# Genipin Attachment of Conjugated Gold Nanoparticles to a Decellularized Tissue Scaffold
## 4 Discussion
The enhanced cellular viability was demonstrated almost immediately with both biocompatibility assays showing a difference between the controls and the genipin treated scaffolds as early as 24 h after plating the cells. This led us to believe the enhanced cellular capabilities are the results of improved cell adhesion. To confirm this, SEM images were acquired at day 1 and day 3 after the scaffolds were plated with fibroblast cells. The cell plated on the scaffolds crosslinked with genipin alone are undoubtedly flusher to the scaffold than the other two treatment types. The treatment of genipin allows the cells to more quickly adhere and regain their spindle like appearance in comparison to the scaffolds left uncrosslinked or the scaffolds treated with genipin and gold. Cell adhesion is a dynamic process involving interactions between cell cytoskeleton, extracellular matrix proteins, and peripheral membrane proteins. These adhesion protein complexes are crucial for the assembly of individual cells into the three-dimensional tissues and play an important role in further cell proliferation, viability, and differentiation [37]. It is well documented that physical surface properties, including stiffness, can significantly influence cell attachment. Forces generated by the cytoskeleton are applied to membrane attachment sites. This can deform materials that lack a degree of stiffness but cannot move an attachment site on a rigid surface. Consequently, cell morphology and functions hinge on substrate stiffness [38]. [PERSON] et al. previously found that the use of genipin crosslinking increased surface roughness and stiffness on a hydrogel surface. This in turn resulted in better cell attachment, and better cell adhesion was associated with higher cell viability and proliferation [39]. As shown in Figure 2, there was an abrupt increase in 10 days 24 h Gen and 24 h AuGen samples. In correlation, the DSC results showed the samples immersed in the genipin solution for 24 h had a higher degree of crosslinking. This crosslinking most likely resulted in increased scaffold stiffness, and this may have contributed to the increased cell growth at 24 h as shown in Figure 2.  
There is evidence that the improved cell attachment may be the result of direct genipin interactions with the cells. The switch from spherical to flattened shapes was not only demonstrated on the fibroblasts growing on the scaffolds, but this phenomenon was also witnessed in the cells attached to the well plate directly adjacent to the scaffolds crosslinked with genipin for 24 h. This is most likely the result of leaching of genipin, or other products of the crosslinking reaction, from the scaffold to the nearby cells. The exact mechanism for this is unclear, and there were no other cases of this phenomena cited in the literature. On the contrary, [PERSON] et al. hypothesized that genipin may impair cell adhesion as it halved the mRNA expression of essential cell adhesion protein integrin \(\beta\)1 in chondrocytes [40]. However, our results clearly demonstrate genipin supporting fibroblasts as they attach, spread out, and flatten both on the scaffold and neighboring to it.