# Genipin Attachment of Conjugated Gold Nanoparticles to a Decellularized Tissue Scaffold
## 4 Discussion  
Genipin was first utilized as a crosslinker in 1988 and since then its use has been studied for a wide array of applications [29]. In materials as diverse as the trachea, pericardium, cartilage, chitosan and gelatin gels, genipin has been successfully utilized to increase the mechanical strength and reduce  
Figure 4: Scanning electron microscopy images taken 1 and 3 days after fibroblasts were plated on the of the scaffold (**A**). Uncrosslinked samples plated for 1 day (**B**). 24 h crosslinking with 3 mM genipin and 20 nm gold nanoparticles plated for 1 day (**C**). 24 h crosslinking with 3 mM genipin plated for 1 day without nanoparticles (**D**,**G**). Uncrosslinked samples plated for 3 days (**E**,**H**). 24 h crosslinking with 3 mM genipin and 20 nm gold nanoparticles plated for 3 days (**F**,**J**). 24 h crosslinking with 3 mM genipin without nanoparticles plated for 3 days. Images (**G–I**) feature the same treatment type as (**D–F**) respectively, but with increased magnification for clarity. antigenicity [8; 30; 31; 32; 33]. Our study correlates with others on the ability of genipin to attach functionalized AuNPs to the tissue. The results of the study demonstrated the ability of genipin to conjugate gold nanoparticles to a decellularized porcine tissue scaffold while also maintaining good biocompatibility. The gold nanoparticles, functionalized with amino groups, allowed the genipin to covalently cross-link between the amino residues on the nanoparticles and the amino groups on the tissue. The modified cyclic form of genipin resides stably within the extracellular collagen matrix adding bridges from adjacent fibers to the functionalized AuNPs [34]. The NAA results demonstrated a correlation between the crosslinking time and the amount of AuNPs attached to the scaffold. An interesting result is noted in that there is an increase in the amount of AuNPs from 15 min to the 4 h immersion times. However, there is no significant increase from the 4 h to 8 h time point followed by a significant increase at 24 h. These biphasic results can be explained via the mechanism of genipin crosslinking. As explained by [PERSON] et al., genipin crosslinking occurs via two separate reactions involving different sites on the genipin molecule. The first reaction is a nucleophilic attack of the genipin C3 carbon atom from a primary amine group which occurs almost immediately. The second slower reaction is the nucleophilic substitution of genipin's ester group to form a secondary amide [34]. The results clearly demonstrated genipin's biphasic reaction. In addition, the results also demonstrated that the functionalized AuNPs will bind to the tissue without the use of a crosslinker; however, the amount is significantly lower. The DSC results provided additional confirmation on the binding ability of genipin. As shown in Figure 1, genipin demonstrated higher denaturation temperatures with the increased crosslinking times (15 min vs. 24 h) and with the higher genipin concentration (3 mM vs. 10 mM). The results also demonstrated the \"two stage\" binding ability of genipin in which the 4 h and 8 h crosslinking times displayed very similar denaturation temperatures. These results correlated with what other researchers reported. [PERSON] et al. utilized genipin to crosslink a collagen chitosan scaffold. The results showed that the longer crosslinking times and higher genipin concentrations led to increased mechanical strength in their scaffold. However, it was also seen that when genipin concentrations reached above 1% (_u/v_) the mechanical strength decreased [35]. Our biocompatibility results demonstrated that genipin is not cytotoxic as confirmed by previous results [36]. On the contrary, the scaffolds with longer incubation times showed both an increase in cell numbers (Figure 3) and cell proliferation (Figure 2) when compared to untreated samples. We saw this result with or without the addition of the AuNPs meaning the genipin alone is associated with this increased cell growth.