## 2 Materials and Methods  
**Gel Preparation:** Solubilized collagen type I was obtained from Advanced BioMatrix, USA, with a stock concentration of 3.1 mg/mL. On ice, the solubilized collagen was mixed with 1 M HEPES buffer and 10X phosphate buffered saline (PBS), and diluted with deionized water, in order to reach final concentrations of 2.5 mg/mL collagen, 25 mM HEPES, and 1X PBS. Using a 0.5 M NaOH solution, the gel solution was titrated to a pH of 7.4. This base gel solution was used in both the control and two treatments. This neutralized collagen solution was split between use for material characterization and cell culture. In order to conserve the amount of genipin available, the treatment for these gels was split between pre-glycation and post-glycation, the difference between the two being that pre-glycated gels are crosslinked while still in solution and before polymerization, whereas post-glycation gels are crosslinked after being polymerized. To form the post-glycated collagen gels, genipin solutions were made by dissolving genipin in 10 mL 1X PBS to concentrations of 1 mM and 10 mM. After the base gels were polymerized, they were incubated in the respective genipin solution for 24 h to maximize glycation. To form the pre-glycated gels, solid genipin was added to the collagen solution and mixed thoroughly to ensure even distribution of genipin, making 13 and 22 mM gels. The solutions were then added to the wells and incubated at 37 \({}^{\circ}\)C, 5% CO\({}_{2}\). To make a higher concentration post-glycation solution requires more genipin than was available at the time of this experiment, so pre-glycation was used for higher concentration gels. A test sample of four 10 mM gels was created through both pre- and post-glycation and were tested to confirm the negligible difference in modulus value between glycation method. To wash out residual genipin, a series of washing steps was introduced. These steps involved four 60-min washes in 3% glycine, three 90-min washes in 70% ethanol, and four 30-min 1X PBS washes. The gels were washed on a rocker at room temperature, with two rinses with deionized water between each wash. After washing, the gels were incubated with EGM-2 medium at 37 \({}^{\circ}\)C, 5% CO\({}_{2}\) for one hour prior to seeding the cells. **Rheological characterization:** All rheological measurements were performed on a Discovery Hybrid Rheometer (DHR-2, TA Instruments, DE, USA). Separate collagen gels were prepared as described above and used for rheological purposes only. Samples were loaded on parallel plate geometries, and an oscillatory time sweep was used. This compressed the gels under 10% strain and an angular frequency of 1 Hz. The time sweep was run for one minute, acquiring a total of 10 measurements. Storage modulus and loss modulus were measured. Storage modulus is an elastic portion of collagen gels and loss modulus is a viscous portion of collagen gels. **Advanced Glycation End-product (AGE) Fluorescence:** To detect the presence of AGEs, the gels were prepared and polymerized in plastic cuvettes before being placed in a BioMate 3S UV-Visible Spectrophotometer (Thermo Fisher, Waltham, MA, USA). Using an excitation wavelength of 365 nm, an absorbance scan was run on the spectrophotometer using light beams spanning wavelengths from 200 to 400 nanometers (nm), using a blank cuvette as a baseline. Each sample was scanned in triplicate to ensure accuracy of the measurements. Data were saved in tabular form in Microsoft Excel and then recompiled into a graph using GraphPad Prism software. **Scanning Electron Microscopy (SEM):** In order to acquire proper scaffolds for SEM visualization, the gels were prepared in a 24-well plate and polymerized. They were then frozen at \(-20\)\({}^{\circ}\)C for three hours, before being lyophilized using a FreeZone 2.5 Plus freezedrier (LabConco, USA) for 14 h. To prepare the gels for imaging, a piece of each gel was cut and placed onto the SEM stage.