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

Jennifer M Hahn,Kevin L Mcfarland,Steven T Boyce,Christopher M Lloyd,Dorothy M Supp,Kelly A Combs,Michael R Hamblin

doi:10.1371/journal.pone.0213325

Jennifer M Hahn, Kevin L Mcfarland + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0213325

Copy DOI

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.

Highlights

Burns are relatively common injuries, accounting for over 350,000 emergency room visits in the United States annually [1]
A collagen-chitosan scaffold populated with fibroblasts and keratinocytes was found to be innervated 60 days after transplantation to mice, and incorporation of Schwann cells in vitro accelerated innervation after grafting [61, 62]
The co-localization of Merkel cell markers and neuroendocrine proteins in cells of the basal epidermis of engineered skin substitutes (ESS) in vivo strongly suggests that these cells are Merkel cells

Summary

Introduction

Burns are relatively common injuries, accounting for over 350,000 emergency room visits in the United States annually [1]. Itch is considered a major problem that contributes significantly to reduced quality of life in burn survivors [4, 5]. Neuronal damage and nervous system morbidity, including sensory loss, is commonly reported in patients after burn injury [8,9,10]. Grafted burns exhibit significant sensory deficits and reductions in innervation density compared with uninjured skin, which may persist for years after initial injury [11, 15, 16]. In one study of survivors of large burns affecting over 30% total body surface area (TBSA), one third of subjects reported sensory loss at 20–30 years after injury [15]. Sensory loss was reported in patients with smaller burns, even in “good quality” scars [14]

Methods

Results

Discussion

Conclusion