A novel antibacterial hydrogel containing aminophylline as a versatile platform for neural differentiation of hWJMSCs through the CREB pathway

Abstract

This study aims to develop a novel antibacterial hydrogel scaffold composed of gelatin (Gel), amniotic membrane extract (AME), and aminophylline (AMP) for neural regeneration. We investigate its ability to sustain AMP release, inhibit bacterial growth, and promote neural differentiation of human Wharton's jelly mesenchymal stem cells (hWJMSCs) via the CREB pathway, addressing unmet needs in neural tissue engineering. The composite hydrogels were synthesized and characterized using various methods and techniques, including X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy (FTIR), porosity, contact angle, water uptake, thermogravimetric analysis (TGA), biodegradation, tensile strength, drug release, and antibacterial activity. Biocompatibility tests (MTT assay, AO/EB staining) confirmed > 95% viability of hWJMSCs over six days and their differentiation to the neural cells was analyzed through immunocytochemistry (ICC) staining and real-time reverse transcription-polymerase chain reaction (RT-PCR) at different time points. The results demonstrate the successful synthesis of porous hydrogels with desirable properties, including hydrophilicity, thermal stability, biodegradability, and mechanical strength. The hydrogels support the sustained release of AMP (53.18% over 336 h) and exhibit antibacterial activity against Pseudomonas aeruginosa (90.52 ± 0.26%) and Staphylococcus aureus (93.06 ± 0.34%) due to the presence of penicillin and streptomycin (P-S) antibiotics. The biocompatibility results show that the hydrogels do not have a cytotoxic effect on the viability of human WJMSCs. The neural differentiation of human WJMSCs seeded on surface hydrogels was confirmed by evaluating specific neural markers at both protein and gene levels. In conclusion, the new antibacterial gel-based hydrogel can support the release of AMP and after further evaluation, can be introduced as a new candidate for neural repair applications.

Fig. 1

Relative cell viability of human WJMSCs via MTT assay in the presence of (A) different concentrations of AMP (0.5, 1, 10, 25, 50, 75, 100, and 200 μg/ml) and (B) cultured on Gel-Glu, Gel-Glu-AME, Gel-Glu-AME-P-S, and Gel-Glu-AME-P-S-AMP (0.5%, 1%, and 1.5% (v/v)) hydrogel scaffolds after 2, 4, and 6 days of cell seeding. The data were presented as mean ± SD and two-way ANOVA was performed with Tukey’s multiple comparisons test. Fluorescence micrographs of AO/EB stained human WJMSCs in the presence of (C) different concentrations of AMP (0.5, 1, 10, 25, 50, 75, 100, and 200 μg/ml) after 2, 4, and 6 days of cell culture and (D) cultured on different hydrogel scaffolds ((a) Gel-Glu, (b) Gel-Glu-AME, (c) Gel-Glu-AME-P-S, (d) and Gel-Glu-AME-P-S-AMP 0.5%, (e) Gel-Glu-AME-P-S-AMP 1%, and (f) Gel-Glu-AME-P-S-AMP 1.5%) after 6 days of cell culture. For AO/EB staining, living cells emitted green fluorescence, while dead cells displayed red fluorescence. The results of the MTT assay and AO/EB staining show that all the hydrogel polymers are biocompatible and that there is no significant cytotoxic effect of all composite hydrogels and AMP on the human WJMSCs over 6 days. AMP at 10 μg/ml concentration and Gel-Glu-AME-P-S-AMP1% composite hydrogel were selected for further investigations.

Fig. 2

The FTIR spectra of (A) pure (Gel, AME, AMP), and hydrogel scaffolds (Gel-Glu, Gel-Glu-AME, Gel-Glu-AME-P-S, and Gel-Glu-AME-P-S-AMP 1%. The XRD spectrum (B) of (a) pure (Gel, AME, AMP), and (b) hydrogel scaffolds (Gel-Glu, Gel-Glu-AME, Gel-Glu-AME-P-S, and Gel-Glu-AME-P-S-AMP 1%. FESEM micrographs of (C) seeded human WJMSCs on (a) Gel-Glu (SEM MAG: 350 kX; SEM kV: 15 kV), (b) Gel-Glu-AME, (c) Gel-Glu-AME-P-S, and (d) Gel-Glu-AME-P-S-AMP 1% (SEM MAG: 5 kX; SEM kV: 15 kV) hydrogel scaffolds. FESEM micrographs of (D) interconnected porous structure of Gel-Glu-AME-P-S-AMP 1% hydrogel scaffold (SEM MAG: 100X; SEM kV: 15 kV).

Fig. 3

Surface hydrophilicity and contact angle of (A) different hydrogel scaffolds (a) Gel-Glu, (b) Gel-Glu-AME, (c) Gel-Glu-AME-P-S, and (d) Gel-Glu-AME-P-S-AMP 1%. The porosity rate of (B) Gel-Glu, Gel-Glu-AME, Gel-Glu-AME-P-S, and Gel-Glu-AME-P-S-AMP 1% hydrogel scaffolds. The water uptake rate of (C) the hydrogel scaffolds (Gel-Glu, Gel-Glu-AME, Gel-Glu-AME-P-S, and Gel-Glu-AME-P-S-AMP 1%.) at 96h. The remaining mass profile of (D) the hydrogel scaffolds (Gel-Glu, Gel-Glu-AME, Gel-Glu-AME-P-S, and Gel-Glu-AME-P-S-AMP 1%) was immersed in PBS at pH 7.4.

Fig. 4

TGA curves of (A) the Gel-Glu and Gel-Glu-AME-P-S-AMP 1% hydrogel scaffolds. The stress-elongation curve of (B) Gel-Glu-AME-P-S-AMP 1% hydrogel scaffold. In vitro releases of (C) AMP from Gel-Glu-AME-P-S-AMP 1% hydrogel scaffold in PBS at pH = 7.4. Antibacterial assays of the Gel-Glu and Gel-Glu-AME-P-S-AMP 1% hydrogel scaffolds against (D) S. aureus and (E) P. aeruginosa. The data are presented as mean ± SD (*P < 0.05, **P < 0.01).

Fig. 5

The protein expression of neural markers (MAP-2, β-tubulin III, and Gamma-enolase) in neural-differentiated cells in the presence of (A) AMP (10 μg/ml) and (B) hydrogel scaffolds ((a) Gel-Glu, (b) Gel-Glu-AME, (c) Gel-Glu-AME-P-S, and (d) Gel-Glu-AME-P-S-AMP 1%) after 14 days of neural induction. Green and blue (DAPI) staining indicate protein and nuclei of differentiated cells, respectively.

Fig. 6

Relative gene expression of (A) TUBB3, (B) MAP-2, (C) GFAP, (D) ENO2, and (E) CREB in neural differentiated cells in the presence of AMP (10 μg/ml) and hydrogel scaffolds (Gel-Glu, Gel-Glu-AME, Gel-Glu-AME-P-S, and Gel-Glu-AME-P-S-AMP 1%) at 7 and 14 days. The data are shown as mean ± SD (****P < 0.0001).