HYDROGELS FOR USE IN SKIN TISSUE ENGINEERING

Abstract

The present invention relates to hydrogels that include native skin components, as well as to bio-inks, compositions and dressings based on the same. Furthermore, the invention relates to their uses in regenerative medicine, in particular, in the regeneration, repair and replacement of skin tissue, and a method for obtaining the hydrogels of the invention.

Claims

1. A monolaminar hydrogel comprising: Type I collagen (Col I), dermatan sulphate (DS), hyaluronic acid (HA), and agarose (Ag).

2. The monolaminar hydrogel according to claim 1, which further comprises elastin (EL).

3. A bilaminar hydrogel comprising two layers of the monolaminar hydrogel according to claim 2.

4. The bilaminar hydrogel according to claim 3, comprising (i) a first layer comprising the monolaminar hydrogel wherein said monolaminar hydrogel comprises mesenchymal stem cells (MSCs), and (ii) a second layer, arranged on top of the first layer, comprising the monolaminar hydrogel wherein said monolaminar hydrogel comprises dermal fibroblasts (DFs).

5. (canceled)

6. The bilaminar hydrogel according to claim 3, wherein: the second layer comprises Col I at a concentration from 1 to 3.5 mg/ml, DS at a concentration from 7 to 10 mg/ml, HA at a concentration from 0.5 to 1.5 mg/ml, Ag at a concentration from 0.010 to 0.030 mg/ml, and EL at a concentration from 0.5 to 1.5 mg/ml; and the first layer comprises Col I at a concentration from 1 to 3.5 mg/ml, DS at a concentration from 7 to 10 mg/ml, HA at a concentration from 0.5 to 1.5 mg/ml, Ag at a concentration from 0.010 to 0.030 mg/ml, and EL at a concentration from 0.5 to 1.5 mg/ml.

7. (canceled)

8. A trilaminar hydrogel comprising: a bottom layer and a middle layer, each comprising the monolaminar hydrogel according to claim 1, and a top layer comprising a hydrogel comprising Col I, keratin (Kt), and sphingolipids (Sph).

9. (canceled)

10. The trilaminar hydrogel according to claim 8, wherein the top layer further comprises EKs, the middle layer further comprises DFs and the bottom layer further comprises MSCs.

11. (canceled)

12. The trilaminar hydrogel according to claim 8, wherein: the top layer comprises Col I at a concentration from 3.5 to 5.5 mg/ml, Kt at a concentration from 10 to 20 mg/ml and Sph at a concentration from 2.5 to 7.5 mg/ml; the middle layer comprises Col I at a concentration from 1 to 3.5 mg/ml, DS at a concentration from 7 to 10 mg/ml, HA at a concentration from 0.5 to 1.5 mg/ml, Ag at a concentration from 10 to 20 mg/ml, and EL at a concentration from 0.5 to 1.5 mg/ml; and the bottom layer comprises Col I at a concentration from 1 to 3.5 mg/ml, DS at a concentration from 7 to 10 mg/ml, HA at a concentration from 0.5 to 1.5 mg/ml, and Ag at a concentration from 10 to 20 mg/ml.

13. (canceled)

14. The trilaminar hydrogel according to claim 8, wherein the trilaminar hydrogel is lyophilised.

15. A bio-ink comprising the hydrogel according to a monolaminar hydrogel according to claim 1.

16-17. (canceled)

18. A dressing or implant, comprising the hydrogel according to claim 8.

19. A pharmaceutical composition comprising the hydrogel according to claim 8.

20-26. (canceled)

27. A method for obtaining a bilaminar hydrogel, comprising the following steps: a) mixing Col I, agarose, DS, HA and EL, obtaining a solution (i), b) mixing Col I, agarose, DS, HA, obtaining a solution (ii), and c) putting the solution (i) and the solution (ii) in contact.

28-31. (canceled)

32. The method according to claim 27, wherein step a) further comprises adding DFs, and wherein step b) further comprises adding MSCs.

33. A method for obtaining a trilaminar hydrogel using the method for obtaining the bilaminar hydrogel according to claim 27, which further comprises the following additional steps: (d) mixing Col I with Kt and Sph, obtaining a solution (iii), and (e) putting the solution (iii) in contact with the bilaminar hydrogel obtained in c).

34-38. (canceled)

39. The method for obtaining the trilaminar hydrogel according to claim 33, which further comprises a step (f) of dehydrating the hydrogel.

40. The method for obtaining the trilaminar hydrogel according to claim 39, which further comprises a subsequent step of lyophilising the hydrogel.

41. (canceled)

42. The method for obtaining the trilaminar hydrogel according to claim 33, wherein step a) further comprises adding DFs, wherein step b) further comprises adding MSCs, and which further comprises adding EKs to the trilaminar hydrogel obtained in step e).

43. A method for treating skin lesions or wounds in a subject, comprising the administration of the hydrogel according to claim 8.

44. A method for regenerating, repairing or replacing skin tissue in a subject, comprising the administration of the hydrogel according to claim 8.

Description

DESCRIPTION OF THE FIGURES

[0228] FIG. 1. (A) Design of the BT Skin hydrogel compared to native skin. Cells are represented as keratinocytes, fibroblasts and mesenchymal stem cells (MSCs), and biological components such as keratin, sphingolipids, type I collagen, dermatan sulphate, hyaluronic acid and elastin. (B) Tube inversion test images and (C) gel time results of each hypodermal, dermal and epidermal bio-ink. (D) Minimum force required to extrude the bio-inks through a 3 ml bioprinter syringe. Statistical significance: * P<0.05; *** P<0.005.

[0229] FIG. 2. Swelling (A), degradation (B) and rheological (C and D) assays. (A) Swelling behaviour and (B) percentage of degradation of the BT Skin hydrogel during 21 days. Young's modulus (C) and viscoelastic moduli (D) of BT Skin hydrogels, with or without cells, before and after the partial dehydration process, after 21 days in culture, compared to native skin. Statistical significance: * P<0.05, *** P<0.005.

[0230] FIG. 3. Cell proliferation (A) and viability (B) of hypodermal bio-ink. Cell proliferation (C) and viability (D) of the dermal bio-ink. (E) Representative microscopy images of the hypodermal and dermal bio-inks on days 0, 7, 14, and 21. A live/dead assay was used, using calcein and ethidium homodimer; live cells were stained with calcein while dead cells were stained with ethidium homodimer. The white arrows point to dead cells, and the unmarked cells are live cells. Scale bars: 200 μm.

[0231] FIG. 4. Cell proliferation (A) and viability (B and C) of the BT Skin hydrogel (the white arrows point to dead cells, and the unmarked cells are live cells. Scale bars: 200 μm). Macroscopic images of the BT Skin hydrogel (D; black arrow: epidermal layer), before (E) and after (F) partial dehydration (scale bars=5 mm). (G) Confocal fluorescence image of the BT Skin hydrogel after 21 days of culture (hEKs labelled at the top, hDFs labelled in Cell Tracker Red in the middle and hMSCs labelled in Cell Tracker Green at the bottom).

[0232] FIG. 5. Macroscopic images of the wound healing process over time (A). The type of treatment is indicated in each row, while the progression time (week 0, 2, 4, 6, 8) is represented in each column. Scale bars are represented on each image: 1 cm. Quantitative assessment of wound/scar surface area over time for all the groups (B) and in separate groups (C). Statistical significance compared to the control: * P<0.05; ** P<0.01; *** P<0.005. Statistical significance compared to day 14: #P<0.05; ##P<0.01.

[0233] FIG. 6. Analysis of homeostasis parameters by week and group. The graphs show the results of each treatment group compared to the native skin group. (A, B, C, D, E, F and G) Control group; (H, I, J, K, L, M and N) Autograft group; (0, P, Q, R, S, T and U) Cell-Free BT Skin; (V, W, X, Y, Z, AA and AB) BT Skin. The results per week were calculated as the mean value of all the measurements of the mice at each moment of the study; Control, autograft, cell-free BT Skin and BT Skin groups (n weeks 2, 4, 6, 8=8, 8, 4, 4); Native skin group (n weeks 2, 4, 6, 8=32, 32, 16, 16). Statistical significance: * P<0.05; ** P<0.01; *** P<0.005.

[0234] FIG. 7. Histological stains with hematoxylin and eosin (H&E) and Masson's Trichrome from biopsies of the wound/scar area of mice and native skin after 4 and 8 weeks. Scale bar: 50 μm.

[0235] FIG. 8. Fibronectin and cytokeratin profiles of the wound/scar area of mice and native skin biopsies after 4 and 8 weeks. Fluorescent microscopy observations. Scale bar: 100 μm.

[0236] FIG. 9. Injectability of the base bio-inks (Col I and Agarose) and studied bio-inks (epidermal, hypodermal and dermal).

[0237] FIG. 10. Analysis of homeostasis by parameter. The graphs show the results of each treatment group compared to the native skin group. The results per week were calculated as the mean value of all the measurements of the mice at each moment of the study; Control, autograft, cell-free BT Skin and BT Skin groups (n weeks 2, 4, 6, 8=8, 8, 4, 4); Native skin group (n weeks 2, 4, 6, 8=32, 32, 16, 16). Statistical significance: *** P<0.005.

[0238] FIG. 11. Histological stains with hematoxylin and eosin (H&E) and Masson's Trichrome from biopsies of the wound/scar area of mice and native skin after 4 and 8 weeks. Scale bar: 500 μm.

[0239] FIG. 12. Lyophilised (F-D). (A) Compression and (B) Viscoelasticity. Cell proliferation (C) and viability (D). (E) Confocal fluorescence images after 21 days. D: days.

[0240] FIG. 13. (A) Macroscopic images of the wound healing process over time. Type of treatment indicated in each row, time progression indicated in each column (weeks 0, 2, 4, and 8). The scale bars represented in each image: 1 cm. (B) Quantitative assessment of wound/scar area over time for all conditions, and (C) by condition. Statistical significance compared to the control: ***P<0.005. Statistical significance compared to day 14: #P<0.05; ##P<0.01.

[0241] FIG. 14. Analysis of homeostasis parameters by week and group. The graphs show the results of each group compared to those of native skin. (A, B, C, D, E, P, Q) Lyophilised dressing group, (F, G, H, I, J, R, S) autograft group, and (K, L, M, N, O, T, U) control group. The results per week were calculated as the mean of all mouse measurements at each time point; Lyophilised, autograft, control (n per week 2, 4, 6, 8=24, 24, 12, 12); native skin (n per week 2, 4, 6, 8=8, 8, 4, 4). Statistical significance: *P<0.05; **P<0.01; ***P<0.005.

[0242] FIG. 15. Histological stains of hematoxylin & eosin (H&E) and Masson's Trichrome from mouse skin biopsies in the wound/scar area and native skin after 4 and 8 weeks. Scale: 50 μm.

[0243] FIG. 16. Fibronectin and cytokeratin profiles from mouse skin biopsies in the wound/scar area and native skin after 4 and 8 weeks. Fluorescent microscopy observations. Scale: 100 μm.

[0244] FIG. 17. Cell viability of AC.sub.low and AC hydrogel formulations. (A) Confocal images of the fibroblast-laden hydrogels after 7 and 14 days. Calcein marks live cells and ethidium heterodimer marks dead cells. The white arrows point to dead cells, and the unmarked cells are live cells. Scale=500 μm. (B) Cell viability (%) in the hydrogels after 7 and 14 days. A two-tailed Student's t-test analysis was performed to calculate the significance of the samples from the AC.sub.low and AC hydrogels at levels of significance of: * p<0.05.

[0245] FIG. 18. Cell viability assay of AC, ACD, ACDH and ACDHE hydrogel samples. (A) Confocal microscopy images of hDF-laden hydrogels at 0, 7, 14, and 21 days. Calcein stains live cells, while EthD-1 stains dead cells. The white arrows point to dead cells, and the unmarked cells are live cells. Scale bar=500 μm. (B) Cell viability (%) on the hydrogel scaffolds after 0, 1, 7, 14 and 21 days. Two-tailed Student's t-test analyses were performed for ACD, ACDH, ACDHE samples compared to AC samples at each time point at levels of significance of: * p<0.05 and ** p<0.01; and for ACDHE samples compared to ACD, ACDH samples at each time point at levels of significance of: #p<0.05 and ##p<0.01.

[0246] FIG. 19. Tube inversion test and ultrastructure of the hydrogels. (A) Representation of AC hydrogels (white cap) and ACDHE hydrogels (black cap) withstanding the tube inversion test. (B) ESEM images (scale bar=1 μm) and (C) pore size characterisation of ACD, ACDH and ACDHE hydrogels. Two-tailed Student's t-test analyses were performed for the ACDH and ACDHE samples compared to the ACD samples with a level of significance of: *** p<0.05.

[0247] FIG. 20. Swelling, degradation and mechanical assays. (A) Swelling behaviour and (B) degradation rates of the ACDHE hydrogel over a 21-day time span. (C-E) Mechanical measurements: (C) Young's Moduli, (D) storage moduli, and (E) loss moduli of AC and ACDHE hydrogels cell-free and with cells at 1, 14 and 21 days under culture conditions, compared to native human skin. Two-tailed Student's t-test analyses were performed for human skin samples compared to AC and ACDHE samples on day 21 at levels of significance of: * p<0.05 and *** p<0.005; and for AC samples compared to ACDHE samples at each time point at a level of significance of: #p<0.05.

[0248] FIG. 21. Stress range analysed to obtain the Young's modulus of hydrogels with and without AC and ACDHE cells, and from human skin samples.

[0249] FIG. 22. In vitro wound healing assay of hDFs and hMSCs treated with DHE-supplemented media. A culture medium without supplements was used as a control for the untreated cells. The images (A) and the analysed results (B, C) showed wound closure at 0, 6, 12, 24, 48 and 72 hours. Statistical significance: * p<0.05; *** p<0.005. Abbreviations: DHE, dermatan sulphate, hyaluronic acid and elastin; MSC, mesenchymal stem cells.

[0250] FIG. 23. ACDHE bilaminar hydrogel. (A) Macroscopic appearance of bilaminar ACDHE hydrogels. (B) Vertical cross-sectional image of the bilaminar hydrogel acquired by confocal microscopy (the white arrows point to dead cells, and the unmarked cells are live cells. Scale bars: 200 μm). Side and bottom views are also included on the right and bottom sides of the figure. hDFs and hMSCs are stained with CellTracker™ Green CMFDA (CTG) and CellTracker™ Red CMTPX (CTR), respectively. Scale bar=1000 μm. (C) Confocal microscopy images of cell viability of the ACDHE hydrogel on days 0, 7, 14, and 21. Additionally, the enlarged image of the bilaminar ACDHE hydrogel is shown on day 21, the white dotted line separates the fibroblast layer (top) from the hMSCs layer (bottom). Scale bar=500 μm. (D) Results of proliferation of the ACDHE hydrogels up to 21 days.

[0251] FIG. 24. ESEM of bilaminar ACDHE hydrogel. (A-F) ESEM images (scale bar=1 μm) of ACDHE hydrogels. Scale bars: (A) 20 μm; (B) 10 μm; (C and E) 3 μm; (D and F) 2 μm. (E and F). ESEM images showing cells and structures similar to ECM produced by cells.

EXAMPLES

[0252] Next, the invention will be illustrated by means of assays carried out by the inventors which demonstrate the effectiveness of the invention.

[0253] 1. Materials and Methods

[0254] 1.1. Trilaminar Model

[0255] 1.1.1. Cell Cultures

[0256] Human mesenchymal stem cells (hMSC) were isolated from human adipose tissue and characterised using a method already described (Lopez-Ruiz E, et al., Eur Cells Mater., 35:209-24 (2018); Gálvez-Martin P, et al., Eur J Pharm Biopharm., 86(3):459-68 (2014)). All human samples used in this study were obtained with informed consent and the approval of the Institutional Review Board (ethical permission number: 02/022010 Virgen de la Victoria Hospital, Malaga, Spain), and transported to the laboratory in Dulbecco's Modified Eagle Medium (DMEM; Sigma, St. Louis, MO, USA) with 1% penicillin and streptomycin (P/S; Invitrogen). hMSCs were cultured in high-glucose DMEM supplemented with 10% foetal bovine serum (FBS) and 1% P/S, at 37° C. in a humidified atmosphere containing 5% CO.sub.2. The medium was renewed every 3 days. At 80% confluence, the cells were subcultured and used between passages 4 and 6 for all experiments.

[0257] Human dermal fibroblasts (hDF) and human epidermal keratinocytes (hEK) were isolated from human skin samples obtained from donors using a previously published method (Sierra-Sanchez A, et al., J Eur Acad Dermatology Venereol. (2020)). Once the skin samples were removed, they were transported to the laboratory in sterile containers containing high-glucose DMEM without phenol red supplemented with antibiotics. All human skin samples were obtained in accordance with the ethical standards of the committee responsible for human experimentation (Provincial Ethical Committees of Granada) and with the Declaration of Helsinki of 1975, revised in 1983. Once isolated, the hDFs and hEKs were cultured (Carrie) V. et al., Cells Tissues Organs, 196196:1-121(2012)).

[0258] 1.1.2. Preparation of Bio-Inks and the BT Skin Substitute

[0259] Hydrogel-based bio-inks were prepared to create a trilaminar skin substitute (also called BT Skin).

[0260] First, an agarose (Ag) solution (UltraPure™ Low Melting Point Agarose Thermo Scientific™) was prepared in phosphate-buffered saline (PBS), sterilised in an autoclave at 120° C. for 2 hours and stored at 4° C. until use. The Ag was preheated and kept at 37° C. in a water bath to avoid gelling and to stabilise its temperature at 37° C. The Col I solution (3.3 mg/ml rat tail collagen I, Corning®) was neutralised with 0.8M NaHCO.sub.3. Next, three solutions were prepared using Kt (Kerapro S, Proalan S.A., Barcelona, Spain), Sph, DS, HA and EL (Bioibérica S.A.U, Barcelona, Spain) diluted in PBS: i) Kt+Sph (KS); ii) DS+HA (DH); iii) DS+HA+EL (DHE). To include DS and HA, Dermial®, a product that has both active ingredients, was used. Each solution was mixed with Col I to the final concentration as shown in Table 1. All component solutions were filtered through a 0.22 μm membrane (Merck Millipore) before use.

TABLE-US-00001 TABLE 1 Concentrations of agarose, collagen I, dermatan sulphate (DS), hyaluronic acid (HA), elastin (EL), keratin (Kt) and sphingolipids (Sph) in the different bio-inks. Composition (mg/ml) Agarose Collagen I DS HA EL Kt Sph Epidermal bio-ink — 4.4 — — — 15.2 5.0 Dermal bio-ink 15 2.2 8.4 1.0 1.0 — — Hypodermal bio-ink 15 2.2 8.4 1.0 — — —

[0261] To biomanufacture the BT Skin substitute, cell-laden dermal and hypodermal bio-inks were prepared by mixing Col I+DHE with hDFs and Col I+DH with hMSCs, respectively, and preheated Ag was added to each mixture, obtaining a final concentration of 1.Math.10.sup.6 cells/ml. Once both the hypodermal and dermal layers were obtained, the epidermal bio-ink (Col I+KS) was placed on top and left to gel in an incubator at 37° C. for 15 minutes. Once the epidermal layer gelled, 2.106 hEKs were sown on the BT Skin hydrogel and cultured for 1 week. After this time, the samples were partially dehydrated by applying 100 g of pressure for 2 minutes, giving the hydrogels greater resistance and enhancing their stiffness and mechanical properties.

[0262] 1.1.3. Physico-Chemical Characterisation of Trilaminar Hydrogels

[0263] Tube Inversion Test

[0264] The tube inversion test was used to define the gel time of the bio-inks. The bio-inks were poured into glass vials, inverting the vials upside down every 1 minute to check for the formation of stable gels. The gel time was considered to be the time when the samples formed a stable gel that remained on the bottom of the vials when inverted.

[0265] pH

[0266] The pH of the hydrogel samples was determined using a Hach Sension+calibrated digital pH meter (Hach Lange S.L., Spain) at room temperature (RT).

[0267] Injectability Test

[0268] The force required to extrude the different biomaterial mixtures was measured using an MCR302 torsional rheometer (Anton Paar, Austria). The equipment used for the assay was arranged as shown in FIG. 1D, where a 3 ml syringe was kept surrounded by hot water at a temperature of 37° C. to prevent rapid gelification and to reproduce the environment of a heated syringe with a 3D bioprinter. The samples were placed in the syringe and the force required to extrude the bio-inks was controlled.

[0269] Swelling Test

[0270] The swelling rates of the lyophilised samples were analysed. Briefly, the samples were previously weighed and immersed in PBS. The swelling rate of the hydrogel samples was calculated at different times as follows:

[00001]$\mathrm{Swelling}\mathrm{rate}\left(\%\right)=\left(\frac{\left(W_t-W_0\right)}{W_0}\right)x100$

[0271] W.sub.0 represents the initial weight of the samples on day zero and Wt represents the wet weight of the samples at the corresponding time.

[0272] Degradation Test

[0273] The degradation rate was analysed by quantifying the weight loss of the samples over time. The hydrogels were incubated with gentle stirring at 37° C., recovered at different times and centrifuged at 5000 rpm for 2 minutes. The supernatant was removed and the samples were weighed. The degradation rate (%) was calculated as a measure of weight loss as follows:

[00002]$\mathrm{Degradation}\mathrm{rate}\left(\%\right)=\left(\frac{\left(W_t-W_i\right)}{W_i}\right)x100$

[0274] W.sub.i represents the initial weight of the samples and W.sub.t represents the wet weight of the hydrogels at the corresponding time.

[0275] Mechanical Analysis

[0276] Rheological tests were carried out using an MCR302 torsional rheometer. The hydrogel samples were moulded using a 20 mm-diameter and 5 mm-height mould. A three-step assay was designed to obtain data on the compression and shear characteristics of the hydrogels. In the first step, the samples were placed on the base of the rheometer, approaching the head of the rheometer at 10 μm/s up to a normal force of 0.5 N. Secondly, the samples were subjected to oscillatory shearing with a strain amplitude of 0.00001% at a frequency of 1 Hz and a normal force of 0.5 N to analyse the viscoelastic shear moduli, and finally the top plate was withdrawn at a constant speed of 10 μm/s.

[0277] 1.1.4. Cell Viability Assay

[0278] The LIVE/DEAD™ Viability/Cytotoxicity Kit (Invitrogen, Massachusetts, USA) was used to analyse cell viability. The hydrogels were stained using a solution of Calcein-AM (2 μM; green) and ethidium homodimer (4 μM; red) diluted in PBS at 37° C. for 30 minutes. The samples were observed using confocal microscopy (Nikon Eclipse Ti-E A1) at different times and analysed using NIS-Elements software. The live and dead cells were quantified using ImageJ software (Fiji), determining the percentage of cell viability as follows:

[00003]$\mathrm{Cell}\mathrm{viability}\left(\%\right)=\frac{\mathrm{Live}\mathrm{cells}}{\left(\mathrm{Live}\mathrm{cells}+\mathrm{Dead}\mathrm{cells}\right)}x100$

[0279] 1.1.5. Cell Proliferation Assays

[0280] The AlamarBlue HS® assay (Invitrogen) was used to analyse cell proliferation of the samples after 1, 3, 5, 7, 14 and 21 days of culture. Fluorescence was measured after incubation, which gave rise to a signal that may be related to the metabolic activity of the cells. The samples were incubated with the AlamarBlue HS® solution at 37° C. for 1 hour. The fluorescence of the reduced solution was determined at excitation/emission wavelengths of 530/590 nm.

[0281] 1.1.6. In Vivo Assay

[0282] 1.1.6.1. Wound Healing Animal Model, Surgical Procedures and Experimental Groups

[0283] A total of 32 male and female ATHYM-Foxn1 nu/nu, immunocompromised, athymic, hairless and albino 4-week-old mice were used for the in vivo assay. All animal handling procedures followed national and European Union legislation (RD 53/2013 and EU Directive 2010/63) on the protection of animals used for scientific purposes and in accordance with the Ethical Principles and Guidelines for the Use of Animals approved by the Provincial Ethical Committees of Granada.

[0284] Surgery was performed to remove a 2 cm.sup.2 area of skin from the upper dorsal part, in a position longitudinal to the mouse's spinal column, using surgical scissors. A 3D-printed splint using 1,4-butanediol thermoplastic polyurethane elastomer (b-TPUe), porous, donut-shaped, and designed with a hinged cover was centred over the wound and secured with seven interrupted sutures. Then, the mice were transplanted with BT Skin, cell-free BT Skin, lower back skin graft (Autograft), or left untreated as control conditions (Control) (n=8 per group). Lastly, the cover of the splint was closed with eight interrupted sutures. Samples and splints were grafted for all the groups, and an antibiotic ointment was applied (Mupirocin 20 mg/g; ISDIN). Likewise, an analgesic (Bupredine 0.3 mg/ml 10 ml; Fatro Ibérica, Desvern, Barcelona, Spain) and an antibiotic (Ganadexil Enrofloxacin 5% 100 ml; Industrial Veterinaria, S.A. Invesa) were injected subcutaneously as post-operative treatment.

[0285] 1.1.6.2. Healing Monitoring

[0286] Follow-up was carried out for 8 weeks, gathering clinical information, such as scar/wound surface area, and several homeostasis parameters. Furthermore, after 8 weeks, the scars were assessed using an adaptation of the Patient and Observer Scar Assessment Scale (POSAS). Every 2 weeks, mice were anaesthetised by isoflurane inhalation to avoid unnecessary stress and cutaneous homeostasis parameters were measured by the Microcaya probe system (Microcaya SL, Bilbao, Spain), allowing the monitoring of different cutaneous parameters: the Thermometer® probe allowed skin temperature to be measured in ° C.; the Skin pH-Meter® probe measured skin pH; the Tewameter® probe determined transepidermal water loss (TEWL), such as the evaporation of water in g/h/m.sup.2; the Cutometer® probe analysed the elasticity of skin (μm) with suction (450 mbar of negative pressure—2 s); the Corneometer® probe determined skin hydration through the capacitance of a dielectric medium; and the Mexameter® probe, based on the absorption/reflection of light in three wavelengths, was able to measure erythema and skin pigmentation, obtaining indirect information on vascularisation (haemoglobin levels) and pigmentation (melanin), respectively. The values of healthy skin were obtained by measuring a healthy area of skin (native skin) from each mouse in the study.

[0287] 1.1.6.3. Histological Analysis and Immunostaining

[0288] Four and eight weeks later, the mice were sacrificed by cervical dislocation once anaesthetised by isoflurane inhalation. Graft biopsies and native skin samples were collected, fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin or in an optimal cutting temperature (OCT) compound (Tissue-Tek®, Sakura Finetek) and cut into 5 μm or 8 μm sections using a microtome and cryostat, respectively.

[0289] The paraffin sections were deparaffinised, rehydrated and stained with hematoxylin and eosin (H&E) and Masson's Trichrome to reveal the histological structure. For immunofluorescence analysis, the cryosections were incubated with primary antibodies against fibronectin (Santa Cruz Biotechnology, 1:100) and cytokeratin (Invitrogen, 1:100). Next, the sections were incubated with Alexa-488 conjugated anti-rabbit secondary antibody (ThermoFisher, 1:500) and counterstained with Hoechst. The images were obtained using a Leica DM 5500B microscope and analysed using ImageJ software.

[0290] 1.2. Lyophilised Trilaminar Model

[0291] To manufacture the lyophilised trilaminar model, dermal and hypodermal bio-inks were prepared by mixing Col I+DHE and Col I+DH, and prewarmed Ag was added to each mixture. Once both the hypodermal and dermal layers were obtained, the epidermal bio-ink (Col I+KS) was placed on top and left to gel in an incubator at 37° C. for 15 minutes. Once the epidermal layer gelled, the samples were partially dehydrated by applying 100 g of pressure for 2 minutes, giving the hydrogels greater resistance and enhancing their stiffness and mechanical properties. Lastly, the samples were frozen at −20° C. and they were introduced into the lyophiliser which cold trap was previously cooled to −70° C. The lyophilisation chamber was closed, keeping the samples at room temperature and a vacuum was applied using a pressurised air pump. The samples were kept in the lyophilisation process until it could be macroscopically observed that they had been properly dehydrated (between 2-16 hours).

[0292] 1.2.1. Biological and Mechanical Characterisation of Lyophilised Dressings

[0293] For this lyophilised trilaminar model, mechanical analyses, cell viability assays, and cell proliferation assays were carried out under the same experimental conditions as for the non-lyophilised trilaminar model.

[0294] 1.2.2. In Vivo Assays

[0295] Furthermore, as in the non-lyophilised trilaminar model, a series of in vivo assays were carried out.

[0296] Wound Healing Animal Model, Surgical Procedures and Experimental Groups

[0297] A total of 24 male and female ATHYM-Foxn1 nu/nu, immunocompromised, athymic, hairless and albino 4-week-old mice were used for the in vivo assay.

[0298] Surgery was performed to remove a 2 cm.sup.2 area of skin from the upper dorsal part, in a position longitudinal to the mouse's spinal column, using surgical scissors. A 3D-printed splint using 1,4-butanediol thermoplastic polyurethane elastomer (b-TPUe), porous, donut-shaped, and designed with a hinged cover was centred over the wound and secured with seven interrupted sutures. Then, the mice were transplanted with the lyophilised dressing, lower back skin graft (Autograft), or left untreated as control conditions (Control) (n=8 per group). Lastly, the cover of the splint was closed with eight interrupted sutures. Samples and splints were grafted for all the groups, and an antibiotic ointment was applied (Mupirocin 20 mg/g; ISDIN). Likewise, an analgesic (Bupredine 0.3 mg/ml 10 ml; Fatro Ibérica, Desvern, Barcelona, Spain) and an antibiotic (Ganadexil Enrofloxacin 5% 100 ml; Industrial Veterinaria, S.A. Invesa) were injected subcutaneously as post-operative treatment.

[0299] Healing Monitoring

[0300] Follow-up was carried out for 8 weeks, gathering clinical information, such as scar/wound surface area, and several homeostasis parameters. Every 2 weeks, mice were anaesthetised by isoflurane inhalation to avoid unnecessary stress and cutaneous homeostasis parameters were measured by the Microcaya probe system (Microcaya SL, Bilbao, Spain), allowing the monitoring of different cutaneous parameters: the Thermometer® probe allowed skin temperature to be measured in ° C.; the Skin pH-Meter® probe measured skin pH; the Tewameter® probe determined transepidermal water loss (TEWL), such as the evaporation of water in g/h/m2; the Cutometer® probe analysed the elasticity of skin (μm) with suction (450 mbar of negative pressure—2 s); the Corneometer® probe determined skin hydration through the capacitance of a dielectric medium; and the Mexameter® probe, based on the absorption/reflection of light in three wavelengths, was able to measure erythema and skin pigmentation, obtaining indirect information on vascularisation (haemoglobin levels) and pigmentation (melanin), respectively. The values of healthy skin were obtained by measuring a healthy area of skin (native skin) from each mouse in the study.

[0301] Histological Analysis and Immunostaining

[0302] Four and eight weeks later, the mice were sacrificed by cervical dislocation once anaesthetised by isoflurane inhalation. Graft biopsies and native skin samples were collected, fixed in 4% paraformaldehyde (PFA), dehydrated, embedded in paraffin or in an optimal cutting temperature (OCT) compound (Tissue-Tek®, Sakura Finetek) and cut into 5 μm or 8 μm sections using a microtome and cryostat, respectively.

[0303] The paraffin sections were deparaffinised, rehydrated and stained with hematoxylin and eosin (H&E) and Masson's Trichrome to reveal the histological structure. For immunofluorescence analysis, the cryosections were incubated with primary antibodies against fibronectin (Santa Cruz Biotechnology, 1:100) and cytokeratin (Invitrogen, 1:100). Next, the sections were incubated with Alexa-488 conjugated anti-rabbit secondary antibody (ThermoFisher, 1:500) and counterstained with Hoechst. The images were obtained using a Leica DM 5500B microscope and analysed using ImageJ software.

[0304] 1.3. Bilaminar Model

[0305] 1.3.1. Cell Cultures

[0306] hDFs were obtained from the American Type Culture Collection (ATCC® PCS-201-012) and cultured in Dulbecco's Modified Eagle Medium (DMEM; Sigma) containing 10% foetal bovine serum (FBS; Sigma), 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen) at 37° C. in a 5% CO.sub.2 humidified atmosphere. The medium was changed every 3 days. When 80% confluence was reached, the cells were released with Tryple Express (Gibco) and subcultured. hDFs were used between passages 4 and 6 for all experiments.

[0307] hMSCs were obtained from human adipose tissue and characterised using a previously described method (Lopez-Ruiz E, et al., Eur Cells Mater., 35:209-24 (2018); Gálvez-Martin P, et al., Eur J Pharm Biopharm., 86(3):459-68 (2014)). hMSCs were cultured in high-glucose DMEM (Sigma) supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen) at 37° C. in a humidified atmosphere containing 5% CO.sub.2. The medium was changed every 3 days. At 80% confluence, the cells were subcultured. hMSCs were used between passages 4 and 6 for all experiments.

[0308] 1.3.2. Hydrogel Formulation

[0309] To prepare the hydrogel, two different concentrations of Ag were dissolved, a previously reported Ag-Col I (AC) hydrogel formulation (Ag 1.5% w/v) and an AC hydrogel with a reduced agarose concentration (1.2% w/v) (AClow). The Ag solutions were prepared by dissolving UltraPure Low Melting Point Agarose (Thermo Scientific) in a phosphate buffer (PBS). This solution was autoclaved at 120° C. for 2 hours and stored at 4° C. Before its use, Ag was preheated to 70° C. and kept in a 37° C. water bath to warm and prevent gelling. The Col I solution (4.42 mg/ml rat tail collagen I, Corning) was kept on ice before use and neutralised with 0.8 M NaHCO.sub.3. The pH of the Col I solution after neutralisation was 7.4. Next, three stock solutions were prepared using DS, HA and EL (Bioibérica SAU) diluted in PBS (DS; DS+HA; DS+HA+EL) and mixed with Col I at the final concentrations shown in Table 2. The Col I solutions were filter sterilised through a 0.22 μm pore size filter (Merck Millipore) before use. Similarly, DS, HA and EL lyophilised powder was sterilised using UV radiation for at least 30 minutes. Finally, cell-laden hydrogels were prepared by mixing the aforementioned solutions with hDFs or hMSCs and prewarmed Ag was added to the mixture, obtaining 1.1 ml of hydrogel with 1.Math.10.sup.6 cells/ml.

TABLE-US-00002 TABLE 2 Concentration of the components in the bilaminar hydrogel. Col- Dermatan Hyaluronic (mg/ml) Agarose lagen sulphate acid Elastin AC 0.015 2.2 — — — ACD 0.015 2.2 8.4 — — ACDH 0.015 2.2 8.4 1.0 — ACDHE 0.015 2.2 8.4 1.0 1.0 AC: agarose + collagen; ACD: agarose + collagen + dermatan sulphate; ACDH: agarose + collagen + dermatan sulphate + hyaluronic acid; ACDHE: agarose + collagen + dermatan sulphate + hyaluronic acid + elastin.

[0310] 1.3.3. Physical Characterisation of the ACDHE Hydrogel

[0311] Inversion Test

[0312] To analyse the gelling of the hydrogel, the tube inversion test was performed and the AC and ACDHE formulations were assayed. The hydrogel mixture was poured into a glass vial for the preparation of the hydrogels. To check the formation of a stable gel, the vial was inverted upside down every 1 minute and the gel time was noted.

[0313] pH Analysis

[0314] The pH values of the hydrogels generated at room temperature were determined using a Hach Sension+calibrated digital pH meter (Hach Lange S.L.) at 25.0±0.5° C. Measurements were made by direct contact of the pH meter electrode in hydrogels.

[0315] Environmental Scanning Electron Microscopy (ESEM)

[0316] ESEM was used to observe the morphology of the surface of the hydrogels and the internal cell distribution. The hydrogel samples were fixed with 2.5% w/v glutaraldehyde (Merck) for 1 hour at 4° C., then they were washed and maintained in 0.1 M sodium cacodylate buffer (EMS, Electron Microscopy Science). Before their analysis, the samples were processed using the critical point drying technique. Four samples were fixed with 1% w/v osmium tetroxide, dehydrated in a series of ethanol solutions of increasing concentration (50, 70, 90 and 100%) for 15 minutes each, and dried at the critical point in a Leica EM CPD300 dryer. Lastly, the samples were covered with carbon using the EMITECH K975X carbon evaporator. Images were acquired with ESEM QEMSCAN 650F and hydrogel pore sizes were analysed with ImageJ software (Fiji).

[0317] Swelling Assay

[0318] Pre-weighed lyophilised hydrogels were immersed in PBS. The weight of the prepared hydrogels was calculated at different times. The swelling ratio was calculated with the following equation:

[00004]$\mathrm{Swelling}\mathrm{rate}\left(\%\right)=\left(\frac{\left(W_t-W_0\right)}{W_0}\right)x100$

[0319] Where W0 represents the dry weight of the sample on day zero and Wt represents the wet weight of the samples at a specific time.

[0320] Degradation Test

[0321] The degradation behaviour of the hydrogels was analysed by weighing known amounts of hydrogel samples. Next, the hydrogel samples were incubated with gentle stirring in a hybridisation oven at 37° C. The samples were recovered at different times from the hybridisation oven and centrifuged at 5000 rpm for 2 minutes. After removing the supernatant, the hydrogel samples were finally weighed. The degradation rate (%) as a measure of weight loss was calculated with the following equation:

[00005]$\mathrm{Degradation}\mathrm{rate}\left(\%\right)=\left(\frac{\left(W_t-W_0\right)}{W_0}\right)x100$

[0322] Where W.sub.0 represents the initial weight of the sample and W.sub.t represents the wet weight of the samples at a specific time.

[0323] Mechanical Assays

[0324] Mechanical analyses were performed in an MCR302 torsional rheometer (Anton Paar) at 25° C. Cell-free and hDF-laden hydrogels were cast into cylindrical shapes in moulds (20×5 mm). A three-step test was designed to obtain information on the compression and shear characteristics of the samples. First, the samples were placed on the base of the rheometer. Then, the rheometer head was approached at a constant speed (10 μm/s) up to a normal force of 0.5 N. Next, the sample was oscillatory sheared according to a strain amplitude of 0.00001% at a frequency of 1 Hz and a normal force of 0.5 N to determine the viscoelastic shear moduli and, lastly, the top plate was separated at a constant speed (10 μm/s).

[0325] 1.3.4. Wound Closure Assay

[0326] hDFs and hMSCs were seeded in 12-well multiwell plates and cultured at 37° C. for 72 hours. The culture medium was removed, and the cells were rinsed with PBS and incubated with a culture medium supplemented with 8.4 mg/ml DS, 1.0 mg/ml HA and 1.0 mg/ml elastin for 24 hours. A culture medium without supplements was used as an untreated control condition. After 24 hours, the medium was removed, the cells were washed with PBS and a trace was made on the cell monolayer by manual scratching with a 200 μl pipette tip. The supplemented and control media were replaced, and images of the wounds were taken at 0, 6, 12, 24, 48 and 72 hours. The samples were kept at 37° C. in a 5% CO.sub.2 atmosphere. The images were taken with a Leica DMi8 microscope (Leica Microsystems) with Leica Application Suite (LAS) X software and analysed with ImageJ software (Fiji).

[0327] 1.3.5. Cell Viability Assay

[0328] Cell viability was determined using the LIVE/DEAD™ Viability/Cytotoxicity Kit (Invitrogen). The samples were incubated in PBS containing calcein-AM (2 μM) and ethidium homodimer (4 μM) at 37° C. for 30 minutes to stain live cells (green) and dead cells (red). Images of hydrogels were obtained by confocal microscopy at different times and analysed with NIS-Elements software (Nikon Eclipse Ti-E A1). The live and dead cells were counted using ImageJ software (Fiji), and cell viability was determined as follows:

[00006]$\mathrm{Cell}\mathrm{viability}\left(\%\right)=\frac{\mathrm{live}\mathrm{cells}}{\left(\mathrm{live}\mathrm{cells}+\mathrm{dead}\mathrm{cells}\right)}x100$

[0329] 1.3.6. Cell Proliferation Assay

[0330] Cell proliferation was analysed using the AlamarBlue® assay (Invitrogen) on days 1, 3, 5, 7, 14 and 21. This reagent is a solution of resazurin, a non-fluorescent cell-permeable blue compound that is modified by the reducing environment of viable cells in resorufin, a fluorescent red compound. Fluorescence can be measured after incubation and, therefore, the generated signal related to the metabolic activity of the samples. The hydrogels were incubated with AlamarBlue® solution at 37° C. for 3 hours. The fluorescence of reduced AlamarBlue® was determined at excitation/emission wavelengths of 530/590 nm.

[0331] 1.3.7. Bilaminar Hydrogel Design

[0332] Separate hMSC- or hDF-laden hydrogels were prepared in conical tubes and loaded into sterile 3 ml syringes. Then, the hydrogel layers were sequentially stacked, placing the hMSC layer at the bottom and the hDF layer at the top. The bilayer hydrogels were left to gel and then placed in a 4-well rectangular multiwell plate with DMEM containing 10% FBS and 1% penicillin/streptomycin and cultured at 37° C. in 5% CO.sub.2 atmosphere.

[0333] 1.3.8. Morphological Characterisation of Bilaminar Hydrogels

[0334] Maintenance of the hydrogel shape was monitored during a culture period of 21 days. To observe the size of the hydrogels over time, three hydrogel samples were prepared and seeded with hDF and hMSC, immersed in culture medium and kept in an incubator. At pre-established time intervals (0, 7, 14 and 21 days), the hydrogels were recovered from the solution, excess medium was removed with filter paper, and the length and width of the samples were measured.

[0335] 1.4. Statistical Analysis

[0336] The results of this work are represented as mean±standard deviation (SD). The differences between two sets of data were tested using the two-tailed Student's t-test for unpaired samples. The differences were considered statistically significant at P<0.05 (*/#), P<0.01 (**/##) and P<0.005 (***/###).

[0337] 2. Results

[0338] 2.1. Trilaminar Model

[0339] 2.1.1. Physico-Chemical Properties of the Trilaminar Hydrogel

[0340] Tube Inversion Test and pH

[0341] The gel time was analysed by applying the tube inversion test for the hypodermal, dermal and epidermal bio-inks. FIG. 1B shows representative images of the bio-inks in their liquid and gel forms. The average gel times of the bio-inks were 0.76±0.03, 0.91±0.03 and 5.22±0.09 minutes (FIG. 10), respectively. The pH value of the complete BT Skin hydrogel was 7.59±0.12.

[0342] Bio-Ink Injectability

[0343] To characterise the injectability of bio-inks, the force required to extrude the solutions from a 3 ml syringe was measured. The results varied between 1.0 and 2.3 N (FIG. 1D and FIG. 9), where the epidermal bio-ink (0.34±0.03 N) showed the lowest value, even less than the force required to extrude Col I (1.04±0.04 N). Regarding the hypodermal bio-ink (1.82±0.57 N) and dermal bio-ink (2.30±0.49 N), although no differences were shown between them or compared to their base mixture, Ag (1.99±0.17 N) and Ag+Col I (2.22±0.19 N), they showed a higher range of N (1.25-2.79 N) than Col I and the epidermal bio-ink (0.31-1.08 N).

[0344] Swelling and Degradation Behaviour of the BT Skin Hydrogel

[0345] Lyophilised BT Skin hydrogels were immersed in PBS (pH 7.4) to observe their swelling behaviour. The swelling kinetics of BT Skin over an elapsed time of 21 days is shown in FIG. 2A. The mean swelling of the BT Skin hydrogel was 70±7%. BT Skin hydrogel (lyophilised) was shown to reach a plateau step after 7 days. To analyse the resistance of BT Skin hydrogel over time, a degradation assay was carried out measuring the change in weight up to 21 days (FIG. 2B). The results showed that the maximum degradation rate was reached after 14 days with 8.8±1.5% mass loss.

[0346] Mechanical Properties of BT Skin Hydrogel

[0347] BT Skin hydrogels with and without cells before (Pre BT Skin and Pre Cell-free BT Skin) and after (BT Skin and Cell-free BT Skin) were generated and maintained at 37° C. in a 5% CO.sub.2 atmosphere. The Young's moduli of the hydrogels maintained up to 21 days compared to native human skin biopsies are represented in FIG. 2C. Although variations were found in Pre BT Skin (7.49±0.62 kPa) and Cell-free BT Skin (13.24±0.30 kPa) compared to native skin (9.46±0.79 kPa), BT Skin (9.06±0.21 kPa) did not show significant differences compared to the reference tissue.

[0348] The viscoelastic moduli of cell-laden and cell-free BT Skin hydrogels were also measured before and after dehydration and are shown in FIG. 2D. Although the four tested conditions were found to share the same loss modulus range of native skin (2.92±0.91 kPa), only the loss modulus of BT Skin hydrogels (0.28±0.10 kPa) did not show significant differences with respect to the loss modulus of natural skin (0.83±0.33 kPa).

[0349] 2.1.2. Biological Characterisation of Bio-Inks and BT Skin Hydrogel

[0350] A biological characterisation of the hypodermal and dermal bio-inks was carried out with cell proliferation and viability assays. Since the handling of the epidermal bio-ink was complicated, since it was fragile once gelled, this bio-ink was incorporated into the BT Skin hydrogel and the biological characterisation of the complete hydrogel was carried out. The cell proliferation rates of the hydrogels of the hypodermal and dermal bio-inks on days 0, 1, 3, 5, 7, 10, 14, 18 and 21 are shown in FIGS. 3A and C, respectively. The hypodermal hydrogels showed an increase in cell proliferation on day 5, maintaining a plateau step until the end of the experiment. Likewise, the dermal hydrogels increased their cell proliferation from day 14, maintaining their level until the end of the experiment. Both conditions were able to maintain the level of cell viability above 97% for the duration of the assay (FIGS. 3B, D and E), as demonstrated by the images acquired at 0, 7, 14 and 21 days.

[0351] The BT Skin hydrogel was prepared in three layers with the hypodermal layer at the bottom, a middle dermal layer, and the epidermal layer on the top (FIG. 1A), and it was partially dehydrated. FIG. 4D shows the macroscopic appearance of the BT Skin (black arrow indicating the epidermal layer), and FIGS. 4E and F show the height difference of the hydrogel before and after the partial dehydration process, where the height of the hydrogel is reduced by 2 mm.

[0352] The skin cell proliferation rate and viability up to 21 days are shown in FIGS. 4A and B, respectively. Similarly, for hypodermal and dermal bio-ink hydrogels, the BT Skin showed an increase in the cell proliferation rate from day 5, remaining at a plateau step until the end of the experiment. The cells were able to adhere and grow in contact with the surrounding cells in some areas of the hydrogels, as can be seen in FIG. 4C. Furthermore, BT Skin showed cell viability rates between 90.9 and 98%. To observe the distribution of the three cell types within the hydrogel, hMSCs, hDFs and hEKs were stained with CellTracker™ Green CMFDA, CellTracker™ Red CMTPX and CellTracker™ Green CMFDA, respectively. It could be seen that the three layers of the hydrogel were well-differentiated (FIG. 4G).

[0353] 2.1.3. In Vivo Assays

[0354] 2.1.3.1. Clinical Analysis

[0355] Suitable wound stabilisation was observed for all the mouse groups at 4 weeks, without complications (FIG. 5A). Wound resolution was faster in the case of the groups treated with Autograft, Cell-free BT Skin and BT Skin than in the Control group. Similarly, wound repair was faster and more effective in the case of the BT Skin group, while the Control, Autograft and Cell-free BT Skin groups experienced slower improvement. In the case of BT Skin, total skin repair was observed after 8 weeks.

[0356] The evaluation of the results by visual clinical observation (FIG. 5A) was correlated with the quantitative analysis of the wound/scar surface area (FIGS. 5B and C), where significant differences were found between the Cell-free BT Skin and BT Skin groups compared to the control group at week 2, indicating a positive effect of these conditions on the wound healing process, but at the end of the experiment, only the BT Skin group showed significant differences with the Control group.

[0357] 2.1.3.2. Study of Homeostasis Parameters

[0358] Temperature, pH, TEWL, elasticity and moisture were monitored (FIG. 6 and FIG. 10), comparing all the groups with the native skin of mice.

[0359] The results of temperature (FIGS. 6A, H, O and V) and pH (FIGS. 6B, I, P and W) showed a homogeneous evolution in all the groups in general of the study, without differences compared to native skin. The temperature values of all the groups during the study ranged from 31.8 to 36.6° C., overlapping the temperature range of native skin (34.0-36.0° C.). Similarly, the pH ranges of all the groups (5.2-7.8) and native skin (6.4-7.3) also generally overlapped throughout the duration of the experiment.

[0360] TEWL (FIGS. 6C, J, Q and X) showed a significant decrease after 2 weeks in all the groups (FIG. 10), reaching native skin levels. Regarding elasticity (FIGS. 6D, K, R and Y), although the Control and BT Skin groups showed oscillatory behaviour throughout the experiment, there were no significant differences between all the groups and Native Skin in general at 8 weeks. Lastly, similar to the results of TEWL, the monitoring of moisture (FIGS. 6E, L, S and Z) showed a recovery after 2 weeks, restoring native skin levels at 4 weeks in all the groups.

[0361] 2.1.3.3. Erythema and Pigmentation

[0362] The evaluation of erythema (FIGS. 6F, M, T and AA) from the BT Skin groups of control, autograft and without cells did not show significant differences with respect to the native skin; however, BT Skin showed significantly higher levels during the 8 weeks of the experiment.

[0363] Regarding pigmentation (FIGS. 6G, N, U and AB), while the Control and Autograft groups failed to achieve native skin melanin levels up to 4 weeks, after 2 weeks, the Cell-free BT Skin and BT Skin groups had already restored those levels.

[0364] 2.1.3.4. Histological Analysis and Immunostaining

[0365] H&E and Masson's Trichrome stains of wound biopsies (FIG. 7 and FIG. 11) showed correct regeneration of the epidermis and dermis after 4 and 8 weeks in all the groups; however, the Autograft, Cell-free BT Skin and BT Skin groups exhibited a more complex dermal matrix structure closer to native skin than the Control group, which showed a less dense dermal matrix structure after 4 weeks of surgical procedure.

[0366] As can be seen in FIG. 8, the immunostaining analysis showed the expression of fibronectin, a typical protein found in the abundantly expressed dermal extracellular matrix (ECM). Likewise, the expression of cytokeratin, a specific epidermal differentiation marker, was observed in all the groups.

[0367] 2.2. Lyophilised Trilaminar Model

[0368] 2.2.1. Mechanical Properties

[0369] The samples with and without cells were generated and maintained at 37° C. in a 5% CO.sub.2 atmosphere. The Young's moduli of the samples kept up to 21 days are represented in FIG. 12A. Although variations were found between the 2 conditions in the period of 21 days, no significant differences were observed.

[0370] The viscoelastic moduli of the cell-laden and cell-free samples were also measured and are shown in FIG. 12B. It was observed that the two tested conditions shared a similar range of viscoelastic moduli, without showing significant differences therebetween during the 21 days.

[0371] 2.2.2. Biological Properties

[0372] A biological characterisation of the samples was carried out with cell proliferation and viability assays. The cell proliferation rates of the lyophilised dressings on days 1, 3, 5, 7, 14, and 21 are shown in FIG. 12C. The lyophilised dressings showed increased cell proliferation from day 3, maintaining a plateau step until the end of the experiment from day 5. The lyophilised dressings were able to maintain the level of cell viability above 93% for the duration of the assay (FIGS. 12D and E), as demonstrated by the images acquired at 1, 10, and 21 days.

[0373] The BT Skin hydrogel was prepared in three layers with the hypodermal layer at the bottom, a middle dermal layer, and the epidermal layer on the top, and it was partially dehydrated. FIG. 4D shows the macroscopic appearance of the BT Skin (black arrow indicating the epidermal layer), and FIGS. 4E and F show the height difference of the hydrogel before and after the partial dehydration process, where the height of the hydrogel is reduced by 2 mm.

[0374] 2.2.3. In Vivo Assays

[0375] Evaluation of the Healing Process in Mouse Skin

[0376] Suitable wound stabilisation was observed for all the mouse groups at 4 weeks, without complications (FIG. 13A). Wound resolution was faster in the group treated with lyophilised dressing than in the Control group. Similarly, wound repair was faster and more effective in the case of this group, while the Control, Autograft and Cell-free BT Skin groups experienced slower improvement. The evaluation of the results by visual observation (FIG. 13A) was correlated with the quantitative analysis of the wound/scar surface area (FIGS. 13B and C), where significant differences were found between the skin groups with lyophilised dressing compared to the control group at week 2, indicating a positive effect of this condition on the wound healing process.

[0377] Study of Homeostasis Parameters

[0378] Temperature, pH, TEWL, elasticity and moisture (FIG. 3) were monitored, comparing all the groups with the native skin of mice. The results of temperature (FIGS. 14A, F, and K) and pH (FIGS. 14, G, and L) showed a homogeneous evolution in all the groups in general of the study, without differences compared to native skin. The temperature values of all the groups during the study ranged from 31.8 to 36.6° C., overlapping the temperature range of native skin (34.0-36.0° C.). Similarly, the pH ranges of all the groups (5.2-7.8) and native skin (6.4-7.3) also generally overlapped throughout the duration of the experiment.

[0379] TEWL (FIGS. 14C, H, and M) showed a significant decrease after 2 weeks in all the groups, reaching native skin levels. Regarding elasticity (FIGS. 14D, I, and N), although an oscillatory behaviour was observed in the Control group throughout the experiment, there were no significant differences between all the groups and Native Skin in general at 8 weeks. Lastly, similar to the results of TEWL, the monitoring of moisture (FIGS. 14E, J, and O) showed a recovery after 2 weeks, restoring native skin levels at 4 weeks in all the groups.

[0380] Erythema and Pigmentation

[0381] The evaluation of erythema (FIGS. 14P, R, and T) of the 3 groups did not show significant differences with respect to the native skin. Regarding pigmentation (FIGS. 14Q, S, and U), the 3 groups reached native skin melanin levels after 4 weeks.

[0382] Histological Analysis and Immunostaining

[0383] H&E and Masson's Trichrome stains of wound biopsies (FIG. 15) showed correct regeneration of the epidermis and dermis after 4 and 8 weeks in all the groups; however, the Autograft and lyophilised groups exhibited a more complex dermal matrix structure closer to native skin than the Control group, which showed a less dense dermal matrix structure after 4 weeks of surgical procedure.

[0384] As can be seen in FIG. 16, the immunostaining analysis showed the expression of fibronectin, a typical protein found in the abundantly expressed dermal extracellular matrix. Likewise, the expression of cytokeratin, a specific epidermal differentiation marker, was observed in all the groups.

[0385] 2.3. Bilaminar Model

[0386] 2.3.1. Hydrogel Formulation

[0387] To determine the most suitable concentration of agarose, the cell viability of the AClow and AC hydrogels was investigated with the Live/Dead assay. Confocal images were used to calculate the percentage of cell viability. Although both concentrations showed good viability rates, cell viability in AClow hydrogels was significantly lower compared to AC hydrogels on days 7 and 14 (FIG. 17).

[0388] Furthermore, a loss of structural integrity was observed in AClow hydrogels after 7 days in culture. Thus, the referenced concentration of Ag (Köpf et al., 8(2):025011 (2016)) was maintained in subsequent experiments.

[0389] To create a hydrogel that mimics the composition of the extracellular matrix (ECM) of the skin, AC-based hydrogels were supplemented with skin components. In other words, DS, a glycosaminoglycan found in native human skin; HA, an ECM polysaccharide from natural skin; and EL, a protein related to the ECM elasticity of the skin, were added to the AC formulation, generating three sequential formulations: ACD, ACDH and ACDHE.

[0390] The hDF-laden hydrogel solutions were pipetted onto the surface of a Petri dish, left to gel, gently transferred to a multiwell culture plate, and cultured for 21 days. All the conditions were mainly populated by live cells, showing a uniform distribution within the hydrogels (FIG. 18A).

[0391] The results of all the formulations did not show negative effects on the viability of the hDFs up to 21 days. The control and hydrogel formulations enriched with skin-related materials were able to maintain levels of viability greater than 78 and 86%, respectively (FIG. 18B), for at least 21 days. These results are consistent with the feasibility specifications for pharmaceutical products prior to their administration to patients, based on the primary quality criteria established by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) 70% and 80%, respectively). ACDHE hydrogels showed higher levels of viability after 21 days compared to AC, ACD and ACDH hydrogels. The cell viability rates of the ACDHE condition were maintained above 93% throughout the experiment. Thus, the ACDHE hydrogel formulation with all ECM components was used for subsequent experiments.

[0392] 2.3.2. Physico-Chemical Properties of the ACDHE Hydrogel

[0393] Inversion Test and pH

[0394] The time taken for the solution to turn into a gel was recorded using the tube inversion test. The mean gel times of the AC and ACDHE hydrogels were 3.4±0.2 and 4.1±0.2 minutes, respectively. FIG. 19A shows evidence of AC hydrogels (white cap vial) and ACDHE hydrogels (black cap vial) in their liquid and gel form. The pH value of the ACDHE hydrogels was 7.36±0.05.

[0395] Hydrogel Ultrastructure

[0396] To physically characterise the hydrogels, ESEM analysis was performed for the ACD, ACDH and ACDHE formulations. The three hydrogel formulations showed a uniform and homogeneous lattice organisation, with an interconnected porous network (FIG. 19B). The pore size increased proportionally with the complexity of the hydrogel formulation, the ACDHE hydrogel showing the largest pore size (FIG. 19C).

[0397] Swelling and Degradation Behaviours of the ACDHE Hydrogel

[0398] Lyophilised ACDHE hydrogels were immersed in PBS (pH 7.4) to study the swelling behaviour of this formulation. The results of the swelling kinetics of the hydrogel are shown in FIG. 20A. The average swelling rate of the ACDHE hydrogel was found to be 42±8%. The swelling kinetics of the ACDHE hydrogel indicated that a stationary phase was reached around 3 days after starting the assay, with a maximum peak on day 14. To study the stability of the ACDHE formulation, hydrogel degradation was determined by measuring the variation of weight over time up to 21 days (FIG. 20B). The maximum degradation rate was found after 14 days with almost 7.14±0.22%.

[0399] Hydrogel Mechanical Properties

[0400] The mechanical properties of AC and ACDHE hydrogels were determined by mechanical assays. As stated in the materials and methods section, hydrogels without and with cells (hDFs) were placed in cylindrical moulds (20 mm in diameter and 5 mm in height) and kept at 37° C. in a 5% CO.sub.2 atmosphere. FIG. 20C shows the Young's moduli obtained from the compression assays of AC and ACDHE hydrogels up to 21 days in culture and they are compared with human skin.

[0401] Although slight variations were found between the Young's moduli of cell-free AC hydrogels and ACDHE hydrogels over time, the cell-free AC hydrogel after 21 days in culture (10.14±0.72 kPa) showed significant differences compared to the range of elasticity observed in native human skin samples (5.22±0.39 kPa), while no significant differences were found on day 21 of culture in cell-free ACDHE hydrogels (7.50±0.94 kPa) compared to native human skin. On the other hand, in addition to the fact that the Young's moduli of the cell-laden hydrogels also showed variations over time, after 21 days of culture, neither the AC hydrogels (5.68±0.00 kPa) nor the ACDHE hydrogels (5.86±0.38 kPa) showed significant differences compared to native human skin (5.22±0.39 kPa). This may be due to the fact that human skin tissue and hydrogels share a similar range of stiffness located in the kPa order of magnitude. The stress range analysed for the Young's moduli obtained in hydrogels and skin samples is shown in FIG. 21.

[0402] The viscoelastic moduli of the AC and ACDHE hydrogels, with cells and cell-free, are shown in FIG. 20D and FIG. 20E. Although the storage moduli of the AC and ACDHE hydrogels, either cell-free (3.19±0.61 and 2.56±0.29 kPa, respectively) or with cells (2.49±0.03 and 1.89±0.12 kPa, respectively) showed a slight variation over time, no significant differences were found after 21 days compared to native human skin samples (2.94±0.27 kPa) (FIG. 20D).

[0403] On the other hand, as can be seen in FIG. 20E when analysing the loss moduli, significant differences were found on day 21 for the AC and ACDHE hydrogels, either cell-free (0.19±0.04 and 0.17±0.00 kPa, respectively) or hydrogels with cells (0.14±0.00 and 0.12±0.00 kPa, respectively) compared to native human skin samples (0.63±0.13 kPa).

[0404] 2.3.3. Wound Closure Assay

[0405] To study the wound healing effect of supplemented soluble biological compounds in the hydrogel formulation, a scratch wound assay was performed. Next, the dermatan/hyaluronic acid/elastin (DHE) solution was added to the culture media and studied in hDF and hMSC (FIG. 22A). Curiously, the hDFs cultured with DHE-supplemented media showed an important wound-healing effect, showing a higher rate of wound closure after 6 and 12 hours, even achieving 100% wound closure 24 hours earlier than the control group (FIG. 22B). Furthermore, the wound closure effect of the DHE formulation on hMSC showed a significantly higher wound healing rate at 48 hours compared to the control group; however, complete wound closure was achieved at 72 hours in both treated and control conditions (FIG. 22C). These results indicate that DHE supplementation can significantly promote the wound healing rate, especially for hDFs.

[0406] 2.3.4. Cellular Bilaminar ACDHE Hydrogel

[0407] Two types of cells were used for the biomanufacture of a bilayer hydrogel. On the one hand, hDFs, an essential component of the skin dermis, and on the other, hMSCs, which can provide growth factors, cytokines and chemokines that promote cell survival and regulate tissue regeneration. The ACDHE hydrogel formulation was prepared in a bilaminar manner with hDFs located in the top layer and hMSCs in the bottom layer. An hMSC-laden ACDHE layer was placed on the surface of a Petri dish and left to gel while the hDF-laden ACDHE solution was prepared. Then, the hDF-laden layer was added on the top of the hMSC-laden layer. Due to the viscosity of the ACDHE formulation, the two layers were not mixed during the process, but rather remained adhered during gelification. Once fully gelled, the samples were transferred to multiwell culture plates and immersed in cell culture medium. FIG. 23A shows the macroscopic appearance of the ACDHE bilayer hydrogel on days 0 and 21, showing that it could hold its shape over time.

[0408] To assess the distribution of cells within the hydrogels, hDFs and hMSCs were previously stained with CellTracker™ Green CMFDA and CellTracker™ Red CMTPX, respectively. Two well-differentiated layers could be observed with the two different cell types (FIG. 23B). The bottom view of the cross section made it possible to see the cells evenly distributed throughout the bilayer structure. Furthermore, this structure maintained its distribution, being able to observe both layers during the entire culture time. The cell viability images acquired on days 0, 7, 14, and 21 showed that most cells were viable, with few dead cells. The ACDHE bilayer hydrogel samples were shown to support cell viability for up to 21 days (FIG. 23C), keeping the clearly differentiated bilaminar distribution, with hDF showing a fibroblastic cell morphology (top) and hMSC showing a spherical shape (bottom). The proliferation rate of the bilaminar hydrogels on days 0, 1, 3, 5, 7, 10, 14, 18, and 21 showed an initial decrease in the proliferation rate (on days 1 to 5) and a subsequent significant increase (from day 10 to 21) (FIG. 23D).

[0409] Furthermore, the cell-laden ACDHE bilaminar hydrogels were also analysed using ESEM (FIG. 24). Encapsulated cells could be observed within the hydrogel matrix (FIGS. 24A and B), and it even seemed that they could produce ECM-like structures (FIGS. 24C and D). FIGS. 24E and 24F show cells and deposited ECM.