Injectable Crosslinked Genipin Hybrid Gelatin-PVA Hydrogels for Future Use as Bioinks in Expediting Cutaneous Healing Capacity: Physicochemical Characterisation and Cytotoxicity Evaluation

Abstract

The irregular shape and depth of wounds could be the major hurdles in wound healing for the common three-dimensional foam, sheet, or film treatment design. The injectable hydrogel is a splendid alternate technique to enhance healing efficiency post-implantation via injectable or 3D-bioprinting technologies. The authentic combination of natural and synthetic polymers could potentially enhance the injectability and biocompatibility properties. Thus, the purpose of this study was to characterise a hybrid gelatin−PVA hydrogel crosslinked with genipin (GNP; natural crosslinker). In brief, gelatin (GE) and PVA were prepared in various concentrations (w/v): GE, GPVA3 (3% PVA), and GPVA5 (5% PVA), followed by a 0.1% (w/v) genipin (GNP) crosslink, to achieve polymerisation in three minutes. The physicochemical and biocompatibility properties were further evaluated. GPVA3_GNP and GPVA5_GNP with GNP demonstrated excellent physicochemical properties compared to GE_GNP and non-crosslinked hydrogels. GPVA5_GNP significantly displayed the optimum swelling ratio (621.1 ± 93.18%) and excellent hydrophilicity (38.51 ± 2.58°). In addition, GPVA5_GNP showed an optimum biodegradation rate (0.02 ± 0.005 mg/h) and the highest mechanical strength with the highest compression modulus (2.14 ± 0.06 MPa). In addition, the surface and cross-sectional view for scanning electron microscopy (SEM) displayed that all of the GPVA hydrogels have optimum average pore sizes (100−199 μm) with interconnected pores. There were no substantial changes in chemical analysis, including FTIR, XRD, and EDX, after PVA and GNP intervention. Furthermore, GPVA hydrogels influenced the cell biocompatibility, which successfully indicated >85% of cell viability. In conclusion, gelatin−PVA hydrogels crosslinked with GNP were proven to have excellent physicochemical, mechanical, and biocompatibility properties, as required for potential bioinks for chronic wound healing.

Keywords: 3D-bioprinting; PVA; bioinks; gelatin; injectable hydrogel; skin tissue; wound healing.

Figure 1

Experimental design for human skin cell isolation: (i) collection of human skin sample, (ii) cell isolation: primary HDFs and KCs, (iii) cell differential prior primary HDFs expansion, (iv) primary HDFs expansion in T75 cell culture flasks, (v) in vitro cell seeding (primary HDFs) on hydrogel.

Figure 2

(a) Schematic of the hydrogel fabrication steps, (b) gross appearance (top view and cross-sectional) of hydrogel, and (c) polymerisation time for non-crosslinked and crosslinked hydrogels, where * indicates p < 0.05.

Figure 3

Physicochemical analysis of GE, GPVA3, and GPVA5 (a) % of swelling ratio (°), (b) contact angle, (c) water vapor transmission rate (WVTR) (gm⁻²h⁻¹), and (d) biodegradation rate. * indicates p < 0.05.

Figure 4

Mechanical properties analysis of gelatin, GPVA3, and GPVA5: (a) compression modulus, (b) concentration of amine groups, (c) percentage of resilience, (d) viscosity, and (e) TGA analysis, where * indicates p <0.05.

Figure 5

Chemical characterisation of hydrogel. (a) FTIR spectra of pure gelatin, PVA, genipin, and fabricated hydrogels, and (b) crystallinity of hydrogels via X-ray diffraction analysis (XRD).

Figure 5

Chemical characterisation of hydrogel. (a) FTIR spectra of pure gelatin, PVA, genipin, and fabricated hydrogels, and (b) crystallinity of hydrogels via X-ray diffraction analysis (XRD).

Figure 6

SEM images showing the cross-sectional microporous structure of the hydrogels: (a) GE_NC, (b) GPVA3_NC, (c) GPVA5_NC, (d) GE_GNP, (e) GPVA3_GNP, and (f) GPVA5_GNP under 100× magnification; (g) average pore size (μm); (h) porosity percentage; (i,j) AFM analysis for surface roughness. * Indicates significant difference.

Figure 6

SEM images showing the cross-sectional microporous structure of the hydrogels: (a) GE_NC, (b) GPVA3_NC, (c) GPVA5_NC, (d) GE_GNP, (e) GPVA3_GNP, and (f) GPVA5_GNP under 100× magnification; (g) average pore size (μm); (h) porosity percentage; (i,j) AFM analysis for surface roughness. * Indicates significant difference.

Figure 7

HDF cytotoxicity and interaction with hydrogels. (a,b) Live and dead assay and cytotoxicity evaluation of HDFs with hydrogel through seeding, pre-mix, and 3D-bioprinting techniques under 100× magnification. (c) Cell morphology on the surface of hydrogels, and after pre-mixing in the hydrogels, under 10× magnification.

Figure 8

Assessment of the potential bioinks using the 3D-bioprinting fabrication technique: (a) printability assessment, (b) gross appearance of the 3D-printed hydrogels, (c) SEM images of the 3D-printed hydrogels, and (d) average pore sizes of the 3D-printed hydrogels. Whereas, * indicates the significant difference.