1-ethyl-3-(3-dimethylaminopropyl) carbodimide)-NHS(N-hydroxysuccinimide) is a common collagen crosslinker offering a non-cytotoxic, zero-length crosslinking alternative to GTA. The zero-length crosslinking results in access being limited to 'neighbouring' free amines [12], following a reaction mechanism, as illustrated in Figure 2. These crosslinks occur between glutamates and arginines/lysines, in either case affecting the GXOGER sequence that is recognised by the integrins which mediate cell binding on collagen, where the amino acid X is most commonly phenylalanine (F). With EDC-NHS, the standard '100% concentration' [2; 15] is often defined as a precise molar ratio of 5:2:1 EDC:NHS:COO\({}^{-}\), where COO\({}^{-}\) refers to the carboxylate groups in the protein. Assuming a ratio of 120 COO\({}^{-}\) for a collagen chain with a molecular weight of 100,000 [2], this molar ratio can be re-written as a mass ratio corresponding to 1.150:0.276:1 EDC:NHS:Collagen, i.e., 1.150 _w/w_% EDC and 0.276 _w/w_% NHS. A scaling from this standard concentration would then be represented by the proportion of EDC and NHS to collagen. For instance, a 50% standard concentration would refer to a 0.575:0.138:1 mass ratio of EDC:NHS:COO\({}^{-}\). For consistency, concentrations will be expressed in terms of this industry standard. Where modifications are made to this standardised ratio, concentrations will be reported in terms of the mass ratio (_w/w_%) so that comparisons can be made with other chemical crosslinkers. EDC-NHS crosslinking allows for a lower crosslink density than traditional crosslink such as GTA, but provides a more hydrophilic surface, that has been shown to be beneficial for fibronectin activity and a greater degree of swelling compared with traditional crosslinkers on Type I bovine dermal swollen gel-derived collagen fibres (with chondro-6-sulfate additives) [12]. Furthermore, EDC-NHS was also shown to induce the self-assembly of collagen bundles roughly 300-nm in width with both acid-soluble and insoluble Type I bovine dermal collagen, suggesting the possibility of both intra-fibril and inter-fibril binding, in spite of being a zero-length crosslinker. The insoluble  
Figure 2: Crosslinking mechanism of EDC-NHS. Illustration redrawn from proposed mechanisms by [PERSON] et al. [13] Image modified from [PERSON] et al. [14] licensed under CC-BY 4.0.  
collagen fragments revealed an enhancement and localisation of the piezoelectric response along these self-assembled fibre bundles [16]. Historically, up to 200% (i.e., 10:4:1 EDC:NHS:COO\({}^{-}\)) of the standard condition has been used in crosslinking collagen, but extensive research on EDC-NHS composition and its effect on both cell migration and mechanics [13; 15; 17; 18; 19] suggest that much lower concentrations (10-100%) can retain the improvements in the mechanical properties imparted by crosslinking. For instance, [PERSON] et al. investigated extruded collagen fibres (Type I bovine dermal acid-swollen gel collagen) which were crosslinked at three different EDC-NHS concentrations (0.02 _w/w_ EDC%--0.006 _w/w_% NHS, 0.002 _w/w_ EDC%--0.0006 _w/w_% NHS and 0.0002 _w/w_ EDC%--0.00006 _w/w_% NHS) [18]. Of the three mechanical properties of interest to their study, the ultimate tensile strengths, in particular, were observed to be unaffected by the crosslinking concentrations used. In the same study by [PERSON] et al., human tenocytes derived from anterior cruciate ligaments and seeded on these extruded collagen fibres demonstrated lower levels of attachment on fibres that were heavily crosslinked after 24 h [18]. Analysis of the cell population after three weeks of seeding also indicated lower levels of tenocyte proliferation on the heavily crosslinked fibres. From these results, the authors speculated that the crosslinking conditions could be reduced by nearly two orders of magnitude without affecting the tensile properties desired for tendon repair [18].