## 4 Summary  
An ideal tissue engineering construct will possess the mechanical properties required to withstand the global forces, and biochemical cues to promote controlled cell invasion and proliferation at the site of injury. Due to the mechanisms employed by chemical crosslinkers, it is impossible to modify the mechanical properties without altering the chemical structure of collagen. The biological and chemical behaviour exhibited by crosslinked collagen is summarised in Table 1.  
Figure 6: Crosslinking mechanism of tissue transglutaminase. Illustration of proposed mechanisms by [PERSON] et al. [39]. Image modified from [PERSON] et al. [14] licensed under CC-BY 4.0. As the number of crosslinking methods and cell types evaluated in the literature continues to grow, determining the ideal crosslinker choice for a given application has become a non-trivial task. One approach to addressing these issues draws from established representations of materials properties through Ashby Materials Selection plots. In such plots, several of the underlying interactions are abstracted, allowing distinct classes of materials to be compared along the same property space. An attempt to create a materials selection map is illustrated in Figure 7, representing schematic trends for the 'bioactivity' (through attachment and cytotoxicity) and'mechanics' based on the literature reviewed in this chapter. Within these observations, there is significant complexity and diversity in both mechanical properties (tensile, compressive, shear as well as strength, strain, viscoelasticity, etc.) and biological responses (cell lines, monoculture vs. co-cultures, attachment vs. proliferation vs. migration). As a result, this materials selection map is intentionally left without exact values since the trends were limited to observations made in systematic and comparative studies of crosslinked collagen. Regardless, the schematic demonstrates the potential to extract and compare multiple properties upon crosslinking, with protocol standardisation and systematic comparisons of crosslinkers in future studies. \begin{table}
\begin{tabular}{c l l} \hline \hline
**Crosslinker** & **Advantages** & **Disadvantages** \\ \hline \multirow{2}{*}{GTA} & \(\bullet\) Increased elastic modulus [1, 13] & \(\bullet\) Non-zero length crosslinker [1] \\  & \(\bullet\) Cytotoxic [8] \\ \hline \multirow{2}{*}{EDC-NHS} & \(\bullet\) Increased elastic modulus [13, 17] & \(\bullet\) Loss of some integrin-specific \\  & \(\bullet\) Zero length crosslinker [2, 12] & binding [7] \\ \hline \multirow{4}{*}{UV} & \(\bullet\) Non-specific reaction sites [24, 25] \\  & \(\bullet\) Integrin binding unaffected [24] & \(\bullet\) Low crosslink densities [24] \\  & \(\bullet\) Zero length crosslinking [24, 25] & \(\bullet\) Collagen fragmentation [26] \\  & & \(\bullet\) Some denaturing [26] without use \\  & & of glucose [24, 27] \\ \hline \multirow{2}{*}{DHT} & \(\bullet\) Integrin binding unaffected [13] & \(\bullet\) Denaturation of collagen [21] \\  & \(\bullet\) Zero length crosslinking & \\ \hline \multirow{4}{*}{Genipin} & \(\bullet\) Non-cytotoxic [34] & \(\bullet\) Not a zero-length crosslinker [31] \\  & \(\bullet\) Mechanisms should not affect & \(\bullet\) Imparts a blue colour [33] \\  & GXOGER motifs in collagen [31] & \\ \hline \multirow{4}{*}{Riboflavin} & \(\bullet\) Mechanism should not affect & \\  & GXOGER motifs in collagen [36] & \(\bullet\) Some, albeit limited, loss of \(\alpha_{2}\beta_{1}\) \\  & \(\bullet\) Comparable ultimate tensile & mediated binding \\  & strength to EDC-NHS [36] & \\ \hline \multirow{2}{*}{TG2} & \(\bullet\) Mechanism do not affect & \(\bullet\) Can cause morphological changes \\  & GXOGER motifs in collagen [39] & \(\bullet\) Can result in a reduction of \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Summary of the advantages and disadvantages of current crosslinking systems used in collagen-based constructs.