Dermal Equivalent Research Articles

Engineering Shape to Overcome Contraction: The Role of Polymer-Collagen Hybrids in Advanced Dermal Substitutes.

Collagen gels are the standard dermal equivalents par excellence, however the problem of rapid cell-mediated contraction remains unresolved. Therefore, the development of hybrid constructs (HCs) based on collagen and polymeric scaffolds is proposed to address the mechanical instability that usually limits the formation of new, functional tissue. Equally important, these synthetic structures should be temporary (degradable) while ensuring that cells are well-adapted to the new extracellular environment. In this study, we screened a library of scaffolds made of various polymers, including homopolymers of polycaprolactone (PCL) and poly D,L-lactide (PLA50), their blends (PCL/PLA50), and copolymers (poly(D,L-lactide-co-caprolactone), PCLLA50) to prepare HCs in a layer-by-layer fashion. The properties of polymers and copolymers along with their processability by electrospinning and 3D-printing were evaluated. Then, we assessed the HCs resistance toward cell-mediated contraction as well as the degradation of the polymeric scaffolds. Our results indicate that scaffolds with higher PLA50 content (e.g., PLA50 100%, PCL/PLA50 or PCLLA50, both at 50/50 caprolactone-to-D,L-lactide molar ratio) presented more drawbacks in terms of handleability and processing, while those with greater PCL presence showed structural steadiness and ease to use. All the scaffolds integrated well with the collagen gel to form the corresponding HCs. With few exceptions, the HCs demonstrated good resistance to cell-derived contraction over 3 weeks. Notably, HCs based on PCLLA50 90/10 (both versions, electrospun or 3D-printed) performed best, showing only a 5%-17% area reduction compared to the 93% observed in collagen-only gels. This copolymer displayed hydrolytic degradation depending on its shape, with up to 45% and 65% loss of molecular weight for the electrospun and 3D-printed forms, respectively, correlating with their progressive change in mechanical features. HCs containing PCLLA50 90/10 also exhibited a better fibroblast distribution, enhanced myofibroblastic differentiation, and a three-fold increase in cell proliferation (when the electrospun type was used) compared to collagen controls. These findings were instrumental in selecting a potential HC that might be used for future experiments in vivo.

Vδ1 T Cells Integrated in Full-Thickness Skin Equivalents: A Model for the Role of Human Skin–Resident γδT Cells

Vδ1 T cells are a subpopulation of γδT cells found in human dermis. In contrast to murine skin-resident γδT cells, much less is known regarding their role and function in skin health and disease. Here we report the successful integration of Vδ1 T cells into long-term fibroblast-derived matrix skin equivalents (SE). We isolated Vδ1 T cells from human blood, where they are rare, and established conditions for the integration and maintenance of the freshly isolated Vδ1 T cells in the SEs. Plated on top of the dermal equivalents (DEs), almost all Vδ1 T cells migrated into the dermal matrix where they exerted their influence on both the DE and the epithelium. Vδ1 T cells contributed to epidermal differentiation as indicated by histology, expression of epidermal differentiation markers and RNAseq expression profile. When complemented with the carcinoma-derived SCC13 cells instead of HaCaT, our data suggest a role for Vδ1 T cells in slowing growth of the tumor cells, as indicated by reduced stratification and changes in gene expression profiles. Together, we demonstrate the successful establishment of human Vδ1 T cell-competent skin and skin carcinoma equivalents (SE, SCE) and provide evidence for molecular and functional consequences of the Vδ1 T cells on their respective environment.

THE PROBLEM OF REGULATING FUNGICIDES ON THE SKIN SURFACE FOR OCCUPATIONAL SKIN DISEASE IN AGRICULTURAL WORKERS’ RISK PREDICTION

The aim. To establish the norms of fungicides on the skin surface and predicting the risk of occupational skin pathology in agricultural workers to develop measures to prevent diseases caused by dermal exposure to pesticides. Materials and methods. The natural experiment was performed in accordance with modern requirements (European Food Safety Authority (2022), and the risk assessment was carried out according to the Recommendations (Approved by the Ministry of Health of Ukraine No. 324 issued on 13.05.2009) with different methods of spraying techniques: rod, air blast fan spraying, pre-sowing, aviation, knapsack treatment. 17 series of natural experiments were performed, and the air of the working zone area (n =56) and dermal exposure (n =56) were analyzed for each worker with different pesticide application methods. Quantitative determination of pesticide content was carried out by gas-liquid and high-performance liquid chromatography methods. The results were statistically processed using a package of licensed statistical programs MedStat v.5.2 (Copyright © 2003-2019) and Microsoft® Excel® for Microsoft 365 MSO. Results. It was established that there is pesticide contamination, mainly of the gloves of tank filling operators when preparing working solutions. No contamination of the skin surface was detected; only pesticide residues were present on the surface of the workers’ overalls. A comparison of the exposure dose in case of percutaneous exposure during the performance of technological operations with different methods of processing, dermal equivalents of Acceptable Operator Exposure Level of pesticide to workers’ skin (DE AOEL) and the allowable dermal dose for professional contingents (ADderm) and the hazard coefficients by dermal exposure (HCderm) showed that that dermal exposure during individual technological operations with different processing methods had a reliable difference between processing methods, but there is no difference within one application method. Conclusions. During the comparison of the hazard coefficients for dermal exposure, calculated according to different models, no significant difference was found between the risks calculated according to DE AOEL and ADderm for individual technological operations in various treatment methods, except for the operator who performed knapsack treatment and the risk calculated according to ADderm is significantly lower compared to the DE AOEL risk.

3D Approaches to Culturing Bovine Skin: Explant Culture versus Organotypic Skin Model

Introduction: Digital dermatitis (DD) in cattle appears with high prevalence; nevertheless, the knowledge on its pathogenesis is still limited. In this context, in vitro skin models represent a valuable tool to facilitate the study of DD. Methods: Two in vitro skin models were established using bovine distal limb skin: a skin explant model and an organotypic skin model. For the skin explant model, skin samples were cultured with an air-liquid interface for up to 7 days. Besides routine histopathological examination, readout parameters were Ki-67 and cleaved Caspase-3 stainings. For the organotypic model, primary keratinocytes were layered on top of a dermal equivalent containing mainly mitotically inactive fibroblasts and maintained for up to 21 days. At regular intervals (days 7, 14, and 21), cultured skin samples were taken for (immuno)histological analysis. Results: Both cultures could be maintained for the entire duration of the intended culture period. In the histopathological assessment, explant skin cultures showed ballooning degeneration of keratinocytes and segmental necrosis starting at day 5 of culturing. Initially, basal keratinocytes in the organotypic model differentiated as demonstrated by positive Keratin 14, Desmoglein-1, Loricrin, and Involucrin immunofluorescent stainings. Ki-67 was observed occasionally and suprabasally still after 21 days of culture. Conclusion: Both in vitro models proved dependable and constitute a viable option for replacing experiments on live animals, each with its own benefits. Whereas skin explants include all cell types available in vivo and can therefore reflect realistic cell-cell interactions and signaling pathways, the organotypic model offers a higher standardization and reproducibility. Depending on the focus of future studies, both models can be used for specific experimental purposes of bovine dermatological research in general or specialized questions concerning (infectious) claw diseases as, e.g., DD.

Molecular Insights Into the Effects of PLLA-SCA on Gene Expression and Collagen Synthesis in Human 3D Skin Models Containing Macrophages.

Injectable poly-L-lactic acid (PLLA-SCA) is used for the correction of shallow to deep nasolabial fold contour deficiencies, cheek wrinkles, and other facial wrinkles. In contrast to hyaluronan (HA) fillers, PLLA-SCA has a biostimulatory effect by activating resident fibroblasts to produce collagen, but the mechanisms are not known in detail at the molecular level. Therefore, our aim was to investigate the molecular effects of PLLA-SCA in a comprehensive in vitro study. Since PLLA-SCA-dependent collagen production in fibroblasts depends on the interaction with macrophages, we generated novel macrophage-containing 3D skin models. According to the clinical application, PLLA-SCA was injected once into the dermal equivalent of the 3D skin model. Histological analysis showed a significant increase in epidermal thickness in these models after 5 and 14 days. Gene expression profiling revealed an upregulation of integrins and laminins (e.g., LAMA3, ITGA6), which are essential components of the dermal-epidermal junction. In addition, we found an upregulation of cytokines and chemokines (TGFB2, CXCL6, IL1B) at day 14 after PLLA-SCA injection. Interestingly, immunohistochemical analyses exhibited a significantly stimulated collagen I production in our models. These effects might be attributed, at least in part, to the upregulation of IL1B and subsequently CXCL6, which stimulates collagen I synthesis in human dermal fibroblasts as we could demonstrate. Taken together, our data provide for the first time molecular insights into the biostimulatory effects of PLLA-SCA on collagen I production in novel human 3D skin models comprising macrophages. J Drugs Dermatol. 2024;23(4):7791.    doi:10.36849/JDD.7791.

The Anti-Diabetic Pinitol Improves Damaged Fibroblasts.

Pinitol (3-O-Methyl-D-chiro-inositol) has been reported to possess insulin-like effects and is known as one of the anti-diabetic agents to improve muscle, liver, and endothelial cells. However, the beneficial effects of pinitol on the skin are not well known. Here, we investigated whether pinitol had effects on human dermal fibroblasts (HDFs), and human dermal equivalents (HDEs) irradiated with ultraviolet A (UVA), which causes various damages including photodamage in the skin. We observed that pinitol enhanced wound healing in UVA-damaged HDFs. We also found that pinitol significantly antagonized the UVA-induced up-regulation of matrix metalloproteinase 1 (MMP1), and the UVA-induced down-regulation of collagen type I and tissue inhibitor of metalloproteinases 1 (TIMP1) in HDEs. Electron microscopy analysis also revealed that pinitol remarkably increased the number of collagen fibrils with regular banding patterns in the dermis of UVA-irradiated human skin equivalents. Pinitol significantly reversed the UVA-induced phosphorylation levels of ERK and JNK but not p38, suggesting that this regulation may be the mechanism underlying the pinitol-mediated effects on UVA-irradiated HDEs. We also observed that pinitol specifically increased Smad3 phosphorylation, which is representative of the TGF-β signaling pathway for collagen synthesis. These data suggest that pinitol exerts several beneficial effects on UVA-induced damaged skin and can be used as a therapeutic agent to improve skin-related diseases.

Development of chitosan-based cerium and titanium oxide loaded polycaprolactone for cutaneous wound healing and antibacterial applications

Cerium dioxide (CeO2) based nanomaterials have emerged as promising dermal equivalents, promoting fibroblast infiltration and tissues regeneration. To enhance the antibacterial and wound healing activity, herein chitosan (CS)-CeO2 combined nano titanium dioxide (TiO2) complex loaded polycaprolactone (PCL) nanohybrid (CS-CeO2/TiO2/PCL) scaffolds were prepared through casting method. The nanohybrid scaffolds' physiochemical, morphological, mechanical, and biological properties were evaluated using advanced analytical techniques. Fourier transform infrared spectroscopy spectrum evidently depicted the various intermolecular interactions on the nanohybrid scaffolds. The developed scaffold exhibited the high swelling behavior and good degradability and permeability which is beneficial for absorbing wound transudation to fasten the healing efficacy. Moreover, CS-CeO2/TiO2/PCL scaffolds owned the better antibacterial activity against bacterial strains E. coli and S. aureus. Also, MTT assay on fibroblast (NIH 3T3) cells and immortalized human keratinocytes (HaCaT) cells indicated improved cell viability and proliferation. In vivo results revealed that the fabricated scaffold full aid to complete wound closure after 14 days which showed CS-CeO2/TiO2/PCL as the significant wound dressing material with potential antibacterial immunity.

Mesenchymal stem cells express epidermal markers in an in vitro reconstructed human skin model.

Introduction: In skin traumas, such as burns, epidermal homeostasis is affected, often requiring clinical approaches. Different therapeutic strategies can be used including transplantation, besides the use of synthetic or natural materials with allogeneic cells. In this context, tissue engineering is an essential tool for skin regeneration, and using mesenchymal stem cells (MSC) from the umbilical cord appears to be a promising strategy in regenerative medicine due to its renewal and differentiation potential and hypo immunogenicity. We evaluated the transdifferentiation of MSC from umbilical cord into keratinocytes in three-dimensional (3D) in vitro skin models, using dermal equivalents composed by type I collagen with dermal fibroblasts and a commercial porcine skin decellularized matrix, both cultured at air-liquid interface (ALI). Methods: The expression of epidermal proteins cytokeratins (CK) 5, 14 and 10, involucrin and filaggrin was investigated by real-time PCR and immunofluorescence, in addition to the activity of epidermal kallikreins (KLK) on the hydrolysis of fluorogenic substrates. Results and discussion: The cultivation of MSCs with differentiation medium on these dermal supports resulted in organotypic cultures characterized by the expression of the epidermal markers CK5, CK14, CK10 and involucrin, mainly on the 7th day of culture, and filaggrin at 10th day in ALI. Also, there was a 3-fold increase in the KLK activity in the epidermal equivalents composed by MSC induced to differentiate into keratinocytes compared to the control (MSC cultivated in the proliferation medium). Specifically, the use of collagen and fibroblasts resulted in a more organized MSC-based organotypic culture in comparison to the decellularized matrix. Despite the non-typical epithelium structure formed by MSC onto dermal equivalents, the expression of important epidermal markers in addition to the paracrine effects of these cells in skin may indicate its potential use to produce skin-based substitutes.

Выживаемость клеток человека в биомедицинских клеточных продуктах при хранении при комнатной температуре

Tissue-engineered constructs (TECs), the dermal equivalent (DE) and the skin equivalent (SE), are allogenic equivalents of the skin and derm used to treat critical skin loss. Selection of storage conditions that contribute to longer shelf life, thereby expanding the possibilities of logistics and use, is one of the major issues related to the TECs development. The study was aimed to determine the shelf life of the DE and SE TECs stored in normal saline at room temperature by assessing morphology and viability of the cells on their surface, along with the levels of endothelial growth factor (VEGF) secreted by these cells. Using the MTT assay and staining with vital dye, we discovered the following: when TECs of both types were stored in normal saline, the cells viability and metabolic activity decreased by more than 50% by days 3–4 of storage. Furthermore, these decreased faster in DEs than in SEs. Morphology of the cells isolated from DEs and SEs after the 3-day storage remained unchanged. Mesenchymal stem cells on the surface of TECs kept producing VEGF after TECs culture medium was changed for saline solution (confirmed by immunofluorescence assay), which could indicate that the cells retained essential secretory activity.

618 Acellular Dermal Matrices in Cutaneous Wound Healing: a Longitudinal Comparative Study

Acellular dermal equivalents provide a temporary three-dimensional scaffold for cell migration and proliferation, therefore promoting the wound healing process. Currently, two of the most widely used substitutes are represented by a native non-cross-linked collagen matrix supplemented by an elastin hydrolysate (MatriDerm®) and a bilayer matrix consisting of a porous dermal replacement layer composed of collagen and glycosaminoglycan and a temporary outer polysiloxane layer (Integra®). However, only few studies comparing the two have been published so far.

Effect of Aronia Extract on Collagen Synthesis in Human Skin Cell and Dermal Equivalent.

The regulation of collagen synthesis, which occurs in fibroblasts in the dermal layer, is a key process in dermis regeneration and skin reconstruction. Herein, we investigated whether Aronia melanocarpa extract affects the human skin condition. We focused on type I collagen synthesis using two different types of model systems: a monolayer of cells and a bioprinted 3D dermal equivalent. The Aronia extract showed no cytotoxicity and increased cell proliferation in neonatal human dermal fibroblasts. Treatment with Aronia extract increased the transcription of COL1A1 mRNA in direct proportion to the extract concentration without causing a decrease in COL1A1 mRNA degradation. Additionally, the Aronia extract inhibited the expression of MMP1 and MMP3, and an increase in type I collagen was observed along with a decrease in MMP1 protein. We also fabricated dermal equivalents from type I collagen (the major component of the dermis) and dermal fibroblasts by bioprinting. In the 3D dermis model, the compressive modulus directly affected by collagen synthesis increased in direct proportion to the Aronia extract concentration, and expression levels of MMP1 and MMP3 decreased in exactly inverse proportion to its concentration. The findings that the Aronia extract increases synthesis of type I collagen and decreases MMP1 and MMP3 expression suggest that this extract may be useful for the treatment of damaged or aged skin.

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.

The brown alga Laminaria ochroleuca: Charming shell pickers of the strait of Messina, fanciful sirens, and treating war wounds and scars in prostitutes

Sir, In one of his precious masterpieces, Portuguese novelist José Saramago narrates in detail the lives of the rice weeders who used to work in this land and, after a day of work, soak their bare feet in a bath with hot water, salt, mallow, and chamomile in order to restore their ankles, swollen and inflated for the alga native to these particular paddy fields, Laminaria ochroleuca (Bachelot de la Pylaie), extremely rich in fucose and alginates, provoking an accelerated and paroxistic synthesis of elastin, fibronectin, and collagen. This brown alga may be retrieved from the waters of France, Spain, Scotland, and the Shetland Islands, yet even in the Azores Islands and Morocco (Alboran Sea) [1–4]. The authors of this paper selected a special Laminaria from the Strait of Messina (Nereocystis luetkeana), a heterokont that is able to grow half a meter per day reaching the surprising and amazing height of 260 ft.). The legend has it that shell collectors, chiefly young girls, day after day, saw their legs together to create the tail of a siren, a cartilaginous appendage rich in collagen and elastin becoming scaly and squamous owing to the seawater. The authors created a dermocosmetic emulsion with high concentrations of acetolyte from Laminaria ochroleuca (12%) and other nourishing active ingredients (obviously, the chief components were petrolatum and lanolin, as per the pharmaceutical art of preparing the cold cream of Galenus). Hence, this golden alga is able to support the skin by providing antioxidants to fight free radicals, supporting the health of collagen and elastin, which helps the skin retain elasticity and radiance, reducing fine lines and wrinkles and helping to moisturize the skin while boosting its barrier. Some researchers [5] claimed that this alga could be useful to build a skin equivalent, as fucoidan significantly stimulates the proliferation of CCD-25Sk human fibroblasts. Also, western blot analysis demonstrates that fucoidan markedly increases the expression of cyclin D1 and decreases the expression of p27. Fucoidan was practically used to reconstruct SE. Immunohistochemical staining reveals that the addition of fucoidan to dermal equivalents increases the expression of proliferating cell nuclear antigen (PCNA) and p63. In addition, the expression of a6-integrin is significantly increased by fucoidan, whereas the expression of b1-integrin, type 1 collagen, elastin, and fibronectin does not markedly change. In the light of the above, the authors may assert that fucoidan shows positive effects on epidermal reconstruction and is, therefore, beneficial in the reconstruction of all types of skin damage. The authors recruited a panel of eight people: 2 veterans from Iraq and Afghanistan (a-b) presenting old war wounds with no chance to cure these after years of treatments and a myriad of surgical operations; 2 elders (one male and one female, 87 and 89 yrs. old, respectively, c-d) presenting severe bedsores; 2 older ladies (e-f) who, in the past, underwent unsuccessful aesthetical operations, presenting puckers, seams, furrows and deep wrinkles; 2 girls (two ex-prostitutes, one black and one white, g-h) who were scarred with a razor by their enemies.

Melanoma spheroid-containing artificial dermis as an alternative approach to in vivo models

Melanoma spheroid-loaded 3D skin models allow for the study of crucial tumor characteristics and factors at a superior level because the neoplastic cells are integrated into essential human skin components, permitting tumor–skin model communication. Herein, we designed a melanoma-containing artificial dermis by inserting multicellular tumor spheroids from the metastatic phase of WM 1617 melanoma cells into an artificial dermis. We cultured multicellular melanoma spheroids by hanging drop method (250 cells per drop) with a size of 420 μm in diameter after incubation for 14 days. These spheroids were integrated into the dermal equivalents that had been previously preparedwith a type-I collagen matrix and healthy fibroblasts. The melanoma spheroid cells invaded and proliferated in the artificial dermis. Spheroids treated with a 1.0 μmol/L aluminum chloride phthalocyanine nanoemulsion in the absence of light showed high cell viability. In contrast, under irradiation with visible red light (660 nm) at 25 J/cm2, melanoma cells were killed and the healthy tissue was preserved, indicating that photodynamic therapy is effective in such a model. Therefore, the 3D skin melanoma model has potential to promote research in full-thickness skin model targeting optimized preclinical assays.

Organotypic Epithelial Raft Cultures as a Three-Dimensional In Vitro Model of Merkel Cell Carcinoma.

Simple SummaryMerkel cell carcinoma (MCC) is an extremely aggressive type of skin cancer. Nevertheless, the development of new therapeutic approaches to treat this malignancy is hampered by the absence of in vitro culture models. Furthermore, prospective clinical trials are scarce due to the rarity of the disease. Thus, the aim of the present study was to generate organotypic epithelial raft cultures (OERCs) of MCC by growing primary human keratinocytes and MCC cell lines on artificial dermal equivalents. Histological and immunohistochemical analyses confirmed the proliferation of MCC cell lines. In addition, gene expression analysis showed genes that were differently expressed in the tumor cells and the keratinocytes. In brief, we developed a three-dimensional cell culture model of MCC that can potentially be used for evaluating the efficacy and selectivity of new drug candidates against this disease.Merkel cell carcinoma (MCC) is a rare type of skin cancer for which an in vitro model is still lacking. MCC tumorigenesis is associated either with the integration of Merkel cell polyomavirus into the host genome, or with the accumulation of somatic mutations upon chronic exposure to UV light. Transgenic animals expressing the viral oncoproteins, which are constitutively expressed in virus-related MCC, do not fully recapitulate MCC. Although cell-line-derived xenografts have been established for the two subtypes of MCC, they still present certain limitations. Here, we generated organotypic epithelial raft cultures (OERCs) of MCC by using primary human keratinocytes and both virus-positive and virus-negative MCC cell lines. The primary human keratinocytes and the tumor cells were grown on top of a dermal equivalent. Histological and immunohistochemical examination of the rafts confirmed the growth of MCC cells. Furthermore, gene expression analysis revealed differences in the expression profiles of the distinct tumor cells and the keratinocytes at the transcriptional level. In summary, considering the limited availability of patient samples, OERCs of MCC may constitute a suitable model for evaluating the efficacy and selectivity of new drug candidates against MCC; moreover, they are a potential tool to study the oncogenic mechanisms of this malignancy.

The use of chitosan as a skin-regeneration agent in burns injuries: A review

Abstract Chitosan is an amino-polysaccharide, traditionally obtained by the partial deacetylation of chitin from exoskeletons of crustaceans. Properties such as biocompatibility, hemostasis, and the ability to absorb physiological fluids are attributed to this biopolymer. Chitosan’s biological properties are regulated by its origin, polymerization degree, and molecular weight. In addition, it possesses antibacterial and antifungal activities. It also has been used to prepare films, hydrogels, coatings, nanofibers, and absorbent sponges, all utilized for the healing of skin wounds. In in vivo studies with second-degree burns, healing has been achieved in at least 80% of the cases between the ninth and twelfth day of treatment with chitosan coatings. The crucial steps in the treatment of severe burns are the early excision of damaged tissue and adequate coverage to minimize the risk of infection. So far, partial-thickness autografting is considered the gold standard for the treatment of full-thickness burns. However, the limitations of donor sites have led to the development of skin substitutes. Therefore, the need for an appropriate dermal equivalent that functions as a regeneration template for the growth and deposition of new skin tissue has been recognized. This review describes the properties of chitosan that validate its potential in the treatment of skin burns.

Benefit of coupling heparin to crosslinked collagen I/III scaffolds for human dermal fibroblast subpopulations' tissue growth.

Currently, there is a lack of models representing the skin dermal heterogeneity for relevant research and skin engineering applications. This is the first study reporting production of dermal equivalents reproducing features of papillary and reticular dermal compartments. Inspired from our current knowledge on the architecture and composition differences between the papillary and reticular dermis, we evaluated different collagen-based porous materials to serve as scaffolds for the three-dimensional expansion of freshly isolated papillary and/or reticular fibroblasts. The scaffolds, composed of either collagen I or collagen I and III mixtures, were prepared by lyophilization. Pore size and hydrolytic stability were controlled by crosslinking with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) or EDC/NHS with covalently bound heparin. The evaluation of the resultant "papillary" and "reticular" dermal equivalents was based on the analysis of characteristic features of each dermal compartment, such as cell density and deposition of newly synthetized extracellular matrix components in histological sections. Crosslinking supported cell growth during dermal tissue formation independent on the fibroblast subpopulation. The presence of collagen III seemed to have some positive but non-specific effect only on the maintenance of the mechanical strength of the scaffolds during dermal formation. Histological analyses demonstrated a significant and specific effect of heparin on generating dermal equivalents reproducing the respective higher papillary than reticular cell densities and supporting distinct extracellular matrix components deposition (three to five times more carbohydrate material deposited by papillary fibroblasts in all scaffolds containing heparin, while higher collagen production was observed only in the presence of heparin).

Tissue dermal equivalent – а cellular product based on human dermal keratinocytes and fibroblasts: the properties of equivalent components and perspectives of practical application

The nature and ways of isolation and cultivation in vitro of keratinocytes and fibroblasts, the main cellular components of skin to prepare a new biomedical product, tissue dermal equivalent were considered. The main attention was payed to optimization of upbuilding dermal cell biomass including selection of medium compositions and conditions of cultivation. The information was given on main parameters of cell cultures as proliferation activity, viability and phenotype of the cells. Genotoxicity of fibroblasts and biocompatibility of the cells with organic matrixes to find the optimal carrier for cellular elements of tissue dermal equivalent were studied. The composition, the process of preparation of tissue dermal equivalent and perspectives of its practical application were discussed.

Fibroblasts Influence Metastatic Melanoma Cell Sensitivity to Combined BRAF and MEK Inhibition.

Simple SummaryPreclinical 3D in vitro coculture models are known to be more complex systems than monolayer cell culture and mimic the physiological environment more closely. Three-dimensional dermal equivalents provide a relevant environment for cutaneous metastatic melanoma cells and are capable of modulating a cancer cell’s response to drugs. We showed that a combined targeted therapy (vemurafenib and cobimetinib) efficiently inhibits cell proliferation and induces apoptosis, especially in the 3D coculture model. A cancer-associated fibroblast population isolated from a cutaneous melanoma was also sensitive to the treatment but with no detectable induction of apoptosis. To better understand the complex crosstalk between melanoma cells and their microenvironment, we compared the influence of conditioned media obtained from healthy or cancer-associated fibroblasts on the response of metastatic melanomas to the drugs. Our data indicate that normal fibroblast supernatants potentialize the therapy’s efficiency, whereas cancer-associated fibroblast secretomes favor melanoma cell survival.The sensitivity of melanoma cells to targeted therapy compounds depends on the tumor microenvironment. Three-dimensional (3D) in vitro coculture systems better reflect the native structural architecture of tissues and are ideal for investigating cellular interactions modulating cell sensitivity to drugs. Metastatic melanoma (MM) cells (SK-MEL-28 BRAF V600E mutant and SK-MEL-2 BRAF wt) were cultured as a monolayer (2D) or cocultured on 3D dermal equivalents (with fibroblasts) and treated with a BRAFi (vemurafenib) combined with a MEK inhibitor (MEKi, cobimetinib). The drug combination efficiently inhibited 2D and 3D MM cell proliferation and survival regardless of their BRAF status. Two-dimensional and three-dimensional cancer-associated fibroblasts (CAFs), isolated from a cutaneous MM biopsy, were also sensitive to the targeted therapy. Conditioned media obtained from healthy dermal fibroblasts or CAFs modulated the MM cell’s response differently to the treatment: while supernatants from healthy fibroblasts potentialized the efficiency of drugs on MM, those from CAFs tended to increase cell survival. Our data indicate that the secretory profiles of fibroblasts influence MM sensitivity to the combined vemurafenib and cobimetinib treatment and highlight the need for 3D in vitro cocultures representing the complex crosstalk between melanoma and CAFs during preclinical studies of drugs.

Phototoxic versus photoprotective effects of tattoo pigments in reconstructed human skin models: In vitro phototoxicity testing of tattoo pigments: 3D versus 2D

The increasing number of tattooed persons urges the development of reliable test systems to assess tattoo associated risks. The alarming prevalence of 60 % phototoxic reactions in tattoos ask for a more comprehensive investigation of phototoxic reactions in tattooed skin.Here, we aimed to compare the cellular responses of human skin cells to ultraviolet (UV)A and UVB irradiation in doses of short to intermitted sun exposure (3–48 J/cm² and 0.05–5 J/cm², respectively) in the presence of tattoo pigments. Therefore, we used fibroblast monolayer culture (2D), our recently developed three dimensional full-thickness skin model with dermal-located tattoo pigments (TatSFT) and its dermal equivalents (TatSDE) that lack keratinocytes. We tested the most frequently used tattoo pigments carbon black, titanium dioxide (TiO2) anatase and rutile as well as Pigment Orange (P.O.)13 in ranges from 0.067 to 2.7 ng/cell in 2D. For TatSDE and TatSFT, concentrations were 1.3 ng/cell for TiO2, 0.67 ng/cell for P.O.13 and 0.067 ng/cell for carbon black. We assessed cell viability and cytokine release in all systems, and cyclobutane pyrimidine dimer (CPD) formation in TatSFT.Phototoxicity of tattoo pigments was exclusively observed in 2D, where especially TiO2 anatase induced phototoxic effects in all concentrations (0.067–2.7 ng/cell). In contrast, fibroblasts were protected from UV irradiation in TatSDE by TiO2 and carbon black. Neither toxic nor protective effects were recorded in TatSFT. P.O.13 showed altered cytokine secretion in 2D (0.067–1.3 ng/cell) and TatSDE, despite the absence of significant effects on viability in all systems. All pigments reduced the number of CPDs in TatSFT compared to the pigment-free controls.In conclusion, our study shows that within a 3D arrangement, intradermal tattoo pigments may act photoprotective despite intrinsic phototoxic properties in 2D. Thus, dermal 3D equivalents should be considered to evaluate acute tattoo pigment toxicology.