Polymerizable Skin Hydrogel for Full Thickness Wound Healing

Abstract

The skin is the largest organ in the human body, comprising the main barrier against the environment. When the skin loses its integrity, it is critical to replace it to prevent water loss and the proliferation of opportunistic infections. For more than 40 years, tissue-engineered skin grafts have been based on the in vitro culture of keratinocytes over different scaffolds, requiring between 3 to 4 weeks of tissue culture before being used clinically. In this study, we describe the development of a polymerizable skin hydrogel consisting of keratinocytes and fibroblast entrapped within a fibrin scaffold. We histologically characterized the construct and evaluated its use on an in vivo wound healing model of skin damage. Our results indicate that the proposed methodology can be used to effectively regenerate skin wounds, avoiding the secondary in vitro culture steps and thus, shortening the time needed until transplantation in comparison with other bilayer skin models. This is achievable due to the instant polymerization of the keratinocytes and fibroblast combination that allows a direct application on the wound. We suggest that the polymerizable skin hydrogel is an inexpensive, easy and rapid treatment that could be transferred into clinical practice in order to improve the treatment of skin wounds.

Keywords: cellular therapy; hydrogel; skin regeneration; tissue engineering.

Figure 1

Analysis of thrombin concentrate and fibrinogen samples. (A) Fibrinogen concentration (g/L) in each FFP used. (B) Enzymatic activity (UI/mL) in each thrombin concentrate produced. (C) Standard curve used to calculate the enzymatic activity of each thrombin concentrate [y (UI/mL) = −3.4503 × x(s) + 161.09]. (D) Fibrinogen concentration (g/L) variation after storage at −80 °C and thawing at different times up to 30 days. Samples did not show a significative depletion (p > 0.05). (E) Enzymatic activity (UI/mL) variation after storage at −80 °C and thawing at different times up to 30 days. Samples did not show a significative depletion (p > 0.05). Data expressed as mean ± SD.

Figure 2

Immunofluorescence study of skin hydrogels fabricated in vitro after 7 days of air-liquid culture. Continuous keratinocyte layer (in red, excitation/emission: 480/527, labelled with anti-human high molecular weight cytokeratin antibody) firmly attached to the underlying fibroblasts-containing hydrogel. Fibroblasts (in green, excitation/emission: 360/470, labelled with anti-vimentin antibody) grew homogeneously across the dermal layer. Scale bar: 50 µm.

Figure 3

In vivo fabrication of polymerizable skin hydrogel. (A) In vivo extrusion of the polymerizable skin hydrogel. (B) Extruded hydrogel on the wound bed at room temperature remained stable without leaking. (C) Devitalized murine skin sutured to the dorsum of the mouse after surgery. (D–F) Wound healing follow-up. Devitalized skin shrank and detached gradually from the back of the mouse. (G) Regenerated human skin in the back of the athymic mouse. Wound completely healed after 21 days.

Figure 4

Immunohistochemistry study of the regenerated skin in athymic mouse. 21 days after treatment with the polymerizable skin hydrogel, the human regenerated skin showed positive labelling for involucrin and cytokeratin 10 in the suprabasal layers. Vimentin labelled full thickness of human dermis and some cells located into the dermis of the grafted area. Scale bars: 50 µm. Vimentin scale bar: 200 µm. In the control group (hydrogel without human cells) wound closure is achieved by murine cells from the wound edges. Biopsies from the treated area showed no labelling for the antibodies used.