Photocrosslinkable Gelatin Hydrogel for Epidermal Tissue Engineering

2.1. Physical Properties of GelMA Hydrogels

The synthesized GelMA was found to have a degree of methacrylation around 75%, consistent with previous report (data not shown).^[25] Upon UV exposure, the prepolymer solution of GelMA could form a crosslinked network (Figure S1A, Supporting Information). It was found that at all concentrations of GelMA ranging from 5% to 20%, the gel precursor solution could be injected via a conventional 27- gauge needle into a PDMS mold to form hydrogels of various shapes (round, square, star and triangle) after UV polymerization (Figure S1B, Supporting Information).

Firstly, we characterized the scaffold's mechanical properties as these are important parameters for engineering optimal skin substitutes. Compressive stress-strain curves illustrated a positive correlation between GelMA concentrations and compressive moduli, ranging from less than 5 kPa (5% GelMA) to ~110 kPa (20% GelMA) (p<0.05, Figure 1A and B), likely due to an increased crosslinking density at higher GelMA concentrations. High compressive moduli (~100 kPa) have previously been demonstrated to be favorable for keratinocyte growth.^[6]

Tensile stress-strain curves followed a similar trend with increased GelMA concentrations resulting in higher elastic moduli and ultimate strength (p<0.05) (Figure 1C). 20% GelMA hydrogels were found to have elastic moduli of up to 200 kPa and ultimate strengths of 30 kPa. Such high values are beneficial considering the significant amount of stretching and bending forces exerted during wound healing.^[26] Although higher GelMA concentrations correlated with reduced elongation at break points from 40% (5% GelMA) to 20% (20% GelMA) possibly due to the increased crosslinking density limiting hydrogel deformation, the 20% extensibility may still be considered appropriate for skin substitutes.^[8] In summary, robust yet tunable mechanical properties of GelMA hydrogels make them ideal candidates as substitute materials for skin regeneration as their properties can mirror the broad range of elasticity found in native skin.^[8]

We further characterized the hydrogel's swelling ratio which indicates water sorption capacity thus predicting the rate of hydrogel degradation.^[27] It was found that increasing GelMA concentrations from 5% to 20% resulted in reduced swelling ratios from 1500% to 500% (p<0.05) (Figure 1E), likely due to increased crosslinking densities at 20% GelMA. This not only limits the rate and amount of water penetration but is also thought to slow down degradation.^[28]

To evaluate degradation, GelMA hydrogels were incubated in collagenase solution. As shown in Figure 1F-G, the degradation rate decreased with increasing GelMA concentrations, with complete degradation by less than 3 days (5% GelMA) to upwards of 8 weeks (20% GelMA). Interestingly, 20% GelMA hydrogels remained present even after 8 weeks of incubation, albeit at significantly reduced size which may be attributable to increased methacrylamide crosslinks in 20% GelMA which are resistant to collagenase. Compared to previously developed collagen hydrogels which usually last for only 1 month,^{[16, 17]} GelMA hydrogels are thought to be better suited for long-term wound healing cases by remaining in the wound bed long-term,^[9] thus ensuring optimal healing whilst avoiding secondary infections.

The above results have suggested that the mechanical and degradation properties of GelMA hydrogels can be readily tuned to a great extent by varying GelMA concentrations. Compressive and elastic moduli could be tuned from a few kPa to a few hundred kPa and the degradation times could be varied from a few days to several months (Table 1), indicating the hydrogel's broad spectrum of properties as skin substitutes in different body sites and for different wound types.

2.2. Cell Viability, Adhesion and Proliferation on GelMA Hydrogels

2.3. In Vitro Epidermal Development on GelMA Hydrogels

After a monolayer of HaCaT cells was developed, the constructs were lifted to air-liquid interface (ALI) to induce HaCaT differentiation and stratification. Macroscopically, lifting the construct to the ALI for 2 weeks resulted in the formation of a cell multi-layer (Figure S2C, Supporting Information), which became thicker in week 6 under ALI conditions (Figure S2D, Supporting Information). Haematoxylin and eosin (H & E) stained images revealed that HaCaTs stratified and flattened on both GelMA (Figure 3A) and control collagen (Figure 3B) hydrogel surfaces after culture at ALI for 6 weeks. Basal keratinocytes showed columnar morphology whereas the keratinocytes further away from the constructs exhibited flattened morphology. With significant increase in thickness over time (p<0.05), the developed epidermis was approximately 20 μm, 50 μm and 100 μm after 2, 4 and 6 weeks, respectively, of ALI culture on both GelMA hydrogels and control collagen scaffolds (compare Figure 3Ai and ii, B i and ii). There was no significant difference in thickness between the epidermis grown on GelMA or collagen (p>0.05). The engineered epidermis on either scaffold was thinner compared to the sample of human abdomen epidermis (120 μm) (Figure 3C and D), but still within the range of native human epidermis (75 ~150 μm).^[13] It is noteworthy that after 6 weeks of ALI culture, GelMA hydrogels were still present as opposed to collagen scaffolds which almost disappeared, indicating long-term substrate stability using the 20% GelMA hydrogels.

Protein expression analysis of the reconstructed epidermis on GelMA (Figure 4A) or control collagen (Figure 4B) hydrogels demonstrated the appearance of both ki67 (proliferation marker) and involucrin (terminal differentiation marker, expressed in the suprabasal layers of stratified squamous epithelium) after 6 weeks of ALI culture. The reconstructed epidermis exhibited slightly disorganized structure possibly due to the immature terminal differentiation of HaCaT cells^{[34, 39]} or to the absence of paracrine signalling from fibroblasts during culture,^{[34-37, 40]} which may be normalized when cultured in vivo.^[36] With significant increase over time, the developed epidermis had 4, 6 and 10 layers after 2, 4 and 6 weeks, respectively, of ALI culture on either GelMA or collagen surfaces. Quantification of the layers of reconstructed epidermis showed no significant difference between the two substrates and their thickness was comparable with the normal human epidermis consisting of 8-12 keratinocyte layers (Figure 4C and D).^[42]

2.4. Barrier Formation of Reconstructed Epidermis

Electrical resistance measurements provide an indication of skin's barrier integrity and thus its relative hydration values.^[43] Statistical analysis demonstrated significant differences between the relative resistances of collagen scaffolds, collagen scaffolds with reconstructed epidermis (epidermis (C)), GelMA hydrogels (20%), and GelMA hydrogels with reconstructed epidermis (epidermis (G)) (p<0.05) (Figure 5A). Collagen scaffolds lacking an epidermis displayed least resistance at ~700 Ω which was more than twice lower compared to the resistance of the corresponding GelMA hydrogels (p<0.05). Barrier function of both scaffolds was predictably increased by the reconstructed epidermal layer compared to the cell-free substrates where the resistance of the epidermis (C) increased two-fold from 700 Ω to ~ 1400 Ω (700 Ω increase) and that of epidermis (G) from 1700 Ω to 2600 Ω (900 Ω increase) when compared to their cell-free equivalents. These results primarily indicated that the collagen scaffold had an inherently lower barrier integrity compared to GelMA hydrogels possibly due to high levels of crosslinking of the latter and lower water content,^[44] and secondly that the addition of an epidermal layer successfully increased barrier properties probably due to functional tight junctions and a stratified architecture of confluent keratinocytes.^{[45, 46]} The resistance values of the reconstructed epidermis were smaller than that of human skin (1,000-10,000 ohm) possibly due to less degree of stratifications resulting from immature terminal differentiation of HaCaT cells^{[34, 39]} or absence of paracrine signalling from fibroblasts during culture,^{[34-37, 39]} which may be normalized when cultured in vivo.^[41]

A tissue engineered skin substitute should ideally be able to control water loss from a wound bed^[47] to prevent excessive dehydration as well as the build-up of exudates.^[48] As expected, scaffolds without an epidermal cover, had significantly higher rates of water loss per day compared to those with an epidermis (p<0.05) (Figure 5B). Collagen scaffolds had the lowest ability to retain water with loss rates approaching 3 g m⁻² day⁻¹ which was almost twice the amount lost by GelMA hydrogels, possibly due to the higher crosslinking density of GelMA which improved physical water retention. The addition of an epidermal layer significantly reduced water loss for both collagen and GelMA scaffolds with water loss rates being almost identical at around 1 g m⁻² day⁻¹, less than the trans-epidermal water loss of normal human skin (~4 g m⁻² day⁻¹), possibly due to the lack of presence of sweat glands.^[45] Interestingly, no difference could be observed between the collagen scaffolds layered with an epidermis and naked GelMA, indicative of a synergistic barrier function between GelMA hydrogels and the epidermis. These results indicated the significant protective barrier function for GelMA based reconstructed epidermis exerted by both the crosslinking and the epidermal cover. This is important as dehydration remains as the major complications of untreated severe skin losses due to extreme trans-epidermal water loss.^[50]

Regenerated skin not only has to prevent loss of water from within the body but also functions as a relative barrier to harmful external insults. To evaluate the resistance of the reconstructed epidermis to external moisture, we studied the permeability of the scaffolds covered with epidermis and their cell-free counterparts to dextran solution. Dextran diffusion studies exhibited highest permeability at almost 100% for naked collagen scaffolds and lowest at 80% after 2 h of diffusion for epidermis (G) (Figure 5C). No difference was observed between epidermis (C) and the naked GelMA, corroborating the results obtained with water loss studies (see above). These results, again, highlighted the importance of both the presence of crosslinked networks and the epidermal cover in preventing diffusion of water molecules either way across the skin. The above results have demonstrated that the reconstructed epidermis had increased resistance and decreased trans-epidermal water permeability, indicative of improved barrier functions.

Our work demonstrates a simple, cost-effective technique to reconstruct functional epidermis using a hydrogel based on photocrosslinkable GelMA with robust mechanical, degradation and biological properties. These hydrogels were able to support the development of multi-layered, renewable keratinocytes with similar organization and differentiation as human epidermis. Changes in GelMA concentration would provide the means to fine-tune its physical and biological properties in order to meet specific requirements as skin substitutes in different body sites and for different wound types.^[51] Moreover, since GelMA is light-polymerizable, it could be easily molded or micropatterned into various shapes and configurations upon light exposure for a broad spectrum of tissue engineering applications.^[22] In terms of skin regeneration, photocrosslinkable hydrogels may be particularly useful in the treatment of trauma wounds which are frequently extensive and irregular. Wound beds of any shape may easily and homogenously be filled using liquid uncrosslinked hydrogel precursors. Upon light exposure, photocrosslinking can thus enable solidification of the precursor. Alternatively, light-polymerizable GelMA could be applied to the regeneration of palmoplantar epidermis with ridges and the interphase between palmoplantar and normal hairy skin, where a gradient in epidermis thickness exists. Furthermore, the hydrogel nature endows GelMA with ready modification of its chemical and physical properties (e.g., incorporation or conjugation of different growth factors). The unique combination of photo-polymerizability, optimal mechanical properties, biodegradability and biocompatibility make GelMA a promising material for skin tissue engineering.^[22] Such exceptional characteristic of GelMA also distinct them from other reported substrates to reconstruct epidermis including polycarbonate membrane^[52] or decellularized porcine intestine^[53] which cannot be tailor made according to the patient's own wounds and whose physical and chemical properties cannot be readily modified. We anticipate that the developed GelMA hydrogel could find applications as epidermal substitutes, wound dressings or substrate to construct in vitro skin models. Future work will include the evaluation of fibroblasts within the dermal layer towards commercialization of this skin equivalent.

Synthesis of GelMA

Synthesis of GelMA was described previously.^[54] Briefly, 10.0 g of type A porcine skin gelatin (Sigma-aldrich, St. Louis, MO) was added into 100 mL of Dulbecco's phosphate-buffered saline (DPBS) (Invitrogen, San Diego, CA) and dissolved by stirring at 60°C using magnetic stirrer. 8.0 mL of methacrylic anhydride was then added to react with the gelatin solution under vigorous stirring for 3 h at 50°C. Then, the reaction was stopped by a 5-fold dilution of the polymer solution with warm (40°C) DPBS. Salts and unreacted methacrylic anhydride were removed from the mixture by 1-week dialysis with 12-14 kDa cut-off in distilled water at 40°C. White porous foam was then obtained by lyophilizing the solution for 1 week and was store at −80°C until further use. The degree of methacrylation was defined as the ratio of the number of methacrylamide groups tagged to gelatin to the number of amine groups in unreacted gelatin. Using ¹H NMR (Varian Inova 500), such value was obtained by the integration of peaks at 7.4 ppm corresponding to the aromatic residues of gelatin, and peaks at 5.5 ppm and 5.7 ppm corresponding to methacrylamide groups.^[25]

Preparation of GelMA Hydrogels

Varying amounts of freeze-dried GelMA macromer was dissolved in DPBS containing 0.5% (w/v) 2-hydroxy-1-(4-(hydroxyethoxy)phenyl)-2-methyl-1-propanone (Irgacure 2959, CIBA Chemicals, Basel, Switzerland) as photoinitiator at 80°C to make final GelMA concentrations at 5%, 7.5%, 10%, 15%, and 20% (w/v). The prepolymer solution was then pipetted into a polydimethylsiloxane (PDMS) mold, covered with 3- (Trimethoxysilyl) propyl methacrylate (TMSPMA)-treated glass slide and exposed to 6.9 mW cm⁻² UV light (360–480 nm) for a certain period of time. For compression test, swelling ratio and degradation study, samples of 6 mm diameter and 3 mm thickness were fabricated upon 180 s of UV exposures whereas for tensile test, samples of 11 mm length, 5.5 mm width and 1 mm thickness were fabricated using 60 s of UV exposures. For biological studies, samples of 6 mm diameter and 150 μm thickness were produced upon 20 s of UV exposure. The UV time for curing samples of different thickness was optimized to allow sufficient crosslinking of GelMA prepolymer solution of various concentrations. The sample thickness of 150 μm for biological studies was selected in present study to allow better epidermis reconstruction and easy handling. Preliminary results have shown that samples with thickness over 200 μm resulted in slightly disorganized multi-layered epidermis (see Figure S3 in supplementary information) where the basal keratinocytes did not exhibit columnar morphology although the keratinocytes further away from the constructs exhibited flattened morphology. This may be due to the insufficient nutrient transport at ALI during culture. In addition, when the thickness of GelMA hydrogel was less than 100 μm, handling of the resultant hydrogel became difficult (e.g., placing the hydrogel on the cell inserts). For all tests, 5 replicates were used unless otherwise stated.

Characterization of Physical Properties of GelMA Hydrogels

Development of HaCaT Monolayer

Reconstruction and Analysis of Epidermis

Statistical Analysis

All data were expressed as mean ± standard deviation (n=3). One-way ANOVA and Scheffe's post hoc test were used to determine statistical significance. p < 0.05 was considered significant.