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

Date Published: March 5, 2019

Publisher: Public Library of Science

Author(s): Jennifer M. Hahn, Kelly A. Combs, Christopher M. Lloyd, Kevin L. McFarland, Steven T. Boyce, Dorothy M. Supp, Michael R Hamblin.


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.

Partial Text

Burns are relatively common injuries, accounting for over 350,000 emergency room visits in the United States annually [1]. Advances in burn care have contributed to reduced mortality rates in recent decades, which has caused a shift in focus from acute burn care towards optimizing the long-term quality of wound healing for improved functional outcomes [2]. Although small and superficial partial-thickness wounds can heal without grafting, deep partial-thickness and full-thickness burns generally require skin grafting to achieve wound closure [3]. The prevailing standard for skin grafting is split-thickness skin autograft taken from uninjured donor skin of the same patient [2, 3]. Burn scars, including scarring of grafted burns and donor sites, can result in substantial morbidity in addition to cosmetic impairment. Pain, itch, abnormal pigmentation, and restricted range of motion due to contraction of burn scars are common problems that impair quality of life for burn survivors [2]. In particular, itch is considered a major problem that contributes significantly to reduced quality of life in burn survivors [4, 5]. Itch is present in most pediatric burn patients at the time of discharge and a majority still experience itching two years after injury [6]; similar trends have been reported in the adult burn population [7]. Neuronal damage and nervous system morbidity, including sensory loss, is also commonly reported in patients after burn injury [8–10]. Clinical studies have documented significant deficits in sensations including touch, heat, and cold in grafted burns [10–15]. In particular, 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 also reported in patients with smaller burns, even in “good quality” scars [14].

Loss of sensation is commonly reported by burn survivors and can persist for decades after burn injury [11–15]. Although deficits in innervation have been reported, relatively little is known about recovery of Merkel cell populations in burn scars. In one study from 1990, Merkel cells were observed in healed autograft but they were not associated with nerves [35]; in a 1994 study, Merkel cells were observed to be present only infrequently in healed skin grafts [34]. In mice, deletion of Piezo2 channels in Merkel cells was associated with increased pain sensitivity, in addition to reduced fine touch sensation, and absence of Merkel cells in mouse skin caused increased itching [30, 31]. It is not yet known whether Merkel cells have similar roles in pain and itch in human skin. If they do, it is possible that the absence of Merkel cell-neurite complexes in burn scars and skin grafts may contribute to not only the decreased fine touch sensation, but also the increased pain and itch experienced by burn survivors. Thus, the presence of Merkel cells in healed ESS would have important implications for functional recovery after burn injury. Hypothetically, Merkel cells in ESS may contribute to restoration of fine touch sensation while reducing pain and itch.

ESS were shown to provide stable wound closure of excised burn wounds in pediatric patients, but innervation status of healed ESS has not been previously examined. Innervation has been studied preclinically in other engineered skin models. For example, 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]. Merkel cells were not described in either of those engineered skin models. The co-localization of Merkel cell markers and neuroendocrine proteins in cells of the basal epidermis of ESS in vivo strongly suggests that these cells are Merkel cells. The current study indicates that innervation of ESS is initiated within four weeks after transplantation, and by four to 6 weeks after grafting, neurons are found in association with Merkel cells. This suggests formation of Merkel cell-neurite complexes in ESS in vivo, which to our knowledge has not been previously demonstrated in a tissue-engineered skin substitute. This observation suggests that fine touch sensation may be restored in healed engineered skin; however, functional studies will be required to confirm the role of Merkel cells in recovery of touch sensation after grafting.




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