[Effects of polyvinyl alcohol-boric acid-based functionalized hydrogels on HSFs and HaCaT cells]

Objective: To investigate the effects of polyvinyl alcohol-boric acid (PVA-BA)-based functionalized hydrogels on human skin fibroblasts (HSFs) and HaCaT cells, providing experimental evidences for subsequent in vivo studies on wound repair and clinical translation of this hydrogel system. Methods: This study was an experimental investigation based on grouped and repeated measures designs. Based on PVA-BA, a freeze-thaw cycling method was employed to prepare PVA-BA hydrogel, PVA-BA-S hydrogel loaded with SB431542 at a final molarity of 5 μmol/L, and PVA-BA-B hydrogel loaded with BML-284 at a final molarity of 1 μmol/L. Fourier transform infrared spectroscopy and X-ray diffraction were employed respectively to characterize the characteristic absorption peaks and diffraction peaks of the above-mentioned three hydrogels. The drug release of PVA-BA-S hydrogel and PVA-BA-B hydrogel in phosphate-buffered saline (PBS) at pH 5.5 and 7.4 was detected by liquid chromatography, and the cumulative drug release rate at 48 hours of immersion was calculated. According to the random number table method, HSFs were divided into control group with conventional culture, activation-only group with conventional culture after treatment with recombinant human transforming growth factor-β₁ (TGF-β₁) protein for 24 hours, as well as PVA-BA group and PVA-BA-S group cultured with PVA-BA hydrogel extract and PVA-BA-S hydrogel extract, respectively, after treatment with recombinant human TGF-β₁ protein for 24 hours. After 24 hours of culture, mRNA expression levels of TGF-β signaling pathway-related factors α-smooth muscle actin (α-SMA), TGF-β, Smad2, Smad3, type Ⅰ collagen (COL Ⅰ), and COL Ⅲ in HSFs were detected using real-time fluorescence quantitative reverse transcription polymerase chain reaction. Protein expression levels of COL Ⅰ and COL Ⅲ in HSFs were assessed via the immunofluorescence method. According to the random number table method, HaCaT cells were divided into control group with conventional culture, PVA-BA group cultured with PVA-BA hydrogel extract, and PVA-BA-B group cultured with PVA-BA-B hydrogel extract. After 24 hours of culture, mRNA expression levels of Wnt signaling pathway-related factors β-catenin, matrix metalloproteinase-9 (MMP-9), E-cadherin, and N-cadherin in HaCaT cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction. The protein expression level of keratin 5 was assessed via the immunofluorescence method. Except for characterization, the sample size for each experiment was 3. Results: The Fourier transform infrared spectroscopy spectra of the PVA-BA hydrogel, PVA-BA-S hydrogel, and PVA-BA-B hydrogel all exhibited stretching vibration peaks corresponding to C-O and B-O bonds within the B-O-C group at wavenumbers of 1 100 and 1 450 cm⁻¹, and the X-ray diffraction patterns all formed a sharp diffraction peak at around 20°. At 48 hours of immersion, the cumulative drug release rate of PVA-BA-S hydrogel in PBS at pH 5.5 was (70.9±2.3)%, which was significantly higher than (60.0±2.2)% in PBS at pH 7.4 (t=6.02, P<0.05); the cumulative drug release rate of PVA-BA-B hydrogel in PBS at pH 5.5 was (83.9±2.2)%, which was significantly higher than (65.2±1.7)% in PBS at pH 7.4 (t=11.63, P<0.05). After 24 hours of culture, the mRNA expression levels of TGF-β, COL Ⅰ, COL Ⅲ, and Smad3 of HSFs in PVA-BA-S group were significantly higher than those in control group (P<0.05), the mRNA expression levels of TGF-β, COL Ⅰ, COL Ⅲ, α-SMA, Smad2, and Smad3 were significantly lower than those in activation-only group and PVA-BA group (P<0.05), and the protein expression levels of COL Ⅰ and COL Ⅲ of HSFs in PVA-BA-S group were significantly lower than those in activation-only group and PVA-BA group (P<0.05). After 24 hours of culture, compared with those in control group and PVA-BA group, the PVA-BA-B group showed significantly increased mRNA expression levels of β-catenin, MMP-9, and N-cadherin (P<0.05) and a significantly decreased mRNA expression level of E-cadherin (with P values both <0.05) in HaCaT cells; the protein expression level of keratin 5 of HaCaT cells in PVA-BA-B group was significantly higher than that in control group and PVA-BA group (with P values both <0.05). Conclusions: The PVA-BA-based PVA-BA-S and PVA-BA-B hydrogels can effectively inhibit the fibrotic phenotype of activated HSFs and enhance the migratory ability of HaCaT cells by regulating the TGF-β signaling pathway and Wnt signaling pathway, respectively, providing a novel drug delivery strategy for functional wound healing.