*2.6. Crystal Violet Assay*

Crystal Violet (CV) staining was performed as follows: Cells were fixed in 50% MeOH-PBS for 3 h at 4 ◦C. For 15 min at room temperature, a 0.1% (m/v) CV, 5% MeOH staining solution was incubated. The staining solution was removed and the stained cells were washed with distilled water. The plate was left to dry for 5 min under a chemical hood. The bound dye was eluted with MeOH 100% for 30 min at 4 ◦C. The optical density of each well was measured at 570 nm using a Victor Multilabel plate-reader (Perkin-Elmer, Wellesley USA).

## *2.7. RNA Extraction and Real-Time PCR*

Prior to RNA extraction, cell retrieval was performed by digesting the collagen scaffold in collagenase solution. Collagenase type I (Sigma-Aldrich) was dissolved in DMEM without FBS at a concentration of 0.5 mg/mL. Samples were incubated in collagenase solution for 10 min at 37 ◦C. Cells suspension was pelleted, and RNA was extracted with an RNeasy® mini kit (Qiagen) following the manufacturer's instruction. A total of 500 ng of RNA was used to obtain cDNA using an iScript™ cDNA Synthesis Kit (BioRad). Real-time PCR was performed using SsoAdvanced Universal SYBR Green Supermix (BioRad), and normalized expression levels were calculated relative to control cells according to the 2−ΔΔCT method. Primers were purchased from Sigma-Aldrich. The sequences are listed in Table 1.


**Table 1.** Primer sequences.

> \* reference gene.
