Pristine Photopolymerizable Gelatin Hydrogels: A Low-Cost and Easily Modifiable Platform for Biomedical Applications

Abstract

The study addresses the challenge of temperature sensitivity in pristine gelatin hydrogels, widely used in biomedical applications due to their biocompatibility, low cost, and cell adhesion properties. Traditional gelatin hydrogels dissolve at physiological temperatures, limiting their utility. Here, we introduce a novel method for creating stable hydrogels at 37 °C using pristine gelatin through photopolymerization without requiring chemical modifications. This approach enhances consistency and simplifies production and functionalization of the gelatin with bioactive molecules. The stabilization mechanism involves the partial retention of the triple-helix structure of gelatin below 25 °C, which provides specific crosslinking sites. Upon activation by visible light, ruthenium (Ru) acts as a photosensitizer that generates sulphate radicals from sodium persulphate (SPS), inducing covalent bonding between tyrosine residues and "locking" the triple-helix conformation. The primary focus of this work is the characterization of the mechanical properties, swelling ratio, and biocompatibility of the photopolymerized gelatin hydrogels. Notably, these hydrogels supported better cell viability and elongation in normal human dermal fibroblasts (NHDFs) compared to GelMA, and similar performance was observed for human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). As a proof of concept for functionalization, gelatin was modified with selenous acid (GelSe), which demonstrated antioxidant and antimicrobial capacities, particularly against E. coli and S. aureus. These results suggest that pristine gelatin hydrogels, enhanced through this new photopolymerization method and functionalized with bioactive molecules, hold potential for advancing regenerative medicine and tissue engineering by providing robust, biocompatible scaffolds for cell culture and therapeutic applications.

Keywords: biomaterial; cardiovascular; chronic wounds; gelatin; hydrogel; selenium; tissue engineering.

Figure 1

Hydrogel formation. Schematic representation of the polymerization process of pristine gelatin.

Figure 2

Gelatin functionalization with selenium (GelSe). (A) Synthesis reaction of GelSe. (B) Schematic representation of GelSe hydrogel formation. (C) Schematic representation of the obtention of gelatin with different degree of functionalization (DoF) by mixing GelSe/GelSe-H (high DoF) with pristine gelatin in proportions 1:1 and 1:2 to obtain GelSe-M (medium DoF) and GelSe-L (low DoF), respectively.

Figure 3

Rheological and photorheological tests (n = 3, 10 rad/s oscillation frequency, 10% shear strain) for the analysis of the viscosity and the viscoelastic properties of the pristine gelatin and polymerize pristine gelatin. (A) Viscosity profile of pristine gelatin at different concentrations (10, 7.5 and 5% w/v). (B) Temperature sweep of pristine gelatin. (C) Time sweep of gelatin at 37 °C (light activated at 20 s, 35 s exposure time, 7.5 cm light probe-to-sample distance). (D) Time sweep of gelatin previously incubated for 5 min at 21 °C (light activated at 20 s, 35 s exposure time, 7.5 cm light probe-to-sample distance). (E) Amplitude sweep of pristine gelatin hydrogel. (F) Frequency sweep of pristine gelatine hydrogel.

Figure 4

Autofluorescence of dityrosine groups in response to UV light exposure. (A) Light-activated pristine gelatin hydrogels. (B) Non-activated pristine gelatin hydrogels. (C) Ru/SPS 1/10 mM solution in PBS. (D) Pristine gelatin 10% w/v in PBS.

Figure 5

Swelling ratio. (A) Representation of the hydrogel diameter in cm after incubation in PBS at 37 °C over 30 days (N = 12). (B) Images showing the evolution of the hydrogel incubated in PBS at 37 °C over 30 days.

Figure 6

Material addition to cell culture. (A) Alamar Blue assay of pristine gelatin and GelMA 10% w/v addition to NHDFs cultured in a 96-well plate. (B) Alamar Blue assay of pristine gelatin and GelMA 10% w/v addition to hiPSC-CMs cultured in a 96-well plate. (N = 3, unpaired t-test, ** p < 0.005, ns: no significant differences).

Figure 7

Cell encapsulation. (A) Alamar Blue assay of NHDFs encapsulated within pristine gelatin and GelMA 10% w/v. (B) Alamar Blue assay of hiPSC-CMs encapsulated within pristine gelatin and GelMA 10% w/v. (N = 3, unpaired t-test, * p < 0.05, ** p < 0.005, ns = no significative differences). (C) Fluorescence images of Live/Dead^® assay of NHDFs encapsulated within pristine gelatin and GelMA 10% w/v. (D) Fluorescence images of Live/Dead assay of hiPSC-CMs encapsulated within pristine gelatin and GelMA 10% w/v. Live cells produced green fluorescence and dead cells showed red fluorescence.

Figure 8

(A) ⁷⁷Se-NMR of GelSe. (B) ¹H-NMR of pristine gelatin (blue) and GelSe (red).

Figure 9

Antioxidant properties of GelSe. DPPH analysis of GelSe-H. Results were calculated relative to a positive control (ascorbic acid 2 mg/mL). The “Ascorbic acid” result is the antioxidant capacity of ascorbic acid at 0.023 mg/mL, the equivalent concentration of selenium found in GelSe-H. (N = 3, unpaired t-test, *** p < 0.001).

Figure 10

Antibacterial properties of GelSe against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). (A) Antibacterial properties of GelSe/GelSe-H. Agar plate culture of a sample from a 96-well plate containing 1 million bacteria of E. coli (left) and S. aureus (right), treated with five different concentrations of GelSe (GelSe-H.1 (13%), H.2 (10%), H.3.3 (6.67%), H.4 (5%), and H.5 (3.33% w/v)). (B) Antibacterial properties of GelSe-M.1 to GelSe-M.5 with E. coli (left) and S. aureus (right). (C) Antibacterial properties of GelSe-L.1 to GelSe-L.5 with E. coli (left) and S. aureus (right), including a control for each bacterial strain using pristine gelatin.

Figure 11

Biocompatibility properties of different concentrations of GelSe-H and GelSe-M added to NHDFs. (A) Alamar blue assay (N = 3, unpaired t-test, *** p < 0.001, ns = no significative differences). (B) Fluorescence images of Live/Dead^® assay. Live cells produced green fluorescence and dead cells showed red fluorescence. Scale bars are 100 µm.

Figure 12

Comparative table of GelSe-H and GelSe-M showing their antibacterial and biocompatibility properties. GelSe-M.3 is proposed as the best candidate, as it demonstrates good cell viability and antibacterial properties. X: not bactericidal/not biocompatible; ✓: bactericidal/biocompatible.