Reconstitution of full-thickness skin by microcolumn grafting

Abstract

In addition to providing a physical barrier, skin also serves a diverse range of physiological functions through different specialized resident cell types/structures, including melanocytes (pigmentation and protection against ultraviolet radiation), Langerhans cells (adaptive immunity), fibroblasts (maintaining extracellular matrix, paracrine regulation of keratinocytes), sweat glands (thermoregulation) and hair follicles (hair growth, sensation and a stem cell reservoir). Restoration of these functional elements has been a long-standing challenge in efforts to engineer skin tissue, while autologous skin grafting is limited by the scarcity of donor site skin and morbidity caused by skin harvesting. We demonstrate an alternative approach of harvesting and then implanting μm-scale, full-thickness columns of human skin tissue, which can be removed from a donor site with minimal morbidity and no scarring. Fresh human skin microcolumns were used to reconstitute skin in wounds on immunodeficient mice. The restored skin recapitulated many key features of normal human skin tissue, including epidermal architecture, diverse skin cell populations, adnexal structures and sweat production in response to cholinergic stimulation. These promising preclinical results suggest that harvesting and grafting of microcolumns may be useful for reconstituting fully functional skin in human wounds, without donor site morbidity. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

Keywords: adnexa; full thickness; healing; regeneration; skin wound.

Figure 1

Harvesting and application of full‐thickness skin columns. (A) Full‐thickness skin columns harvested with a 19 gauge coring needle, each containing epidermis (1), dermis including adnexal structures (2) and some subcutaneous fat (3); each mark on the ruler in the photograph spans 1 mm. (B) Viability of skin columns after harvesting, demonstrated by calcein‐AM staining (green), with only a small portion of the tissue showing signs of cell death, as indicated by ethidium homodimer‐1 staining (red), mostly along the edge of the epidermis (solid arrow); one particular skin column contained a viable sweat gland (hollow arrow); dermal portions of the columns are highlighted by dashed arrows (for high‐power views of the epidermis and sweat gland, see supporting information, Figure S1); scale bar = 2.5 mm. (C) Human skin columns applied in random orientation to a full‐thickness wound on the dorsal skin of a mouse; arrow highlights the epidermal head of one skin column. (D) Photograph of the same wound site taken 8 weeks later; the wound has healed, with some pigmentation due to growth of the human epidermis (arrow). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Restoration of human epidermal features, 8 weeks after murine full‐thickness wounds were repaired using human skin columns: positive immunohistochemical staining is shown in red; human‐specific staining is labelled as such by the suffix (hu) behind the name of the corresponding target. (A) Human keratinocytes, identified by positive staining for human major histocompatibility complex 1 (MHC1), forming a contiguous epidermis with an overlying human stratum corneum; arrow, transition point between murine (left, MHC1^–) and human (right, MHC1⁺) epidermis at the wound margin. (B) Architecture of the restored epidermis, seen under H&E staining; all four layers of the epidermis, basal (1), spinous (2), granular (3) and cornified (4), are present in the correct ascending order; rete ridges are present at the dermal–epidermal junction (yellow arrow). (C) Melanocytes identified by Melan‐A expression (red) reside in the basal epidermal layer, while melanin is visible as brown pigmentation present in a perinuclear pattern in neighbouring cells (arrow). (D) Proliferative cells in the basal epidermal layer, identified by Ki67 expression (red, highlighted by arrows). (E) Dendritic Langerhans cells expressing CD1a (shown in red), populated most densely in the spinous layer of the epidermis. Scale bars = (A) 250 μm; (B, E) 50 μm. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Restoration of human dermal features, 8 weeks after murine full‐thickness wounds were repaired using human skin columns: positive immunohistochemical staining is shown in red; human‐specific staining is labelled as such by the suffix (hu) behind the name of the corresponding target. Human fibroblasts expressing vimentin (A) and elastin (B) fibres were present throughout the restored dermis. Sebaceous glands of human origin, as shown by MHC1 staining (C), were also restored. Sebocytes containing lipid granules were present in the gland lumen (C, arrow). Proliferative cells, labelled by Ki67 (D, arrows), lined the periphery of the sebaceous glands. Hair follicles were associated with human melanocytes (E) and Nestin‐expressing progenitor cells (F). Eccrine sweat glands maintained the characteristic expression of cytokeratins 7 (G) and 8 (H). The restored dermis was innervated by PGP9.5‐expressing neurons through the dermis (I), but especially around the adnexal structures (J). Scale bars = 50 μm. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4

Agonist‐induced sweat test: 8 weeks after full‐thickness wounds treated with human skin columns (~400 μm diameter), the reconstituted skin (A) was covered first with iodine, then a starch–mineral oil mixture (B, blurriness of image caused by thick layer of starch‐mineral mix on top of the skin). Acetylcholine was injected subcutaneously under the region of interest: 10 min after acetylcholine was administered, sweat production could be detected by the formation of dark blue spots (C, arrows). As a negative control, normal mouse back skin, which is devoid of eccrine sweat glands, is shown before (D) and 10 min after (E) acetylcholine stimulation, with no detectable sweat production. As a positive control, the same test caused sweating in the normal mouse paw (F, arrows), which contains eccrine sweat glands. [Colour figure can be viewed at wileyonlinelibrary.com]