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INTRODUCTION: Pelvic Organ Prolapse (POP) is characterized by the descendance of the pelvic organs due to weakening of the pelvic floor. Up to 20% of the women get recurrence after surgery, implying that surgical outcomes are poor due to suboptimal wound healing[1]. Tissue engineering has shown great potential in stimulating regeneration by combining cells, biomaterials and biochemical cues. Polyisocyanopeptide (PIC) hydrogels are synthetic, thermosensitive and highly biomimetic, displaying stress-stiffening behavior similar to other biomacromolecules[2]. Furthermore, the PIC hydrogel can be functionalized with growth factors, like basic fibroblast growth factor (bFGF) to further promote cell proliferation and extracellular matrix (ECM) remodeling[3]. In this study, we developed a bFGF-functionalized hydrogel and investigate its wound healing capabilities in vitro.
Methodology: PIC polymers were synthesized and conjugated with a cell-adhesive peptide GRGDS as previously reported[4]. bFGF was reacted with DBCO-PEG4-NHS and Alexa647-NHS at 3 and 1.5 equivalent respectively in PBS. Next, the bFGF-DBCO was purified over a 10 kDa spin filter and conjugated to the PIC polymer overnight at 4°C. The bioactivity of PIC-bFGF was validated on 3T3 fibroblasts and human adipose-derived stem cells (ADSCs) using the CellTiter-Glo® assay. To assess its wound healing capabilities in vitro, the PIC-bFGF (50 ng/mL), encapsulated with ADSCs was evaluated at day 1, 7, 14 and 28. Cell viability was visualized with a LIVE/DEAD staining. ECM deposition was evaluated by quantifying (Sirius red staining) and visualizing (CNA-OG488) collagen and quantification of elastin (FastinTM Elastin assay).
RESULTS: Dose-response curves were generated to validate the bioactivity of PIC-bFGF (EC50 = 18.3 ng/mL), showing a 3-fold induction in proliferation which is in line with the positive control whereby bFGF is added soluble to the PIC-RGD hydrogel (EC50 = 3.7 ng/mL). ADSCs (EC50 = 17.9 ng/mL) are highly viable in the PIC-bFGF. Furthermore, total collagen amount significantly increases up to day 7 (p<0.001; data not shown) resulting in mature collagen network at day 14. Preliminary data shows no significant increase in total collagen and elastin in the PIC-bFGF versus PIC-RGD yet.
CONCLUSION: PIC-bFGF is bioactive in both 3T3 and ADSCs. Therefore, the PIC-bFGF is very promising and will be studied to investigate whether wound healing can be promoted in vivo. Further research is ongoing to provide more insight in the promoting effect of PIC-bFGF versus PIC-RGD in ADSCs regarding ECM metabolism in vitro.
REFERENCES: 1 Ismail, S. et al., Int Urogynaecol J. 27, 1619-1632 (2016). 2 Das, R. et al., Nat. Mater. 15, 318-325 (2016). 3 van Velthoven, M.J.J. et al., manuscript in preparation (2022). 4Liu, K. et al., ACS. Appl. Mater. Interfaces. 212, 56723-56730 (2020).
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