Fabrication of Bio-Based Gelatin Sponge for Potential Use as A Functional Acellular Skin Substitute

Abstract

Gelatin possesses biological properties that resemble native skin and can potentially be fabricated as a skin substitute for full-thickness wound treatment. The native property of gelatin, whereby it is easily melted and degraded at body temperature, could prevent its biofunctionality for various applications. This study aimed to fabricate and characterise buffalo gelatin (Infanca halal certified) crosslinked with chemical type crosslinker (genipin and genipin fortified with EDC) and physicaly crosslink using the dihydrothermal (DHT) method. A porous gelatin sponge (GS) was fabricated by a freeze-drying process followed by a complete crosslinking via chemical-natural and synthetic-or physical intervention using genipin (GNP), 1-ethyl-3-(3-dimethylaminopropyl) (EDC) and dihydrothermal (DHT) methods, respectively. The physicochemical, biomechanical, cellular biocompatibility and cell-biomaterial interaction of GS towards human epidermal keratinocytes (HEK) and dermal fibroblasts (HDF) were evaluated. Results showed that GS had a uniform porous structure with pore size ranging between 60 and 200 µm with high porosity (>78.6 ± 4.1%), high wettability (<72.2 ± 7.0°), high tensile strain (>13.65 ± 1.10%) and 14 h of degradation rate. An increase in the concentration and double-crosslinking approach demonstrated an increment in the crosslinking degree, enzymatic hydrolysis resistance, thermal stability, porosity, wettability and mechanical strength. The GS can be tuned differently from the control by approaching the GS via a different crosslinking strategy. However, a decreasing trend was observed in the pore size, water retention and water absorption ability. Crosslinking with DHT resulted in large pore sizes (85-300 µm) and low water retention (236.9 ± 18.7 g/m²·day) and a comparable swelling ratio with the control (89.6 ± 7.1%). Moreover no changes in the chemical content and amorphous phase identification were observed. The HEK and HDF revealed slight toxicity with double crosslinking. HEK and HDF attachment and proliferation remain similar to each crosslinking approach. Immunogenicity was observed to be higher in the double-crosslinking compared to the single-crosslinking intervention. The fabricated GS demonstrated a dynamic potential to be tailored according to wound types by manipulating the crosslinking intervention.

Keywords: biocompatibility; carbodiimide; gelatin sponge; genipin.

Figure 1

Crosslinking structure of (A) gelatin with genipin, (B) gelatin with EDC and (C) gelatin with double crosslinking of genipin and EDC.

Figure 2

Effect of crosslinking on gelatin sponge (GS) biostability. (A) Measurement of crosslinking degree using ninhydrin assay. Double crosslinking and a higher concentration of crosslinker resulted in a higher crosslinking degree. Significant difference was observed between GNP0.1 and the DHT group with the other group; */** represent a significant difference (p < 0.05; n = 3, N = 5) between groups. (B) GS degradation under enzymatic hydrolysis. Double crosslinking and higher concentration of crosslinker resulted in longer degradation time. (C) Mass change of GS as a function of temperature. Double crosslinking and higher concentration of crosslinker resulted in reduced total.

Figure 3

The effect of crosslinking on GS porosity. (A) Level of porosity of gelatin scaffold based on density displacement. Crosslinking resulted in reduction of porosity compared to control. (B) Representative SEM images of GS microarchitecture. Pore size range is depicted in yellow font. Double crosslinking and higher concentration resulted in identical microstructure with the control (30X magnification)/scale bar 100 µm. (C) WVTR measures water passage through the GS. Double crosslinking and higher concentration of crosslinker resulted in higher permeability. Only GNP0.1 and DHT demonstrated significantly lower permeability compared to the control as tested with Student’s t-test. (D) A lower contact angle indicates higher surface wettability of the GS. Double crosslinking and higher concentration of crosslinker resulted in higher wettability. Only GNP0.1 and DHT demonstrated significantly lower wettability compared to the control as tested with Student’s t-test. (E) Swelling ratio indicates the water absorption capability of GS. Double crosslinking and higher concentration of crosslinker resulted in lower water absorption. Only GNP0.5EDC30 demonstrated significantly lower water absorption compared to the control as tested with Student’s t-test. * represent a significant difference (p < 0.05; n = 3, N = 5) between groups.

Figure 4

Effect of crosslinking on chemical composition and structure. (A) FTIR peaks indicate the chemical functional group detected in GS. Crosslinking did not alter the chemical functional group composition of gelatin. (B) XRD diffractogram indicates the crystal structure of GS. Crosslinking did not alter the crystal structure of gelatin.

Figure 5

Effects of crosslinking on human epidermal keratinocytes (HEK) and dermal fibroblasts (HDF). Live/dead assay indicated the EDC crosslink scaffolds were toxic towards skin cells compared to genipin (GNP) and dihydrothermal (DHT) crosslink scaffold. (A) HDF cells and (B) HEK cells seeded on GS crosslink with GNP0.5/GNP0.5EDC30 at day 1 (D1) until day 3 (D3) (100X magnification). The white line circle indicates the dead cells. Effects of crosslinking on immunogenicity. (C) Proliferation properties of peripheral blood mononuclear cells (PBMCs) in medium supplemented with GS extracts in the carboxyfluorescein succinimidyl ester (CFSE)-labeling assay. (A–D) Representative FACS histograms of immune cells cultured with supplemented medium. Proliferation response without GS extracts was used as a control. PHA served as a positive control. PHA = phytohemagglutinin. The depicted line defines the level of proliferated immune cells. Results revealed that double crosslinking resulted in significant immune reaction in vitro. * p < 0.05 tested with Student’s t-test.

Figure 6

Cell attachment of (A) HEK and (B) HDF on GS crosslink with GNP0.5/GNP0.5EDC30 at 24 h. Significant difference at first hour of DF attachment are represented. Analysis of cell proliferation of (C) HEK and (D) HDF using MTT assay after 1, 3 and 7 days of cell seeding (n = 3). Cells seeded on both GNP0.5/GNP0.5EDC30 scaffolds proliferate efficiently after day 3. * p ≤ 0.05; ** p ≤ 0.01. Electron micrograph of crosslinked scaffold seeded with human dermal cell. (E) HDF cells and (F) HEK cells (I-P) seeded on GS crosslink with GNP0.5/GNP0.5EDC30 at day 1 (D1) until day 5 (D5) (250X magnification)/scale bar 20 µm are shown. White arrows indicate the cells attached.