Nkemcho Ojeh

Reepithelialization of a full-thickness burn from stem cells of hair follicles micrografted into a tissue-engineered dermal template (integra)

We report a head and neck full-thickness burn injury that was reconstructed with a tissue-engineered dermal template and then early implantation of microdissected hair follicles through the silicone epidermis 12 days after the burn injury. The treatment resulted in complete reepithelialization and a hair-bearing scalp without the need for a split-thickness skin graft. Restoration of the stem cell population, hair growth, and earlier reepithelialization were achieved using this novel micrografting technique, and histologic examination confirmed maturation of a normal skin type over the subsequent 2 years.

Stem Cells in Skin Regeneration, Wound Healing, and Their Clinical Applications

The skin is the largest organ of the body and has an array of functions. Skin compartments, epidermis, and hair follicles house stem cells that are indispensable for skin homeostasis and regeneration. These stem cells also contribute to wound repair, resulting in restoration of tissue integrity and function of damaged tissue. Unsuccessful wound healing processes often lead to non-healing wounds. Chronic wounds are caused by depletion of stem cells and a variety of other cellular and molecular mechanisms, many of which are still poorly understood. Current chronic wound therapies are limited, so the search to develop better therapeutic strategies is ongoing. Adult stem cells are gaining recognition as potential candidates for numerous skin pathologies. In this review, we will discuss epidermal and other stem cells present in the skin, and highlight some of the therapeutic applications of epidermal stem cells and other adult stem cells as tools for cell/scaffold-based therapies for non-healing wounds and other skin disorders. We will also discuss emerging concepts and offer some perspectives on how skin tissue-engineered products can be optimized to provide efficacious therapy in cutaneous repair and regeneration.

Epithelial-mesenchymal transition in tissue repair and fibrosis

AbstractThe epithelial-mesenchymal transition (EMT) describes the global process by which stationary epithelial cells undergo phenotypic changes, including the loss of cell-cell adhesion and apical-basal polarity, and acquire mesenchymal characteristics that confer migratory capacity. EMT and its converse, MET (mesenchymal-epithelial transition), are integral stages of many physiologic processes and, as such, are tightly coordinated by a host of molecular regulators. Converging lines of evidence have identified EMT as a component of cutaneous wound healing, during which otherwise stationary keratinocytes (the resident skin epithelial cells) migrate across the wound bed to restore the epidermal barrier. Moreover, EMT plays a role in the development of scarring and fibrosis, as the matrix-producing myofibroblasts arise from cells of the epithelial lineage in response to injury but are pathologically sustained instead of undergoing MET or apoptosis. In this review, we summarize the role of EMT in physiologic repair and pathologic fibrosis of tissues and organs. We conclude that further investigation into the contribution of EMT to the faulty repair of fibrotic wounds might identify components of EMT signaling as common therapeutic targets for impaired healing in many tissues. Graphical AbstractModel for injury-triggered EMT activation in physiologic wound repair (left) and fibrotic wound healing (right)

An in vitro skin model to study the effect of mesenchymal stem cells in wound healing and epidermal regeneration

The development of new wound therapies, such as bioengineered skin equivalents, is an ongoing process. Multi-potent mesenchymal stem cells (MSCs) give rise to many tissue lineages and have been implicated in wound healing making them a potential candidate for cell-based bioengineered products for injured tissue. In this study, we investigated the mesenchymal/epithelial interactions of cultured MSCs in comparison to cultured fibroblasts on epidermal proliferation, differentiation, and extracellular matrix (ECM) protein expression using a de-epidermalized dermis (DED) skin model. We also studied whether MSCs can transdifferentiate to keratinocytes using the same model. Keratinocytes were cultured on unseeded DED or DED populated with fibroblasts or MSCs at an air-liquid interface to induce epidermal differentiation. Fibroblasts or MSCs were also seeded on the papillary surface of the DED alone or on the reticular surface. General histology and immunostaining was performed on the skin equivalents to examine the expression of pan keratin (K) (K1, K5, K6, and K18) and protein markers for epidermal differentiation (K10), hyperproliferation (K6), proliferation (PCNA), ECM component (collagen type IV), and mesenchymal marker (vimentin). Keratinocyte-fibroblast skin model and keratinocyte-MSC skin model both displayed an epidermal phenotype similar to epidermis in vivo. Positive expression of proliferation, differentiation and ECM protein markers was observed. MSCs failed to adopt an epithelial phenotype in the DED skin model. Our findings highlight the potential use of MSCs in bioengineered tissue for the treatment of wounds.

The effects of caffeine on wound healing.

The purine alkaloid caffeine is a major component of many beverages such as coffee and tea. Caffeine and its metabolites theobromine and xanthine have been shown to have antioxidant properties. Caffeine can also act as adenosine‐receptor antagonist. Although it has been shown that adenosine and antioxidants promote wound healing, the effect of caffeine on wound healing is currently unknown. To investigate the effects of caffeine on processes involved in epithelialisation, we used primary human keratinocytes, HaCaT cell line and ex vivo model of human skin. First, we tested the effects of caffeine on cell proliferation, differentiation, adhesion and migration, processes essential for normal wound epithelialisation and closure. We used 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) proliferation assay to test the effects of seven different caffeine doses ranging from 0·1 to 5 mM. We found that caffeine restricted cell proliferation of keratinocytes in a dose‐dependent manner. Furthermore, scratch wound assays performed on keratinocyte monolayers indicated dose‐dependent delays in cell migration. Interestingly, adhesion and differentiation remained unaffected in monolayer cultures treated with various doses of caffeine. Using a human ex vivo wound healing model, we tested topical application of caffeine and found that it impedes epithelialisation, confirming in vitro data. We conclude that caffeine, which is known to have antioxidant properties, impedes keratinocyte proliferation and migration, suggesting that it may have an inhibitory effect on wound healing and epithelialisation. Therefore, our findings are more in support of a role for caffeine as adenosine‐receptor antagonist that would negate the effect of adenosine in promoting wound healing.

Skin Metabolite, Farnesyl Pyrophosphate, Regulates Epidermal Response to Inflammation, Oxidative Stress, and Migration.

Skin produces cholesterol and a wide array of sterols and non‐sterol mevalonate metabolites, including isoprenoid derivative farnesyl pyrophosphate (FPP). To characterize FPP action in epidermis, we generated transcriptional profiles of primary human keratinocytes treated with zaragozic acid (ZGA), a squalene synthase inhibitor that blocks conversion of FPP to squalene resulting in endogenous accumulation of FPP. The elevated levels of intracellular FPP resulted in regulation of epidermal differentiation and adherens junction signaling, insulin growth factor (IGF) signaling, oxidative stress response and interferon (IFN) signaling. Immunosuppressive properties of FPP were evidenced by STAT‐1 downregulation and prominent suppression of its nuclear translocation by IFNγ. Furthermore, FPP profoundly downregulated genes involved in epidermal differentiation of keratinocytes in vitro and in human skin ex vivo. Elevated levels of FPP resulted in induction of cytoprotective transcriptional factor Nrf2 and its target genes. We have previously shown that FPP functions as ligand for the glucocorticoid receptor (GR), one of the major regulator of epidermal homeostasis. Comparative microarray analyses show significant but not complete overlap between FPP and glucocorticoid regulated genes, suggesting that FPP may have wider transcriptional impact. This was further supported by co‐transfection and chromatin immunoprecipitation experiments where we show that upon binding to GR, FPP recruits β‐catenin and, unlike glucocorticoids, recruits co‐repressor GRIP1 to suppress keratin 6 gene. These findings have many clinical implications related to epidermal lipid metabolism, response to glucocorticoid therapy as well as pleiotropic effects of cholesterol lowering therapeutics, statins. J. Cell. Physiol. 231: 2452–2463, 2016.

In vitro skin models to study epithelial regeneration from the hair follicle

The development of dermal equivalents (DEs) for the treatment of burns has contributed toward efficient wound closure. A collagen-glycosaminoglycan DE (C-GAG) grafted with hair follicles converted a full-thickness wound to partial-thickness resulting in complete wound closure and restored hair. In this study we compared the ability of both intact pilosebaceous units (PSU) or truncated hair follicles (THF) to regenerate a multilayered epidermis in vitro when implanted into de-epidermalized dermis (DED) or C-GAG with the epidermis generated in vivo using C-CAG. Keratinocytes explanted from the outer root sheath of PSU and THF in both DED and C-GAG but only formed a multilayered epidermis with PSU in DED. PSU were more effective at forming multilayered epidermis in DED than THF. Both DED and C-GAG skin expressed proliferation (PCNA), differentiation (K1, K10), hyperproliferation (K6, K16), basal (K14), putative stem cell (p63), extracellular matrix protein (Collagen IV), mesenchymal (vimentin) and adherens junction (β-catenin) markers. These data suggest DEs supported initial maintenance of the implanted hair follicles, in particular PSU, and provide an excellent model with which to investigate the regulation of hair follicle progenitor epithelial cells during epidermal regeneration. Although neither PSU nor THF formed multilayered epidermis in C-CAG in vitro, hair follicles implanted into engrafted C-GAG on a burns patient resulted in epithelial regeneration and expression of proliferation and differentiation markers in a similar manner to that seen in vitro. However, the failure of C-GAG to support epidermal regeneration in vitro suggests in vivo factors are essential for full epidermal regeneration using C-GAG.

Feasibility of an electrostimulation system treatment for wound healing: a case series of patients with chronic ulcers in Barbados

Major advances have been made in the development of new therapies for chronic wounds. Fenzian™, an electrostimulation system (ES), has been clinically used for a variety of conditions. The ES was recently tested in the Barbadian population for tolerability and acceptability by asthma patients, with encouraging results. Barbados has an estimated 170 people with diabetes having some form of lower‐extremity amputation annually. Here, we describe a case series of 21 chronic ulcer patients with diabetes recruited as inpatients (n = 10) and outpatients (n = 11) in a pilot study to evaluate the feasibility and acceptability of ES in the Barbadian population. Results showed statistically significant improvement among those in the inpatient‐ versus outpatient‐recruited group for wound perimeter (P = 0·04), wound surface area (P = 0·03) and wound volume (P = 0·08). We also demonstrate that the improvement continued after cessation of ES treatment. Participants reported increased levels of pain at the end of treatment, and there was no statistically significant change in the reported quality of life. Our results showed greater improvements in reduction of ulcer size for participants from the inpatient‐ versus outpatient‐recruited group.