Co-culture Of Keratinocytes Research Articles

Design and self-assembly of new peptides and peptide bolaamphiphiles as antioxidant biocomposite scaffolds for potential tissue regeneration applications – a replica modeling and experimental study

ABSTRACT In this work, five new peptides derived from natural resources and two peptide bolaamphiphiles were designed. The self-assembling ability of the peptides and the bolaamphiphiles, as well as their predicted antioxidant activity was examined computationally. In particular, replica modeling molecular dynamics studies were carried out at three different temperatures. Results showed that the bolaamphiphiles as well as three of the peptides efficiently formed spherical or fibrous assemblies, particularly at physiological temperatures. In addition, stacking interactions and hydrogen bonds played a critical role in assembly formation. Furthermore, molecular docking studies with extracellular matrix proteins such as the triple helix motif of collagen and the fibronectin (III) motif of tenascin-X displayed binding interactions with the peptides and the bolaamphiphiles. The most optimal peptide bolaamphiphile WMYGGGWMY-CO-NH-(CH2)4-YMWGGGYMW was then synthesized in the laboratory and its ability to form functional scaffolds upon binding to collagen and tenascin-X was examined. The scaffolds were bioprinted with co-cultures of fibroblasts and keratinocytes. The cells not only proliferated over time but also showed strong adherence and spreading within the matrix. Thus, the peptides and the bolaamphiphiles studied in this work, may be potentially developed as scaffold components for tissue regeneration applications.

Food hydroxycinnamic acids alleviate ageing in dermal cells

Dermal damage is inducible by several factors including UV exposure, oxidative stress and inflammation exacerbating skin senescence and degradation of the skin elastic fibers accumulated in ageing accordingly. Which, phenolics of food hydroxycinnamates with a myriad of health benefits are potentially applicable for ageing treatment. Particularly those of food hydroxycinnamic acids, i.e., caffeic, sinapic and rosmarinic acids, that would be efficient against skin ageing. Effectiveness of caffeic, sinapic and rosmarinic acids alleviating ageing was indicated in human dermal fibroblasts (HDF) and co-culture of human keratinocytes (HaCaT) and HDF. Caffeic acid was exhibited as the strongest (p < 0.01) anti-senescent phenolic examined. The studied food hydroxycinnamic acids were shown to induce collagen synthesis in aged HDF with the noted activities inhibiting MMP-1 and IL-6. Their photoaging protections were proved in the co-culture model with significant (p < 0.001) inhibitions against IL-6, IL-8, MMP-1 and MMP-9 (collagen and elastin degrading enzymes). Which, caffeic acid was demonstrated as the most potent photoaging agent among its counterparts. Caffeic, sinapic and rosmarinic acids were proved to be the efficient nutrients for ageing treatment. These functional food hydroxycinnamates are proven on their anti-senescent and photoprotection activities, and capable to maintain homeostasis of dermal cells. Food-derived hydroxycinnamic acids are therefore recommended for innovative product alleviates skin ageing.Graphical

Prevotella melaninogenica disrupted oral epithelial barrier function via myosin light chain kinase.

Our previous studies have found that the composition ratio of Prevotella melaninogenica (Pm) on buccal mucosa surface of oral lichen planus (OLP) patients increased significantly compared with control. Furthermore, Pm could invade the epithelium of OLP patients. This study aimed to further explore the impact of Pm on oral keratinocytes. The Pm-human oral keratinocyte (HOK) co-culture model was established to detect monolayer permeability, zona occludens-1 (ZO-1) expression, and intracellular survival of Pm. We performed RNA-seq followed by identification of differentially expressed genes (DEGs) and Gene Ontology (GO) analysis, with a particular focus on myosin light chain kinase (MLCK). An MLCK inhibitor ML-7 was utilized in Pm-HOK co-culture model to assess its effects on monolayer permeability and ZO-1 expression. HOK monolayer permeability was increased, and ZO-1 expression was decreased after co-culture (p < 0.05). Pm could survive in HOK cells. RNA-seq revealed MLCK was an upregulated common DEG. The expression of MLCK in the Pm-HOK co-culture model was upregulated. Inhibition of MLCK rescued the increased epithelial permeability, and ZO-1 expression was upregulated (p < 0.05). MLCK may be involved in disrupting epithelial barrier function by Pm.

A Platform for Testing the Biocompatibility of Implants: Silicone Induces a Proinflammatory Response in a 3D Skin Equivalent.

Biocompatibility testing of materials is carried out in 2D cell cultures or animal models despite serious limitations. 3D skin equivalents are advanced in vitro models for human skin. Silicone has been shown to be noncytotoxic but capable of eliciting an immune response. Our aim was to (1) establish a 3D skin equivalent to (2) assess the proinflammatory properties of silicone. We developed a coculture of keratinocytes and fibroblasts resulting in a 3D skin equivalent with an implant using samples from a breast implant. Samples with and without the silicone implant were studied histologically and immunohistochemically in comparison to native human skin samples. Cytotoxicity was assessed via LDH-assay, and cytokine response was assessed via ELISA. Histologically, our 3D skin equivalents had a four-layered epidermal and a dermal component. The presence of tight junctions was demonstrated in immunofluorescence. The only difference in 3D skin equivalents with implants was an epidermal thinning. Implanting the silicone samples did not cause more cell death, however, an inflammatory cytokine response was triggered. We were able to establish an organotypical 3D skin equivalent with an implant, which can be utilised for studies on biocompatibility of materials. This first integration of silicone into a 3D skin equivalent confirmed previous findings on silicone being non-cell-toxic but capable of exerting a proinflammatory effect.

Indocyanine green-loaded platelet activated by photodynamic and photothermal effects for selective control of wound repair

ObjectivePrompt and effective wound repair is an essential strategy to promote recovery and prevent infection in patients with various types of trauma. Platelets can release a variety of growth factors upon activation to facilitate revascularization and tissue repair, provided that their activation is uncontrollable. The present study is designed to explore the selective activation of platelets by photodynamic and photothermal effects (PDE/PTE) as well as the trauma repair mediated by PDE/PTE. Materials and MethodsIn the current research, platelets were extracted from the blood of mice. Indocyanine green (ICG) was applied to induce PDE/PTE. The uptake of ICG by platelets was detected by laser confocal microscopy and flow cytometry. The cellular integrity was measured by microscopy. The reactive oxygen species (ROS) generation and temperature of platelets were assayed by 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) and temperature detector. The activation of platelets was measured by western blots (WB), dynamic light scattering (DLS), and scanning electron microscopy (SEM). The release of growth factor was detected by enzyme-linked immuno sorbent assay (Elisa), wherein the in vitro cell proliferation was investigated by 5-Ethynyl-2’-deoxyuridine (EDU) assay. The wound infection rates model and histological examination were constructed to assay the ICG-loaded platelet-mediated wound repair. ResultsPlatelets could load with ICG, a kind of photodynamic and photothermal agent, as carriers and remain intact. Near-infrared (NIR) laser irradiation of ICG-loaded platelets (ICG@PLT) facilitated higher temperature and ROS generation, which immediately activated ICG@PLT, as characterized by increased membrane p-selectin (CD62p), cyclooxygenase-2 (COX-2), thromboxane A2 receptor (TXA2R) expression, elevated hydrated particle size, and prominent aggregation in platelets. Further investigation revealed that massive insulin-like growth factor (IGF) and platelet-derived growth factor (PDGF) were released from the activated ICG@PLT, which also promoted the proliferation of endothelial cells and keratinocytes in co-culture. In consequence, activated platelets and increased neovascularization could be observed in rats with wound infection treated by ICG@PLT in the presence of NIR. More impressively, the hydrogel containing ICG@PLT accelerated wound healing and suppressed inflammation under NIR, exhibiting excellent wound repair properties. ConclusionTaken together, the current work identified that platelets could be activated by PDE/PTE and thereby release growth factor, potentiating wound repair in a controlled manner.

Bacteria‐Epithelial Cell Interaction Influences Cytotoxicity and Antibacterial Effect of Silver‐Gold Alloy Nanoparticles on a Species‐Specific Level

AbstractThe rising number of antibiotic‐resistant bacteria requires alternative antibacterial substances as therapeutics. However, besides a strong effect against bacteria, they should not exhibit cytotoxicity towards human cells. Typically, both properties are tested in separate in vitro experiments that do not take into account naturally occurring interactions. To analyze whether the bacteria‐cell interaction influences the results of antibacterial and cytotoxic testing, surfactant‐free antibacterial silver‐gold alloy nanoparticles produced by laser ablation in liquid were examined both separately and in a straightforward coculture setup. Whereas different commensal bacteria and human skin cells exhibited reduced metabolic activity at comparable concentrations when treated separately, their response completely differed when analyzed in a combined coculture. For the combination of oral keratinocytes and Staphylococcus aureus, reduced cytotoxicity with increasing cell numbers, but a similar antibacterial effect quantified by standard plate counting could be observed. In contrast, coculture of keratinocytes and Porphyromonas gingivalis resulted in a complete absence of cytotoxicity and antibacterial effect, probably due to bacterial invasion of cells. These results clearly showed that the bacteria‐cell interaction greatly influences the results of antibacterial and cytotoxic testing and highlight the importance of more complex in vitro experiments to reliably characterize novel antibacterial substances for efficient clinical translation.

Eicosapentaenoic acid reduces the proportion of IL-17A–producing T cells in a 3D psoriatic skin model

Psoriasis is a skin disease presenting as erythematous lesions with accentuated proliferation of epidermal keratinocytes, infiltration of leukocytes, and dysregulated lipid metabolism. T cells play essential roles in the disease. n-3 polyunsaturated fatty acids are anti-inflammatory metabolites, which exert an immunosuppressive effect on healthy T cells. However, the precise mechanistic processes of n-3 polyunsaturated fatty acids on T cells in psoriasis are still unrevealed. In this study, we aimed to evaluate the action of eicosapentaenoic acid (EPA) on T cells in a psoriatic skin model produced with T cells. A coculture of psoriatic keratinocytes and polarized T cells was prepared using culture media, which was either supplemented with 10 μM EPA or left unsupplemented. Healthy and psoriatic skin substitutes were produced according to the self-assembly method. In the coculture model, EPA reduced the proportion of IL-17A–positive cells, while increasing that of FOXP3-positive cells, suggesting an increase in the polarization of regulatory T cells. In the 3D psoriatic skin model, EPA normalized the proliferation of psoriatic keratinocytes and diminished the levels of IL-17A. The expression of the proteins of the signal transducer and activator of transcription was influenced following EPA supplementation with downregulation of the phosphorylation levels of signal transducer and activator of transcription 3 in the dermis. Finally, the NFκB signaling pathway was modified in the EPA-supplemented substitutes with an increase in Fas amounts. Ultimately, our results suggest that in this psoriatic model, EPA exerts its anti-inflammatory action by decreasing the proportion of IL-17A–producing T cells.

Human Keratinocytes and Fibroblasts Co-Cultured on Silk Fibroin Scaffolds Exosomally Overrelease Angiogenic and Growth Factors.

Objectives: The optimal healing of skin wounds, deep burns, and chronic ulcers is an important clinical problem. Attempts to solve it have been driving the search for skin equivalents based on synthetic or natural polymers. Methods: Consistent with this endeavor, we used regenerated silk fibroin (SF) from Bombyx mori to produce a novel compound scaffold by welding a 3D carded/hydroentangled SF-microfiber-based nonwoven layer (C/H-3D-SFnw; to support dermis engineering) to an electrospun 2D SF nanofiber layer (ESFN; a basal lamina surrogate). Next, we assessed-via scanning electron microscopy, attenuated total reflectance Fourier transform infrared spectroscopy, differential scanning calorimetry, mono- and co-cultures of HaCaT keratinocytes and adult human dermal fibroblasts (HDFs), dsDNA assays, exosome isolation, double-antibody arrays, and angiogenesis assays-whether the C/H-3D-SFnws/ESFNs would allow the reconstitution of a functional human skin analog in vitro. Results: Physical analyses proved that the C/H-3D-SFnws/ESFNs met the requirements for human soft-tissue-like implants. dsDNA assays revealed that co-cultures of HaCaTs (on the 2D ESFN surface) and HDFs (inside the 3D C/H-3D-SFnws) grew more intensely than did the respective monocultures. Double-antibody arrays showed that the CD9+/CD81+ exosomes isolated from the 14-day pooled growth media of HDF and/or HaCaT mono- or co-cultures conveyed 35 distinct angiogenic/growth factors (AGFs). However, versus monocultures' exosomes, HaCaT/HDF co-cultures' exosomes (i) transported larger amounts of 15 AGFs, i.e., PIGF, ANGPT-1, bFGF, Tie-2, Angiogenin, VEGF-A, VEGF-D, TIMP-1/-2, GRO-α/-β/-γ, IL-1β, IL-6, IL-8, MMP-9, and MCP-1, and (ii) significantly more strongly stimulated human dermal microvascular endothelial cells to migrate and assemble tubes/nodes in vitro. Conclusions: Our results showed that both cell-cell and cell-SF interactions boosted the exosomal release of AGFs from HaCaTs/HDFs co-cultured on C/H-3D-SFnws/ESFNs. Hence, such exosomes are an asset for prospective clinical applications as they advance cell growth and neoangiogenesis and consequently graft take and skin healing. Moreover, this new integument analog could be instrumental in preclinical and translational studies on human skin pathophysiology and regeneration.

New human in vitro co-culture model of keratinocytes and sensory neurons like cells releasing substance P with an evaluation of the expression of ZIKV entry receptors: A potent opportunity to test Zika virus entry and to study Zika virus' infection in neurons?

During the course of acute ZIKV infection, pruritus is a cardinal symptom widely documented in the literature. Its frequent association with dysesthesia and several dysautonomic manifestations, suggests a pathophysiological mechanism involving the peripheral nervous system. The aim of this study was to develop a functional human model to potentially able to be infected by ZIKV: by demonstrating the functionality on a new human model of co-culture of keratinocyte and sensory neuron derived from induced pluripotent stem cells using a classical method of capsaicin induction and SP release, and verify the presence of ZIKV entry receptor in these cells. Depending of cellular type, receptors of the TAMs family, TIMs (TIM1, TIM3 and TIM4) and DC-SIGN and RIG1 were present/detected. The cells incubations with capsaicin resulted in an increase of the substance P. Hence, this study demonstrated the possibility to obtain co-cultures of human keratinocytes and human sensory neurons that release substance P in the same way than previously published in animal models which can be used as a model of neurogenic skin inflammation. The demonstration of the expression of ZIKV entry receptors in these cells allows to considerate the potent possibility that ZIKV is able to infect cells.

Evaluation of in vitro coculture of keratinocytes derived from foreskin and adipose-derived mesenchymal stem cells (AMSCs) on a multilayer oxygen-releasing electrospun scaffold based on PU/PCL.Sodium percarbonate (SPC)-gelatine/PU.

Despite significant advancements in tissue engineering and regenerative medicine during the last two decades, the fabrication of proper scaffolds with appropriate cells can still be considered a critical achievement in this field. Hypoxia is a major stumbling block to chronic wound healing, which restrains tissue engineering plans because a lack of oxygen may cause cell death. This study evaluated the cocultured human keratinocytes and human adipose-derived mesenchymal stem cells (AMSCs) on a multilayer oxygen-releasing electrospun scaffold based on PU/PCL.Sodium percarbonate (SPC)-gelatin/PU. The scaffold was characterized using Fourier transform infrared (FTIR) and scanning electron microscopy (SEM) methods. Flow cytometry confirmed mesenchymal stem cells, and then the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay and DAPI staining were used to assess the in vitro biocompatibility of the scaffold. The experimental results showed that the multilayer electrospun scaffold containing 2.5% SPC could efficiently produce oxygen. Furthermore, according to cell viability results, this structure makes a suitable substrate for the coculture of keratinocytes and AMSCs. Gene expression analysis of various markers such as Involucrin, Cytokeratin 10, and Cytokeratin 14 after 14 days confirmed that keratinocytes and AMSCs coculture on PU/PCL.SPC-gelatin/PUelectrospun scaffold promotes dermal differentiation and epithelial proliferation compared to keratinocytes single-cell culture. Therefore, our study supports using oxygen-releasing scaffolds as a potential strategy to hasten skin tissue regeneration. Based on the results, this structure is suggested as a promising candidate for cell-based skin tissue engineering. Given that the developed oxygen-generating polymeric electrospun scaffolds could be used as part of a future strategy for skin tissue engineering, the PU/PCL.SPC-gelatin/PUhybrid electrospun multilayer scaffold in combination with keratinocyte/AMSC coculture is proposed as an effective substrate for skin tissue engineering and regenerative medicine platforms.

Modeling of three-dimensional innervated epidermal like-layer in a microfluidic chip-based coculture system

Reconstruction of skin equivalents with physiologically relevant cellular and matrix architecture is indispensable for basic research and industrial applications. As skin-nerve crosstalk is increasingly recognized as a major element of skin physiological pathology, the development of reliable in vitro models to evaluate the selective communication between epidermal keratinocytes and sensory neurons is being demanded. In this study, we present a three-dimensional innervated epidermal keratinocyte layer as a sensory neuron-epidermal keratinocyte co-culture model on a microfluidic chip using the slope-based air-liquid interfacing culture and spatial compartmentalization. Our co-culture model recapitulates a more organized basal-suprabasal stratification, enhanced barrier function, and physiologically relevant anatomical innervation and demonstrated the feasibility of in situ imaging and functional analysis in a cell-type-specific manner, thereby improving the structural and functional limitations of previous coculture models. This system has the potential as an improved surrogate model and platform for biomedical and pharmaceutical research.

Co-axial electrospinning of PLLA shell, collagen core nanofibers for skin tissue engineering.

The main functions of wound dressing biomaterials are to promote a moist environment in the wound while protecting the area from mechanical injury and microbial contamination. Furthermore, the scaffold used for skin tissue engineering must mimic epidermal and dermal layers as well as support the growth of keratinocytes and fibroblasts. In this study, PLLA (shell) and EGF-encapsulated collagen (core) nanofibers were produced by coaxial electrospinning at 25 kV potential, 12cm collector distance, and 0.125mL/h flow rate of PLLA (15%) and collagen (4%). The bilayer structure was produced by gelling GeIMA in between two nanofiber membranes to imitate the epidermal and dermal layers of skin. Cytocompatibility properties of nanofiber membrane and bilayer structure were characterized by mono- and coculture of keratinocytes (HaCaT) and fibroblasts (3T3), respectively. TEM revealed that the PLLA shell and collagen core thicknesses were about 60 and 115nm, respectively. Oxygen and water vapor could pass through the GeIMA- integrated bilayer nanofiber membranes. The presence of EGF in nanofibers could increase cell proliferation. Fluorescence and SEM imaging showed that HaCaT and 3T3 could cover the membrane after 14days of monoculture. Cocultures showed a reduction in the proliferation of cells in the first week and a recovery during the second and third weeks. In a mechanical bioreactor, cocultured bilayer membranes formed interlocked polygonal keratinocyte cells. These results showed that the bilayer nanofiber membrane and GeIMA combination provided cell compatibility. Furthermore, the use of a mechanical reactor was found to be effective in the formation of a functional keratinocyte layer by stimulating cells.

Study the disassembly of epithelial tight junction under the influence of nanoplastics using an in vitro multicellular model.

More than 350M tons of plastics are produced over the world; eventually, plastic is degraded into microplastics/nanoplastics and released into the environment. Compared to the inert bulk plastics, the impacts of nanoplastics on the health are still largely underexplored. For epithelial tissues, the dynamic of tight junctions is important in maintaining the barrier integrity. In this study, using a microfluidic system, we build up a multi-layer model to reconstruct the epithelial barrier to investigate the influence of nanoplastics on the epithelial barrier. To support long-term culture and real-time monitoring, the microfluidic chip is designed with medium-exchange channels and a side-view observation window. A multi-layer cell model is established via layer-by-layer co-culture of human keratinocytes and fibroblasts representing the epidermis and dermis; the thickness of the on-chip epidermis (0.1–0.2 mm) and the on-chip dermis (0.3–0.6 mm) is close to the clinical observations. Applying nanoplastics to the epithelial barrier, we found the disassembly of tight junctions along the intercellular contacts; nanoplastics further penetrate deeply into the multicellular structure. Moreover, we observed the increasing intracellular reactive oxygen species (ROS), which suggests the potential dysfunction of mitochondria led by nanoplastics internalized into the cells. Our data suggested that the nanoplastics exposure may lead to the destruction of epithelial functions through the tight junction disassembly. Considering the critical roles of tight-junction and ROS in homeostasis and disease progression, besides the nanotoxicity, our findings suggest an alternative impact of nanoplastic on our health through interrupting the junction assembly.

Functional validation of co-culture model of human keratinocytes and neuronal cell line for sensitive skin by using transient receptor potential channel vanilloid subfamily member 1 antagonist.

Sensitive skin is a subjective cutaneous hyper-reactivity that occurs in response to various innocuous stimuli. Keratinocytes have recently been shown to participate in sensory transduction by releasing many neuroactive molecules that bind to intra-epidermal free nerve endings and modulate nociception. In the literature, the characterization of these interactions has been based on the co-culture of keratinocyte and mammalian-origin neuronal cell lines. In this study, we established an in vitro model based on a co-culture of primary human keratinocytes and differentiated SH-SY5Y cells, a human neuronal cell line. Human epidermal keratinocytes and SH-SY5Y cells were monocultured and co-cultured. Changes in calcium influx, substance P, inflammatory cytokines, and neuropeptides between the monoculture and co-culture groups treated with capsaicin only and capsaicin with transient receptor potential channel vanilloid subfamily member 1 (TRPV1) antagonist, trans-4-tert-butylcyclohexanol (TTBC), together. In addition, the difference in stinging sensation was evaluated by applying it to the volunteers. When SH-SY5Y cells were co-cultured with keratinocytes, they had no significant effect on axonal development. Substance P was also released after capsaicin treatment and reduced by TTBC under co-culture conditions. Moreover, the expression of inflammatory cytokines and neuropeptides was significantly increased in co-cultured keratinocytes compared to that under monoculture conditions. In addition, the stinging sensation was significantly induced after the application of capsaicin in vivo and was relieved after the application of the TRPV1 antagonist. We demonstrated that the novel co-culture model is functionally valid through capsaicin and TRPV1 antagonist. We also confirmed that TTBC could be used for the treatment of sensitive skin through a co-culture model and in vivo tests. This co-culture model of keratinocytes and SH-SY5Y cells may be useful in vitro alternatives for studying the close communication between keratinocytes and neuronal cells and for screening therapeutic drugs for sensitive skin.

Melanoma stimulates the proteolytic activity of HaCaT keratinocytes

BackgroundKeratinocytes constitute a major part of the melanoma microenvironment, considering their protective role towards melanocytes in physiological conditions. However, their interactions with tumor cells following melanomagenesis are still unclear.MethodsWe used two in vitro models (melanoma-conditioned media and indirect co-culture of keratinocytes with melanoma cells on Transwell inserts) to activate immortalized keratinocytes towards cancer-associated ones. Western Blotting and qPCR were used to evaluate keratinocyte markers and mediators of cell invasiveness on protein and mRNA expression level respectively. The levels and activity of proteases and cytokines were analysed using gelatin-FITC staining, gelatin zymography, chemiluminescent enzymatic test, as well as protein arrays. Finally, to further study the functional changes influenced by melanoma we assessed the rate of proliferation of keratinocytes and their invasive abilities by employing wound healing assay and the Transwell filter invasion method.ResultsHaCaT keratinocytes activated through incubation with melanoma-conditioned medium or indirect co-culture exhibit properties of less differentiated cells (downregulation of cytokeratin 10), which also prefer to form connections with cancer cells rather than adjacent keratinocytes (decreased level of E-cadherin). While they express only a small number of cytokines, the variety of secreted proteases is quite prominent especially considering that several of them were never reported as a part of secretome of activated keratinocytes’ (e.g., matrix metalloproteinase 3 (MMP3), ADAM metallopeptidase with thrombospondin type 1 motif 1). Activated keratinocytes also seem to exhibit a high level of proteolytic activity mediated by MMP9 and MMP14, reduced expression of TIMPs (tissue inhibitor of metalloproteinases), upregulation of ERK activity and increased levels of MMP expression regulators-RUNX2 and galectin 3. Moreover, cancer-associated keratinocytes show slightly elevated migratory and invasive abilities, however only following co-culture with melanoma cells on Transwell inserts.ConclusionsOur study offers a more in-depth view of keratinocytes residing in the melanoma niche, drawing attention to their unique secretome and mediators of invasive abilities, factors which could be used by cancer cells to support their invasion of surrounding tissues.52obFQmRyJn1nHi_mM3M7bVideo abstract

A Systematic Review of Keratinocyte Secretions: A Regenerative Perspective.

Cell regenerative therapy is a modern solution for difficult-to-heal wounds. Keratinocytes, the most common cell type in the skin, are difficult to obtain without the creation of another wound. Stem cell differentiation towards keratinocytes is a challenging process, and it is difficult to reproduce in chemically defined media. Nevertheless, a co-culture of keratinocytes with stem cells usually achieves efficient differentiation. This systematic review aims to identify the secretions of normal human keratinocytes reported in the literature and correlate them with the differentiation process. An online search revealed 338 references, of which 100 met the selection criteria. A total of 80 different keratinocyte secretions were reported, which can be grouped mainly into cytokines, growth factors, and antimicrobial peptides. The growth-factor group mostly affects stem cell differentiation into keratinocytes, especially epidermal growth factor and members of the transforming growth factor family. Nevertheless, the reported secretions reflected the nature of the involved studies, as most of them focused on keratinocyte interaction with inflammation. This review highlights the secretory function of keratinocytes, as well as the need for intense investigation to characterize these secretions and evaluate their regenerative capacities.

Engineering a 3D In Vitro Model of Human Gingival Tissue Equivalent with Genipin/Cytochalasin D.

Although three-dimensional (3D) co-culture of gingival keratinocytes and fibroblasts-populated collagen gel can mimic 3D structure of in vivo tissue, the uncontrolled contraction of collagen gel restricts its application in clinical and experimental practices. We here established a stable 3D gingival tissue equivalent (GTE) using hTERT-immortalized gingival fibroblasts (hGFBs)-populated collagen gel directly crosslinked with genipin/cytochalasin D and seeding hTERT-immortalized gingival keratinocytes (TIGKs) on the upper surface for a 2-week air–liquid interface co-culture. MTT assay was used to measure the cell viability of GTEs. GTE size was monitored following culture period, and the contraction was analyzed. Immunohistochemical assay was used to analyze GTE structure. qRT-PCR was conducted to examine the mRNA expression of keratinocyte-specific genes. Fifty µM genipin (G50) or combination (G + C) of G50 and 100 nM cytochalasin D significantly inhibited GTE contraction. Additionally, a higher cell viability appeared in GTEs crosslinked with G50 or G + C. GTEs crosslinked with genipin/cytochalasin D showed a distinct multilayered stratified epithelium that expressed keratinocyte-specific genes similar to native gingiva. Collagen directly crosslinked with G50 or G + C significantly reduced GTE contraction without damaging the epithelium. In summary, the TIGKs and hGFBs can successfully form organotypic multilayered cultures, which can be a valuable tool in the research regarding periodontal disease as well as oral mucosa disease. We conclude that genipin is a promising crosslinker with the ability to reduce collagen contraction while maintaining normal cell function in collagen-based oral tissue engineering.

Altered Distribution and Expression of Syndecan-1 and -4 as an Additional Hallmark in Psoriasis.

Syndecans act as independent co-receptors to exert biological activities and their altered function is associated with many pathophysiological conditions. Here, syndecan-1 and -4 were examined in lesional skin of patients with psoriasis. Immunohistochemical staining confirmed altered syndecan-1 distribution and revealed absence of syndecan-4 expression in the epidermis. Fibronectin (FN)—known to influence inflammation and keratinocyte hyperproliferation via α5β1 integrin in psoriasis—was also decreased. Syndecan-1 and -4 expression was analyzed in freshly isolated lesional psoriatic human keratinocytes (PHK) characterized based on their proliferation and differentiation properties. mRNA levels of syndecan-1 were similar between healthy and PHK, while syndecan-4 was significantly decreased. Cell growth and release of the pro-inflammatory Tumor Necrosis Factor-alpha (TNFα) were selectively and significantly induced in PHKs plated on FN. Results from co-culture of healthy keratinocytes and psoriatic fibroblasts led to the speculation that at least one factor released by fibroblasts down-regulate syndecan-1 expression in PHK plated on FN. To assay if biological treatments for psoriasis target keratinocyte proliferation, gelatin-based patches enriched with inteleukin (IL)-17α or TNFα blockers were prepared and tested using a full-thickness healthy epidermal model (Phenion®). Immunohistochemistry analysis showed that both blockers impacted the localisation of syndecan-1 within the refined epidermis. These results provide evidence that syndecans expression are modified in psoriasis, suggesting that they may represent markers of interest in this pathology.

Characterization of Keratinocytes, Fibroblasts and Melanocytes Isolated from Human Skin using Gene Markers

Cells isolated from skin have wide applications in studies of the pathogenesis of skin-related diseases and the construction of 3D skin equivalents. This study aimed to isolate keratinocytes, fibroblasts, and melanocytes from human foreskin and characterize the purity of the cell types. Keratinocytes, fibroblasts, and melanocytes from human foreskin were isolated by differential trypsinization and media selection. The purity of the cell types was characterized based on the expression of gene markers. The assessment of gene marker expression involved RNA extraction, primer design, quantitative polymerase chain reaction (qPCR) and immunocytochemical staining. Our results showed that in cocultures of keratinocytes and fibroblasts isolated from the dermis, fibroblasts could be separated from keratinocytes by quick trypsinization and culture in Dulbecco’s modified Eagle’s medium. The remaining keratinocytes are cultured in Epilife medium. Melanocytes in cocultures of melanocytes and keratinocytes isolated from the epidermis could be selected by changing Epilife medium to M254 medium. Gene marker results suggested that cytokeratin 14 (CK14) is a suitable marker for keratinocytes, elastin (ELN) is a suitable marker for fibroblasts, and tyrosinase (TYR) and tyrosinase-related proteins 1 and 2 (TYRP1 and TYRP2) are suitable markers for melanocytes. In conclusion, keratinocytes, fibroblasts, and melanocytes can be isolated from the same human foreskin sample by differential trypsinization and media selection techniques and characterized by suitable gene markers. This finding will aid in the isolation of pure skin cell types for various applications in regenerative medicine and toxicity studies.

P 60 A fully human sensory neuron – keratinocyte co-culture model for investigating the neuro-cutaneous unit

Introduction: Traditionally, peripheral sensory neurons were seen as the sole transducers of external stimuli. However, the contribution of non-neuronal cells is increasingly recognized. In the epidermis, keratinocytes act as sensors and transmitters of somatosensory and nociceptive input. They form intimate connections with afferent intraepidermal nerve fibers at the neuro-cutaneous unit. A conserved mechanism of nerve fiber ensheathment was described up to human skin. Via release of signaling molecules like ATP, keratinocytes can activate nerve endings, induce action potentials, and thereby tune peripheral input. Material & Methods: We obtained primary human fibroblasts and keratinocytes from diagnostic skin punch biopsies. Induced pluripotent stem cell-derived sensory neurons were generated from fibroblasts. After four-week maturation, neurons were transferred into a two-comparted well chamber system and primary keratinocytes seeded in the opposite well. We tracked the formation of neurite-keratinocyte contacts via live-imaging and subsequently investigated these interactions by confocal and super-resolution fluorescence microscopy. Results: Upon barrier removal, neurites form the neuronal compartment actively grew towards keratinocytes and formed contacts within few days. Via discriminative labeling of keratinocyte and neuronal membranes, we identified several types of interactions. Neurites tunneled through keratinocyte cytoplasm and formed bouton-like structures in contact with keratinocytes. Furthermore, connexin 43, a hemichannel and gap junction forming pore protein expressed in keratinocytes, accumulated at neurite-keratinocyte contact sites. Discussion: We successfully established a fully human co-culture model of sensory neurons and keratinocytes maintaining viability over several days. Our 2D model reduces the multilayered complexity of native human skin, yet conserves ensheathment of sensory neurites as a feature of the NCU and showed Cx43 plaques as a potential signaling route for NCU communication. This approach inherits a high potential for translational investigations, bypassing challenges of heterologous animal co-culture models and facilitating the integration of patient derived neurons and keratinocytes.