Introduction: Pelvic organ prolapse (POP) is a condition in which defects of supportive tissues due to changes in collagen metabolism result in bladder, bowel, and uterus descending into the vagina, which results in a lifetime surgery risk of 10% worldwide . Surgery comprises the plication of healthy connective tissue to cover the defect, which is called native tissue repair (NTR). However, the extracellular matrix (ECM) deposition and remodeling by fibroblasts in the tissue are already comprimised leading to a high failure rate. We hypothesize that the synthetic but strongly biomimetic thermosensitive polyisocyanopeptide (PIC)-based hydrogel  could be applied to the surgical site to deliver wound-healing promoting factors. To that end, PIC was decorated with cell-adhesive ligands containing the Arg-Gly-Asp (RGD) that allow for cell-matrix interactions. Here, we studied wound healing effects in terms of collagen, elastin and collagen-cleaving enzymes (MMP-2) for vaginal fibroblasts seeded on PIC gels, with different physical properties and RGD densities.
Methodology: In vitro experiments (n = 3) were performed using vaginal fibroblasts from POP tissue cultured on PIC hydrogels with RGD in 50 μM (PIC-RGD50) and 180 μM (PIC-RGD180) and without RGD (PIC-RGD0). Cell behavior and functionality were assessed from their morphology (fluorescent imaging of nucleus (Hoechst) and actin filaments (Phalloidin)), proliferation (WST-I assay) and ECM metabolism on the protein level [elastin (Fastin assay), collagen (picrosirius red), and MMP-2 production (zymography)] and gene level (quantitative PCR) up to 28 days of culture. As an illustration, data at day 21 is presented.
Results: After 4 days of culture, cell morphology images showed interconnected cell networks on PIC-RGD180, cell aggregates with elongations on PIC-RGD50, and round clusters appeared on PIC-RGD0. Vaginal fibroblast proliferation increased 2.5-fold on PIC-RGD180 (p < 0.01) and PIC-RGD50 (p < 0.005) compared to PIC-RGD0 at day 21. On PIC-RGD180, collagen (+60%, p < 0.005) and elastin (+40%, p < 0.01) deposition were significantly increased compared to PIC-RGD1% as well as active MMP-2 albeit not significantly (+20%, p > 0.05). Gene expression for collagen I, III, elastin and MMP-2 were in line with the protein results. The cells cultured on PIC-RGD0 did not produce these ECM factors at any detectable level.
Conclusion: The presented results show that PIC-hydrogel with RGD-ligand triggers regenerative behavior by vaginal fibroblasts in terms of collagen, elastin, and MMP-2 production, which are crucial factors in the healing process of pelvic connective tissue after POP surgery. We aim to apply PIC-RGD180 in vivo to study the host response and performance of PIC-hydrogel in NTR.
References: 1. Guler, Z.; Roovers, J.P., Biomolecules. 12, 94 (2022) 2. Kouwer, P.H.J. et al, Nature 493, 651-655 (2013) 3. Liu K, Biomacromolecules 20, 826-834 (2019).