## 3 Results and Discussion
(**B**) Total degradation for each scaffold measured in change in average absorbance between Day 1 and Day 3. (**\(\mathbf{\ast}=p<0.001\)). Figure 2: Storage (G’) and loss (G”) moduli of control, pre- and post-glycated collagen scaffolds (\(\mathbf{\ast}\mathbf{\ast}p<0.0001\) between all samples). The difference in absorbance spectra between pre- and post-glycated scaffolds could potentially be from the pattern of color change exhibited in the gels. While the post-glycated gels maintained a yellow-brown hue, the pre-glycated gels often turned darker colors. Another main constituent of collagen helices is the amino acid proline, which has been reported to elicit a black color when reacting with genipin [14]. The action of genipin upon proline residues on the collagen could result in AGEs unique to pre-glycation, which would explain the formation of new peaks in the absorbance spectra. This exact mechanism is as of yet unknown, but may be due to genipin being dispersed throughout the still-solubilized gel solution, rather than acting upon the surface of already-polymerized gels. **Scanning Electron Microscopy (SEM):** As shown in Figure 4, SEM results show that collagen structural morphology was changed upon glycation with genipin. Both fiber size and network density were altered with increasing concentration of genipin, including a difference in structure depending on whether the scaffold was pre- or post-glycated (Figure 4). The formation of denser fiber networks was an expected result when glycating the scaffolds. The increase in storage modulus as a result of glycation in the collagen scaffolds could offer more plasticity to the fibers, allowing them to stretch--and become thinner as a result--without breaking, as opposed to the control gels. The difference between pre-and post-glycation was consistent, as post-glycated gels retained the somewhat porous morphology of the control gels, while the gels pre-glycated with genipin were essentially crosslinked from within, resulting in the tight-knit \"woven\" morphology observed in the SEM images. The structural morphology of glycated scaffolds was analyzed in terms of fiber diameter and network density using ImageJ. Post-glycated gels had more porous scaffolds with larger interfibrillar spaces, while pre-glycated gels were more tightly woven (Figure 5). Regardless of glycation method, both pre- and post-glycated gels showed significantly smaller collagen fibers than the control gel. **Cell proliferation and viability:** Cell proliferation was one of the markers used to determine the cytotoxicity of the genipin-crosslinked collagen hydrogel. The cells were  
Figure 4: SEM images of (**a**) control, (**b**,**c**) post-glycated of (**b**) 1 mM, (**c**) 10 mM, and (**d**,**e**) pre-glycated of (**d**) 13 mM, and (**e**) 22 mM scaffolds. imaged in triplicate on each day of the 3 days of growth in the 24-well plate. The results show that there was no significant difference in total cell proliferation across gel treatments (Figure 6). According to ISO 10993-5, cell viability above 80% is considered as non-cytotoxic or biocompatible. As shown in Figure 6, pre-glycated collagen (higher genipin concentration) shows more biocompatibility then pre-glycated collagen. Genipin was used as a noncytotoxic crosslinking agent which is safer than other common chemical crosslinkers such as glutaraldehyde [5]. However, most papers explored the actual cytotoxic nature of genipin, often using as small an amount as possible in order to optimize the balance between scaffold strength and cell viability. The effect of matrix hydrogel to host viable cells was not explored enough [8]. While this was the initial impression in this experiment upon glycation using genipin, the introduction of serial washes greatly improved cellular viability on scaffolds with higher concentrations of genipin. Another method of introducing genipin to hydrogels was to dissolve the compound in DMSO [10]. The standard procedure was to dissolve genipin in a 1:3 ratio of DMSO to PBS.