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Identification of Merkel cells associated with neurons in engineered skin substitutes after grafting to full thickness wounds.

ABSTRACT: Engineered skin substitutes (ESS), prepared using primary human fibroblasts and keratinocytes with a biopolymer scaffold, were shown to provide stable closure of excised burns, but relatively little is known about innervation of ESS after grafting. This study investigated innervation of ESS and, specifically, whether Merkel cells are present in healed grafts. Merkel cells are specialized neuroendocrine cells required for fine touch sensation in skin. We discovered cells positive for keratin 20 (KRT20), a general marker for Merkel cells, in the basal epidermis of ESS after transplantation to mice, suggesting the presence of Merkel cells. Cells expressing KRT20 were not observed in ESS in vitro. However, widely separated KRT20-positive cells were observed in basal epidermis of ESS by 2 weeks after grafting. By 4 weeks, these cells increased in number and expressed keratins 18 and 19, additional Merkel cells markers. Putative Merkel cell numbers increased further between weeks 6 and 14; their densities varied widely and no specific pattern of organization was observed, similar to Merkel cell localization in human skin. KRT20-positive cells co-expressed epidermal markers E-cadherin and keratin 15, suggesting derivation from the epidermal lineage, and neuroendocrine markers synaptophysin and chromogranin A, consistent with their identification as Merkel cells. By 4 weeks after grafting, some Merkel cells in engineered skin were associated with immature afferents expressing neurofilament-medium. By 8 weeks, Merkel cells were complexed with more mature neurons expressing neurofilament-heavy. Positive staining for human leukocyte antigen demonstrated that the Merkel cells in ESS were derived from grafted human cells. The results identify, for the first time, Merkel cell-neurite complexes in engineered skin in vivo. This suggests that fine touch sensation may be restored in ESS after grafting, although this must be confirmed with future functional studies.

SUBMITTER: Hahn JM

PROVIDER: S-EPMC6400390 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Identification of Merkel cells associated with neurons in engineered skin substitutes after grafting to full thickness wounds.

Hahn Jennifer M JM Combs Kelly A KA Lloyd Christopher M CM McFarland Kevin L KL Boyce Steven T ST Supp Dorothy M DM

PloS one 20190305 3

Engineered skin substitutes (ESS), prepared using primary human fibroblasts and keratinocytes with a biopolymer scaffold, were shown to provide stable closure of excised burns, but relatively little is known about innervation of ESS after grafting. This study investigated innervation of ESS and, specifically, whether Merkel cells are present in healed grafts. Merkel cells are specialized neuroendocrine cells required for fine touch sensation in skin. We discovered cells positive for keratin 20 ( ...[more]

PMID: 30835771

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Project description:Over 1 million burn injuries are treated annually in the United States, and current tissue engineered skin fails to meet the need for full-thickness replacement. Bioprinting technology has allowed fabrication of full-thickness skin and has demonstrated the ability to close full-thickness wounds. However, analysis of collagen remodeling in wounds treated with bioprinted skin has not been reported. The purpose of this study is to demonstrate the utility of bioprinted skin for epidermal barrier formation and normal collagen remodeling in full-thickness wounds. Human keratinocytes, melanocytes, fibroblasts, dermal microvascular endothelial cells, follicle dermal papilla cells, and adipocytes were suspended in fibrinogen bioink and bioprinted to form a tri-layer skin structure. Bioprinted skin was implanted onto 2.5 × 2.5 cm full-thickness excisional wounds on athymic mice, compared with wounds treated with hydrogel only or untreated wounds. Total wound closure, epithelialization, and contraction were quantified, and skin samples were harvested at 21 days for histology. Picrosirius red staining was used to quantify collagen fiber orientation, length, and width. Immunohistochemical (IHC) staining was performed to confirm epidermal barrier formation, dermal maturation, vascularity, and human cell integration. All bioprinted skin treated wounds closed by day 21, compared with open control wounds. Wound closure in bioprinted skin treated wounds was primarily due to epithelialization. In contrast, control hydrogel and untreated groups had sparse wound coverage and incomplete closure driven primarily by contraction. Picrosirius red staining confirmed a normal basket weave collagen organization in bioprinted skin-treated wounds compared with parallel collagen fibers in hydrogel only and untreated wounds. IHC staining at day 21 demonstrated the presence of human cells in the regenerated dermis, the formation of a stratified epidermis, dermal maturation, and blood vessel formation in bioprinted skin, none of which was present in control hydrogel treated wounds. Bioprinted skin accelerated full-thickness wound closure by promoting epidermal barrier formation, without increasing contraction. This healing process is associated with human cells from the bioprinted skin laying down a healthy, basket-weave collagen network. The remodeled skin is phenotypically similar to human skin and composed of a composite of graft and infiltrating host cells. Impact statement We have demonstrated the ability of bioprinted skin to enhance closure of full-thickness wounds through epithelialization and normal collagen remodeling. To our knowledge, this article is the first to quantify collagen remodeling by bioprinted skin in full-thickness wounds. Our methods and results can be used to guide further investigation of collagen remodeling by tissue engineered skin products to improve ongoing and future bioprinting skin studies. Ultimately, our skin bioprinting technology could translate into a new treatment for full-thickness wounds in human patients with the ability to recapitulate normal collagen remodeling in full-thickness wounds.

Dataset Information

Identification of Merkel cells associated with neurons in engineered skin substitutes after grafting to full thickness wounds.

Publications

Identification of Merkel cells associated with neurons in engineered skin substitutes after grafting to full thickness wounds.

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