Skin Equivalent Research Articles

Investigation into the effect of skin tone modulators and exogenous stress on skin pigmentation utilizing a novel bioengineered skin equivalent.

Human skin equivalents (HSEs) are a popular technology due to limitations in animal testing, particularly as they recapitulate aspects of structure and function of human skin. Many HSEs contain two basic cell types to model dermal and epidermal compartments, however this limits their application, particularly when investigating the effect of exogenous stressors on skin health. We describe the development of a novel platform technology that accurately replicates skin pigmentation in vitro. Through incorporation of melanocytes, specialized pigment producing cells, into the basal layer of the epidermis we are able to re-create skin pigmentation in vitro. We observe apical distribution of melanin within keratinocytes and formation of supranuclear caps (SPNCs), only when the epidermal compartment is co-cultured with a dermal compartment, leading to the conclusion that fibroblast support is essential for correct pigment organization. We also evaluate the commonly observed phenomenon that pigmentation darkens with time in vitro, which we further explore through mechanical exfoliation to remove a build-up of melanin deposits in the stratum corneum. Finally, we demonstrate the application of a pigmented HSE to investigate drug modulation of skin tone and protection from UV-induced damage, highlighting the importance of such a model in the wider context of skin biology.

Activation of Nrf2 in fibroblasts promotes a skin aging phenotype via an Nrf2-miRNA-collagen axis

Aging is associated with progressive skin fragility and a tendency to tear, which can lead to severe clinical complications. The transcription factor NRF2 is a key regulator of the cellular antioxidant response, and pharmacological NRF2 activation is a promising strategy for the prevention of age-related diseases. Using a combination of molecular and cellular biology, histology, imaging and biomechanical studies we show, however, that constitutive genetic activation of Nrf2 in fibroblasts of mice suppresses collagen and elastin expression, resulting in reduced skin strength as seen in aged mice. Mechanistically, the “aging matrisome” results in part from direct Nrf2-mediated overexpression of a network of microRNAs that target mRNAs of major skin collagens and other matrix components. Bioinformatics and functional studies revealed high NRF2 activity in aged human fibroblasts in 3D skin equivalents and human skin biopsies, highlighting the translational relevance of the functional mouse data. Together, these results identify activated NRF2 as a promoter of age-related molecular and biomechanical skin features.

Spatially Guided Construction of Multilayered Epidermal Models Recapturing Structural Hierarchy and Cell-Cell Junctions.

A current challenge in three-dimensional (3D) bioprinting of skin equivalents is to recreate the distinct basal and suprabasal layers and to promote their direct interactions. Such a structural arrangement is essential to establish 3D stratified epidermis disease models, such as for the autoimmune skin disease pemphigus vulgaris (PV), which targets the cell-cell junctions at the interface of the basal and suprabasal layers. Inspired by epithelial regeneration in wound healing, we develop a method that combines 3D bioprinting and spatially guided self-reorganization of keratinocytes to recapture the fine structural hierarchy that lies in the deep layers of the epidermis. Here, keratinocyte-laden fibrin hydrogels are bioprinted to create geographical cues, guiding dynamic self-reorganization of cells through collective migration, keratinocyte differentiation and vertical expansion. This process results in a region of self-organized multilayers (SOMs) that contain the basal to suprabasal transition, marked by the expressed levels of different types of keratins that indicate differentiation. Finally, we demonstrate the reconstructed skin tissue as an in vitro platform to study the pathogenic effects of PV and observe a significant difference in cell-cell junction dissociation from PV antibodies in different epidermis layers, indicating their applications in the preclinical test of possible therapies.

3D skin models along with skin-on-a-chip systems: A critical review

Skin is the largest organ in human body, and it plays an important role in regulating physiological microenvironments and acts as a barrier to protect human body from harmful intrusions. The demand for fully functional skin models (also called skin equivalents, SE) in an in-vivo mimicking culturing microenvironment has been increased dramatically due to the fast development in skin disease treatments and skin care products. Owing to the emerging of the concept and technology of organ-on-chips along with the three-dimensional (3D) bioprinting technology, 3D skin models and their applications have been fast evolving. In this paper, the advances in the development of 3D skin models along with skin-on-a-chip (SOC) are reviewed and commented. One of the findings with this paper is that the SOC together with the 3D bioprinting technology is promising to construct fully functional 3D skin models in the field of pharmaceutical and cosmetic industries.

Biofabrication of Human Skin with Its Appendages.

Much effort has been made to generate human skin organ in the laboratory. Yet, the current models are limited due to the lack of many critical biological and structural features of the skin. Importantly, these in vitro models lack appendages and fail to recapitulate the whole human skin construction. Thus, engineering a human skin with the capacity to generate all components, including appendages, is a major challenge. This review intends to provide an update on the recent efforts underway to regenerate appendage-bearing skin organs based on scaffold-free and scaffold-based bioengineering approaches. Although the mouse skin equivalents containing hair follicles, sebaceous glands, and sweat glands have been established in vitro, there has been limited success in humans. A combination of biofabricated matrices and cell aggregates, such as organoids, can pave the way for generating skin substitutes with human-like biological, structural, and physical features. Accordingly, the formation of human skin organoids and reconstruction of vascularized skin equipped with immune cells prompt calls for more scientific research. The generation of appendage-bearing skin substitutes can be applied in practice for wound healing, hair restoration, and scar treatment.

Analysis of drug efficacy for inflammatory skin on an organ-chip system.

Bacterial skin infections cause a variety of common skin diseases that require drugs that are safer than antibiotics and have fewer side effects. However, for evaluating skin disease drugs, human skin tissue in vitro constructed traditionally on Transwell has inefficient screening ability because of its fragile barrier function. With mechanical forces and dynamic flow, the organ-on-a-chip system became an innovative, automatic, and modular way to construct pathological models and analyze effective pharmaceutical ingredients in vitro. In this research, we integrated skin extracellular matrix and skin cells into a microfluidic chip to construct a biomimetic “interface-controlled-skin-on-chip” system (IC-SoC), which constructed a stable air–liquid interface (ALI) and necessary mechanical signals for the development of human skin equivalents. The results demonstrated that in the microfluidic system with a flowing microenvironment and ALI, the skin tissue formed in vitro could differentiate into more mature tissue morphological structures and improve barrier function. Then, following exposing the skin surface on the IC-SoC to the stimulation of Propionibacterium acnes (P.acnes) and SLS (sodium lauryl sulfate), the barrier function decreased, as well as inflammatory factors such as IL-1α, IL-8, and PEG2 increased in the medium channel of the IC-SoC. After this pathological skin model was treated with dexamethasone and polyphyllin H, the results showed that polyphyllin H had a significant repair effect on the skin barrier and a significant inhibition effect on the release of inflammation-related cytokines, and the effects were more prominent than dexamethasone. This automated microfluidic system delivers an efficient tissue model for toxicological applications and drug evaluation for bacterial-infected damaged skin instead of animals.

34982 Transcriptome analysis reveals intrinsic proinflammatory signaling in healthy African American skin

Background: The disparities in prevalence and clinical features for many inflammatory skin diseases including atopic dermatitis and psoriasis are well recognized; however, the underlying mechanisms are largely unknown. Methods: Using bulk RNA-seq analysis, we compared the baseline gene expression in upper arm full thickness skin biopsies from 17 healthy individuals self-reporting as African American (AA) or White non-Hispanic (WNH) and in 3D human skin equivalents (HSE) made from AA and WNH primary skin keratinocytes. Results: Extensively validated RNA-Seq analysis identified 570 differentially expressed genes (DEG) in AA skin including immunoglobulins and their receptors such as FCER1G; proinflammatory genes such as TNFα, IL-32; EDC (epidermal differentiation cluster) and keratin genes. DEGs were functionally enriched for inflammatory responses, keratinization, cornified envelope formation. RNA-seq analysis of gene expression in AA versus WNH HSEs revealed 360 DEGs (some shared with skin) which were enriched by similar to skin functions. In addition, AA HSE appeared more responsive to TNFα proinflammatory effects. Finally, AA-specific DEGs in skin and HSE significantly overlapped with molecular signatures of lesional skin in AD and psoriasis patients. Interestingly, AA skin DEGs overlapping with AD molecular signature in skin were enriched by IL13 and IL4 signaling central for AD. Conclusions: Overall, these findings suggest the existence of intrinsic proinflammatory circuits in AA keratinocytes/skin that may account for disease disparities and will help to build a foundation for the development of targeted skin disease prevention and treatment.

FLIM for Evaluation of Difference in Metabolic Status between Native and Differentiated from iPSCs Dermal Papilla Cells.

iPSCs and their derivatives are the most promising cell sources for creating skin equivalents. However, their properties are not fully understood. In addition, new approaches and parameters are needed for studying cells in 3D models without destroying their organization. Thus, the aim of our work was to study and compare the metabolic status and pH of dermal spheroids created from dermal papilla cells differentiated from pluripotent stem cells (iDP) and native dermal papilla cells (hDP) using fluorescence microscopy and fluorescence lifetime imaging microscopy (FLIM). For this purpose, fluorescence intensities of NAD(P)H and FAD, fluorescence lifetimes, and the contributions of NAD(P)H, as well as the fluorescence intensities of SypHer-2 and BCECF were measured. iDP in spheroids were characterized by a more glycolytic phenotype and alkaline intra-cellular pH in comparison with hDP cells. Moreover, the metabolic activity of iDP in spheroids depends on the source of stem cells from which they were obtained. So, less differentiated and condensed spheroids from iDP-iPSDP and iDP-iPSKYOU are characterized by a more glycolytic phenotype compared to dense spheroids from iDP-DYP0730 and iDP-hES. FLIM and fluorescent microscopy in combination with the metabolism and pH are promising tools for minimally invasive and long-term analyses of 3D models based on stem cells.

Isolation of an "Early" Transit Amplifying Keratinocyte Population in Human Epidermis: A Role for the Low Affinity Neurotrophin Receptor CD271.

In the interfollicular epidermis (IFE), stem cells (KSC) generate transit amplifying (TA) cells that, after symmetric divisions, produce differentiating daughters. Here, we isolated and characterized the highly proliferative interfollicular epidermal basal cell population "early" TA (ETA) cells, based on their capacity to adhere to type IV collagen. Proliferation and colony-forming efficiency in ETA cells are lower than in KSC but higher than in "late" TA (LTA). Stemness, proliferation, and differentiation markers confirmed that ETA cells display a unique phenotype. Skin reconstructs derived from ETA cells present different features (epidermal thickness, Ki67, and Survivin expression), as compared to skin equivalents generated from either KSC or LTA cells. The low-affinity neurotrophin receptor CD271, which regulates the KSC to TA cell transition in the human epidermis through an on/off switch control mechanism, is predominantly expressed in ETA cells. Skin equivalents generated from siRNA CD271 ETA cells display a more proliferative and less differentiated phenotype, as compared to mock-derived reconstructs. Consistently, CD271 overexpression in LTA cells generates a more proliferative skin equivalent than mock LTA cells. Finally, the CD271 level declines with cellular senescence, while it induces a delay in p16INK4 expression. We conclude that ETA cells represent the first KSC progenitor with exclusive features. CD271 identifies and modulates ETA cells, thus participating in the early differentiation and regenerative capacity of the human epidermis.

Recombinant Human Collagen-Based Bioinks for the 3D Bioprinting of Full-thickness Human Skin Equivalent.

As a major extracellular matrix component within the skin, collagen has been widely used to engineer human skin tissues. However, most collagen is extracted from animals. Here, we introduced recombinant human type III collagen (rhCol3) as a bioactive component to formulate bioinks for the bioprinting of a full-thickness human skin equivalent. Human dermal fibroblasts were encapsulated in the gelatin methacryloyl-rhCol3 composite bioinks and printed on a transwell to form the dermis layer, on which human epidermal keratinocytes were seeded to perform an air-liquid interface culture for 6 weeks. After optimizing the bioink formulation and bioprinting process, we investigated the effect of rhCol3 on skin tissue formation. The results suggest that a higher concentration of rhCol3 would enhance the growth of both cells, resulting in a more confluent (~100%) spreading of the epidermal keratinocytes at an early stage (3 days), compared to the rhCol3-free counterpart. Moreover, in an in vivo experiment, adding rhCol3 in the hydrogel formulation would contribute to the skin wound healing process. Taken together, we conclude that rhCol3 could act as a functional bioink component to promote basic skin cellular processes for skin tissue engineering.

MiR-100-5p Promotes Epidermal Stem Cell Proliferation through Targeting MTMR3 to Activate PIP3/AKT and ERK Signaling Pathways.

Skin epidermal stem cells (EpSCs) play a critical role in wound healing and are ideal seed cells for skin tissue engineering. Exosomes from human adipose-derived stem cells (ADSC-Exos) promote human EpSC proliferation, but the underlying mechanism remains unclear. Here, we investigated the effect of miR-100-5p, one of the most abundant miRNAs in ADSC-Exos, on the proliferation of human EpSCs and explored the mechanisms involved. MTT and BrdU incorporation assays showed that miR-100-5p mimic transfection promoted EpSC proliferation in a time-dependent manner. Cell cycle analysis showed that miR-100-5p mimic transfection significantly decreased the percentage of cells in the G1 phase and increased the percentage of cells in the G2/M phase. Myotubularin-related protein 3 (MTMR3), a lipid phosphatase, was identified as a direct target of miR-100-5p. Knockdown of MTMR3 in EpSCs by RNA interference significantly enhanced cell proliferation, decreased the percentage of cells in the G1 phase and increased the percentage of cells in the S phase. Overexpression of MTMR3 reversed the proproliferative effect of miR-100-5p on EpSCs, indicating that miR-100-5p promoted EpSC proliferation by downregulating MTMR3. Mechanistic studies showed that transfection of EpSCs with miR-100-5p mimics elevated the intracellular PIP3 level, induced AKT and ERK phosphorylation, and upregulated cyclin D1, E1, and A2 expression, which could be attenuated by MTMR3 overexpression. Consistently, intradermal injection of ADSC-Exos or miR-100-5p-enriched ADSC-Exos into cultured human skin tissues significantly reduced MTMR3 expression and increased the thickness of the epidermis and the number of EpSCs in the basal layer of the epidermis. The aforementioned effect of miR-100-5p-enriched ADSC-Exos was stronger than that of ADSC-Exos and was reversed by MTMR3 overexpression. Collectively, our findings indicate that miR-100-5p promotes EpSC proliferation through MTMR3-mediated elevation of PIP3 and activation of AKT and ERK. miR-100-5p-enriched ADSC-Exos can be used to treat skin wound and expand EpSCs for generating epidermal autografts and engineered skin equivalents.

Contribution of keratinocytes to dermal fibrosis.

Purpose of reviewThe cellular pathogenesis of fibrotic disorders including systemic sclerosis (SSc) remains largely speculative. Currently, the altered function of endothelial cells and fibroblasts under the influence of an inappropriate immune response are considered central pathogenic events in SSc. Adding to this complexity, novel evidence here reviewed suggests that keratinocytes may concur in the development of skin fibrosis.Recent findingsEpidermal equivalents (EE) generated from primary SSc keratinocytes display a distinct gene expression program when compared to healthy donor (HD) EE. SSc-EE, among others, exhibited enhanced oxidative and metabolic response pathways. Immunohistochemical studies demonstrated similarities between SSc-EE and SSc epidermis including altered keratinocyte differentiation, enhanced expression of activation markers, and reduced rate of basal keratinocytes proliferation. SSc-EE supernatants more than HD-EE modified the inflammatory and extracellular matrix deposition/resorption program of dermal fibroblasts. Further evidence indicated that the relative lack rather than the excess of interleukin-25 in keratinocytes may contribute to enhanced dermal fibrotic changes. Overall, these data support keratinocyte-intrinsic SSc-related modifications.SummaryImproved methods for engineering epidermal and skin equivalents are helping to address the question whether keratinocyte alterations in SSc are primary and capable to dysregulate dermal homeostasis or secondary following dermal fibrotic changes.

Novel 3D-Printed Cell Culture Inserts for Air-Liquid Interface Cell Culture.

In skin research, widely used in vitro 2D monolayer models do not sufficiently mimic physiological properties. To replace, reduce, and refine animal experimentation in the spirit of ‘3Rs’, new approaches such as 3D skin equivalents (SE) are needed to close the in vitro/in vivo gap. Cell culture inserts to culture SE are commercially available, however, these inserts are expensive and of limited versatility regarding experimental settings. This study aimed to design novel cell culture inserts fabricated on commercially available 3D printers for the generation of full-thickness SE. A computer-aided design model was realized by extrusion-based 3D printing of polylactic acid filaments (PLA). Improvements in the design of the inserts for easier and more efficient handling were confirmed in cell culture experiments. Cytotoxic effects of the final product were excluded by testing the inserts in accordance with ISO-norm procedures. The final versions of the inserts were tested to generate skin-like 3D scaffolds cultured at an air–liquid interface. Stratification of the epidermal component was demonstrated by histological analyses. In conclusion, here we demonstrate a fast and cost-effective method for 3D-printed inserts suitable for the generation of 3D cell cultures. The system can be set-up with common 3D printers and allows high flexibility for generating customer-tailored cell culture plastics.

3D Printing of Skin Equivalents with Hair Follicle Structures and Epidermal-Papillary-Dermal Layers Using Gelatin/Hyaluronic Acid Hydrogels.

Recent advances in three-dimensional (3D) bioprinting technologies have enabled the fabrication of sophisticated live 3D tissue analogs. Despite the existing hydrogel-based bioinks, the development of advanced bioink materials that can accurately reproduce the composition of a native extracellular matrix and mimic the intrinsic properties of laden cells remains challenging. In this study, 3D printed skin equivalents incorporating hair follicle structures and epidermal/papillary dermal layers were fabricated using gelatin methacryloyl/hyaluronic acid methacryloyl (GelMA/HAMA) bioink. The composition of collagen and glycosaminoglycan in native skin was recapitulated by adjusting the combination of GelMA and HAMA. The GelMA/HAMA bioink exhibited excellent viscoelastic and physicochemical properties, 3D printability, cytocompatibility, and functionality to maintain hair-inductive potency while facilitating spontaneous hair pore development. The results indicate that GelMA/HAMA hydrogels are promising candidates as bioinks for the 3D printing of skin equivalents. Furthermore, they may serve as useful models for skin tissue engineering and regeneration.

Robots with a human touch

Robotics offers an unprecedented means to support people's daily lives. However, human-robot interactions remain challenging because of the appearance of robots. Kawai et al. propose to overcome this by growing biological skin equivalents on robots. Their study opens new possibilities to further humanize perception of robots and improve human-robot interactions.

400 Microfluidic culture insert device for fabrication of full-thickness human skin equivalent with perfusable vascular-like channels

Full-thickness skin equivalent(SE) is widely used to assess the safety of chemicals. However, a lack of important components, such as vascular and neuro-immune systems, prevents the use of such models in acute toxicity, repeated dose toxicity, and reproductive developmental toxicity tests. Several approaches to fabricate vascularized SE have been published. However, each of them had its limitations. Some models did not contain vascular-like channels but instead had an underlying channel, which does not allow differentiation between percutaneous penetration and absorption by vessels.

121 Using in vitro human skin equivalents to understand the complexities of skin ageing and identify therapeutic interventions to restore skin health

The ageing global demographic constantly reinforces the need for more age focussed research, in order to better support the population to live healthier for longer. As the interface between the human body and the external environment, the experience of skin is unique, being subjected to both internal and external ageing influences, leading to pathological changes in structure/function. Currently, there are few in vitro ageing skin equivalents to undertake this research, and even fewer which account for both ageing factors.

Design of an Integrated Microvascularized Human Skin-on-a-Chip Tissue Equivalent Model.

Tissue-engineered skin constructs have been under development since the 1980s as a replacement for human skin tissues and animal models for therapeutics and cosmetic testing. These have evolved from simple single-cell assays to increasingly complex models with integrated dermal equivalents and multiple cell types including a dermis, epidermis, and vasculature. The development of micro-engineered platforms and biomaterials has enabled scientists to better recreate and capture the tissue microenvironment in vitro, including the vascularization of tissue models and their integration into microfluidic chips. However, to date, microvascularized human skin equivalents in a microfluidic context have not been reported. Here, we present the design of a novel skin-on-a-chip model integrating human-derived primary and immortalized cells in a full-thickness skin equivalent. The model is housed in a microfluidic device, in which a microvasculature was previously established. We characterize the impact of our chip design on the quality of the microvascular networks formed and evidence that this enables the formation of more homogenous networks. We developed a methodology to harvest tissues from embedded chips, after 14 days of culture, and characterize the impact of culture conditions and vascularization (including with pericyte co-cultures) on the stratification of the epidermis in the resulting skin equivalents. Our results indicate that vascularization enhances stratification and differentiation (thickness, architecture, and expression of terminal differentiation markers such as involucrin and transglutaminase 1), allowing the formation of more mature skin equivalents in microfluidic chips. The skin-on-a-chip tissue equivalents developed, because of their realistic microvasculature, may find applications for testing efficacy and safety of therapeutics delivered systemically, in a human context.

Advanced Online Monitoring of In Vitro Human 3D Full-Thickness Skin Equivalents.

Skin equivalents and skin explants are widely used for dermal penetration studies in the pharmacological development of drugs. Environmental parameters, such as the incubation and culture conditions affect cellular responses and thus the relevance of the experimental outcome. However, available systems such as the Franz diffusion chamber, only measure in the receiving culture medium, rather than assessing the actual conditions for cells in the tissue. We developed a sampling design that combines open flow microperfusion (OFM) sampling technology for continuous concentration measurements directly in the tissue with microfluidic biosensors for online monitoring of culture parameters. We tested our design with real-time measurements of oxygen, glucose, lactate, and pH in full-thickness skin equivalent and skin explants. Furthermore, we compared dermal penetration for acyclovir, lidocaine, and diclofenac in skin equivalents and skin explants. We observed differences in oxygen, glucose, and drug concentrations in skin equivalents compared to the respective culture medium and to skin explants.

Therapeutic base editing and prime editing of COL7A1 mutations in recessive dystrophic epidermolysis bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by loss-of-function mutations in the COL7A1 gene, which encodes type VII collagen (C7), a protein that functions in skin adherence. From 36 Korean RDEB patients, we identified a total of 69 pathogenic mutations (40 variants without recurrence), including point mutations (72.5%) and insertion/deletion mutations (27.5%). For fibroblasts from two patients (Pat1 and Pat2), we applied adenine base editors (ABEs) to correct the pathogenic mutation of COL7A1 or to bypass a premature stop codon in Pat1-derived primary fibroblasts. To expand the targeting scope, we also utilized prime editors(PEs) to correct the COL7A1 mutations in Pat1- and Pat2-derived fibroblasts. Ultimately, we found that transfer of edited patient-derived skin equivalents (i.e., RDEB keratinocytes and PE-corrected RDEB fibroblasts from the RDEB patient) intothe skin of immunodeficient mice led to C7 deposition and anchoring fibril formation within the dermal-epidermal junction, suggesting that base editing and prime editing could be feasible strategies for exvivo gene editing to treat RDEB.