Reconstruction Of Epidermis Research Articles

PLA tissue-engineered scaffolds loaded with sustained-release active substance chitosan nanoparticles: Modeling BSA-bFGF as the active substance

The utilization of basic fibroblast growth factor (bFGF) in the development of tissue-engineered scaffolds is both challenging and imperative. In our pursuit of creating a scaffold that aligns with the natural healing process, we initially fabricated chitosan-bFGF nanoparticles (CS-bFGF NPs) through electrostatic spraying. Subsequently, polylactic acid (PLA) fiber was prepared using electrospinning technique, and the CS-bFGF NPs were uniformly embedded within the pores of porous PLA fibers. Scanning electron micrographs illustrate the smooth surface of the nanoparticles, showing a porous structure intricately attached to PLA fibers. Fourier-transform infrared spectroscopy (FTIR), energy-dispersive X-ray spectroscopy (EDS), and X-ray diffraction (XRD) analyses provided conclusive evidence that the CS-bFGF NPs were uniformly distributed throughout the porous PLA fibers, forming a robust physical bond through electrostatic adsorption. The resultant scaffolds exhibited commendable mechanical properties and hydrophilicity, facilitating a sustained-release for 72 h. Furthermore, the biocompatibility and degradation performance of the scaffolds were substantiated by monitoring conductivity and pH changes in pure water over different time intervals, complemented by scanning electron microscopy (SEM) observations. Cell experiments confirmed the cytocompatibility of the scaffolds. In animal studies, the group treated with 16 % NPs/Scaffold demonstrated the highest epidermal reconstruction rate. In summary, our developed materials present a promising candidate for serving as a tissue engineering scaffold, showcasing exceptional biocompatibility, sustained-release characteristics, and substantial potential for promoting epidermal regeneration.

34785 Epidermal reconstruction during ex vivo confocal microscopy for detection of superficial basal cell carcinoma with 3D-mosaicking and intensity projection

Background: Ex vivo confocal microscopy (EVCM) rapidly images fresh tissues to create digitally colorized H&E-images. It has a high sensitivity (79.8%-100%) and specificity (78%-100%) for the detection of residual basal cell carcinoma (BCC) during Mohs surgery. However, with current tissue mounting techniques, tissue flattening affects the visualization of the entire epidermis, impacting detection of superficial BCC (sBCC).

Hypoxia reveals a new function of Foxn1 in the keratinocyte antioxidant defense system.

Skin exposed to environmental threats, including injuries and oxidative stress, develops an efficient but not fully recognized system of repair and antioxidant protection. Here, using mass spectrometry analysis (LC-MS/MS), followed by in vitro and in vivo experiments, we provided evidence that Foxn1 in keratinocytes regulates elements of the electron transport chain and participates in the thioredoxin system (Txn2, Txnrd3, and Srxn1) induction, particularly in a hypoxic environment. We first showed that Foxn1 in keratinocytes upregulates glutathione thioredoxin reductase 3 (Txnrd3) protein expression, and high levels of Txnrd3 mRNA were detected in injured skin of Foxn1+/+ mice. We also showed that Foxn1 strongly downregulated the Ccn2 protein expression, participating in epidermal reconstruction after injury. An in vitro assay revealed that Foxn1 controls keratinocyte migration, stimulating it under normoxia and suppressing it under hypoxia. Keratinocytes overexpressing Foxn1 and exposed to hypoxia displayed a reduced ability to promote angiogenesis by downregulating Vegfa expression. In conclusion, this study showed a new mechanism in which Foxn1, along with hypoxia, participates in the activation of antioxidant defense and controls the functional properties of keratinocytes.

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.

Comparison of Lipid Foam Cream and Basic Cream on Epidermal Reconstruction in Mild Atopic Eczema

Introduction: Basic therapy is of central importance in the treatment of atopic eczema. Using electron microscopic images, the morphology of epidermal skin barrier and its lipids was investigated after application of a lipid foam cream and basic cream. Methods: Patients with two contralateral comparable atopic eczema (local SCORAD 1–10) on the forearms were tested. Eczema was treated with a lipid foam cream or basic cream twice daily for 28 days. At the beginning, after 14 days, and at the end of application, the local SCORAD, trans-epidermal water loss (TEWL), skin hydration, intercellular lipid length in the intercellular space of the stratum corneum (SC), and skin lipids were determined. Results: After application of the foam cream, the epidermal skin barrier could be completely restored and corresponded to healthy skin, while the epidermal skin barrier could not reach this state after care with the basic cream. The content of lipids in the SC increases significantly by 31% after basic cream treatment, whereas they are significantly increased by 85% after application of the lipid foam cream. The local SCORAD improved for both treatments to about the same extent, and no significant results could be shown for TEWL and skin hydration. Conclusion: In subjects with mild atopic eczema, the lipid foam cream leads to a measurable recovery of the skin barrier which is much more pronounced in comparison to the basic cream.

685 Locomotive ability of human keratinocyte stem cells is an intrinsic property for stem cell expansion and epidermal reconstruction

685 Locomotive ability of human keratinocyte stem cells is an intrinsic property for stem cell expansion and epidermal reconstruction

Modelling atopic dermatitis during the morphogenetic process involved in reconstruction of a human epidermis

Most crucial role of epidermis is to maintain efficient barrier between the organism and its environment. This barrier is however perturbed in inflammatory skin conditions like atopic dermatitis (AD), one common chronic disease. This review depicts characteristics of a model intending to reproduce epidermal features of AD in vitro. Firstly, methyl-β-cyclodextrin (MβCD) during reconstruction of epidermis was used to deplete cholesterol from plasma membrane because this condition reproduces characteristics of AD at transcriptomic level in monolayer cultures. Major changes are confirmed after same treatment inside reconstructed human epidermis (RHE). However, since early treatment do not reveal impairment to reconstruct a functional epidermal barrier and given the importance of the Th2 dysregulated immune response in AD, cholesterol-depleted RHE at day 11 of reconstruction were then incubated with three Th2-related cytokines (IL-4, IL-13 and IL-25) previously reported as playing important roles in the development of AD, as well as altering overall function of epidermal barrier. When combining both treatments, essential epidermal features of AD are observed. Indeed, RHE then exhibit spongiosis, disappearing granular layer, alteration of barrier function, as well as dysregulated expression levels for genes involved in AD pathogenesis. Moreover, while trying to identify individual roles for each component used to create AD-like alterations, incubation with IL-4 following cholesterol depletion from plasma membrane was found inducing most of the reported alterations. This model suggests potential for better investigations of epidermal AD features and may be considered for eventual in vitro screening of cosmetics or therapeutic compounds.

Reconstructing human skin equivalents on fibrin-based dermal matrix: Reproducible human skin equivalents with superior epidermal reconstruction

Reconstructing human skin equivalents on fibrin-based dermal matrix: Reproducible human skin equivalents with superior epidermal reconstruction

Study of proliferation and 3D epidermal reconstruction from foreskin, auricular and trunk keratinocytes in children

ObjectiveSevere burns in children are conventionally treated with split-thickness skin autografts or epidermal sheets. An alternative approach is to graft isolated keratinocytes. We evaluated foreskin and other anatomic sites as donor sources for autologous keratinocyte graft in children. We studied in vitro capacities of isolated keratinocytes to divide and reconstitute epidermal tissue. MethodsKeratinocytes were isolated from foreskin, auricular skin, chest and abdominal skin by enzymatic digestion. Living cell recovery, in vitro proliferation, epidermal reconstruction capacities and differentiation status were analyzed. ResultsIn vitro studies revealed the higher yield of living keratinocyte recovery from foreskin and higher potential in terms of proliferative capacity, regeneration and differentiation.Cultured keratinocytes from foreskin express lower amounts of differentiation markers than those isolated from trunk and ear. Histological analysis of reconstituted human epidermis derived from foreskin and inguinal keratinocytes showed a structured multilayered epithelium, whereas those obtained from ear pinna-derived keratinocytes were unstructured. ConclusionOur studies highlight the potential of foreskin tissue for autograft applications in boys. A suitable alternative donor site for autologous cell transplantation in female paediatric burn patients remains an open question in our department. We tested the hypothesis that in vitro studies and RHE reconstructive capacities of cells from different body sites can be helpful to select an optimal site for keratinocyte isolation before considering graft protocols for girls.

Hyaluronan Metabolism in Human Keratinocytes and Atopic Dermatitis Skin Is Driven by a Balance of Hyaluronan Synthases 1 and 3

Hyaluronan (HA) is a glycosaminoglycan synthesized directly into the extracellular matrix by three hyaluronan synthases (HAS1, HAS2, and HAS3). HA is abundantly synthesized by keratinocytes but its epidermal functions remain unclear. We used culture models to grow human keratinocytes as autocrine monolayers or as reconstructed human epidermis (RHE) to assess HA synthesis and HAS expression levels during the course of keratinocyte differentiation. In both the models, epidermal differentiation downregulates HAS3 mRNA expression while increasing HAS1 without significant changes in hyaluronidase expression. HA production correlates with HAS1 mRNA expression level during normal differentiation. To investigate the regulation of HAS gene expression during inflammatory conditions linked to perturbed differentiation, lesional and non-lesional skin biopsies of atopic dermatitis (AD) patients were analyzed. HAS3 mRNA expression level increases in AD lesions compared with healthy and non-lesional skin. Simultaneously, HAS1 expression decreases. Heparin-binding EGF-like growth factor (HB-EGF) is upregulated in AD epidermis. An AD-like HAS expression pattern is observed in RHE incubated with HB-EGF. These results indicate that HAS1 is the main enzyme responsible for HA production by normal keratinocytes and thus, must be considered as an actor of normal keratinocyte differentiation. In contrast, HAS3 can be induced by HB-EGF and seems mainly involved in AD epidermis.

Fucoidan Promotes the Reconstruction of Skin Equivalents

In this study we investigated the effects of fucoidan on the proliferation of fibroblasts and the reconstruction of a skin equivalent (SE). Fucoidan significantly stimulated the proliferation of CCD-25Sk human fibroblasts and Western blot analysis demonstrated that fucoidan markedly increased the expression of cyclin D1 and decreased the expression of p27. Fucoidan was used to reconstruct SE. Immunohistochemical staining showed that the addition of fucoidan to dermal equivalents increased expression of proliferating cell nuclear antigen (PCNA) and p63. In addition, expression of α6-integrin was significantly increased by fucoidan, whereas expression of β1-integrin, type 1 collagen, elastin, fibronectin did not markedly change. These results suggest that fucoidan has positive effects on epidermal reconstruction and will therefore be beneficial in the reconstruction of SE.

Foreskin-isolated keratinocytes provide successful extemporaneous autologous paediatric skin grafts

Severe burns in children are conventionally treated with split-thickness skin autografts or epidermal sheets. However, neither early complete healing nor quality of epithelialization is satisfactory. An alternative approach is to graft isolated keratinocytes. We evaluated paediatric foreskin and auricular skin as donor sources, autologous keratinocyte transplantation, and compared the graft efficiency to the in vitro capacities of isolated keratinocytes to divide and reconstitute epidermal tissue. Keratinocytes were isolated from surgical samples by enzymatic digestion. Living cell recovery, in vitro proliferation and epidermal reconstruction capacities were evaluated. Differentiation status was analysed, using qRT-PCR and immunolabelling. Eleven children were grafted with foreskin-derived (boys) or auricular (girls) keratinocyte suspensions dripped onto deep severe burns. The aesthetic and functional quality of epithelialization was monitored in a standardized way. Foreskin keratinocyte graft in male children provides for the re-epithelialization of partial deep severe burns and accelerates wound healing, thus allowing successful wound closure, and improves the quality of scars. In accordance, in vitro studies have revealed a high yield of living keratinocyte recovery from foreskin and their potential in terms of regeneration and differentiation. We report a successful method for grafting paediatric males presenting large severe burns through direct spreading of autologous foreskin keratinocytes. This alternative method is easy to implement, improves the quality of skin and minimizes associated donor site morbidity. In vitro studies have highlighted the potential of foreskin tissue for graft applications and could help in tissue selection with the prospect of grafting burns for girls.

Immortalized sebocytes can spontaneously differentiate into a sebaceous‐like phenotype when cultured as a 3D epithelium

Sebocytes originate from the same lineage as keratinocytes, and both cell types may have similarities in terms of growth and differentiation. We were interested in studying the behaviour of human sebocytes when cultured in conditions validated for epidermal reconstruction. For this purpose, we established a HPV16-E6/7-immortalized human sebocyte cell line (SEBO662) growing in keratinocyte defined media. Postconfluent SEBO662 cells in monolayers express the early sebocyte marker, cytokeratin 7 (K7), do not express Epithelia Membrane Antigen (EMA) and do not exhibit strong lipogenic activity. However, when placed at the air-liquid interface, SEBO662 multilayers spontaneously differentiate into a sebaceous-like structure as shown by the strong polarized expression of the late sebaceous marker EMA, the overexpression of some lipogenic markers and lipid production on the upper side of the epithelium. This work highlights the value of simple 3D models for exhibiting spontaneous differentiation and polarization.

Experimental study on repairing of nude mice skin defects with composite skin consisting of xenogeneic dermis and epidermal stem cells and hair follicle dermal papilla cells

Objective To investigate the influence of hair follicle dermal papilla cells (DPCs) on biological features of composite skin. Methods In the test group, xenogeneic acellular dermal matrix was employed as the frame, DPCs were seeded on the subcutaneous side, and epithelial stem cells onto the dermal papilla side of the dermal frame so as to construct a composite skin. In the control group, there was no DPC in the frame. The two kinds of composite skin were employed to cover skin defects on the back of the nude mice. Wound healing was observed 4 weeks after grafting and area was analyzed and contraction rate was calculated. The tissue samples in the grafted area were harvested for HE staining and the state of the composite skin was observed. The stress–strain curve of the sampled skin was measured, so as to calculate the maximal breaking power of the sample. The data were collected and statistically analyzed. Results HE staining indicated that the epithelial depth was increased (more than 10 layers of cells) in test group, with only 6–7 layers in control group. The skin contraction rate in test group on the 4th week after skin grafting (3.94 ± 0.013)% was much lower than that in control group (29.07 ± 0.018)% ( P < 0.05). It was indicated by biomechanical test that the stress–strain curve of the composite skin in the test group was closer to that of normal nude mice skin in comparison to that in control group. The maximal breaking force of the composite skin in test group was (1.835 ± 0.035) N (Newton), while that in control group was (1.075 ± 0.065) N ( P < 0.01). Conclusion Reconstruction of epidermis in composite skin was promoted by dermal DPCs seeded in the dermal matrix frame. As a result, there was less skin contraction in the composite skin with DPCs, so that the biological characteristics of the skin were improved.

Hyperbaric oxygen stimulates epidermal reconstruction in human skin equivalents

The crucial role of oxygen during the complex process of wound healing has been extensively described. In chronic or nonhealing wounds, much evidence has been reported indicating that a lack of oxygen is a major contributing factor. Although still controversial, the therapeutic application of hyperbaric oxygen (HBO) therapy can aid the healing of chronic wounds. However, how HBO affects reepithelization, involving processes such as keratinocyte proliferation and differentiation, remains unclear. We therefore used a three-dimensional human skin-equivalent (HSE) model to investigate the effects of daily 90-minute HBO treatments on the reconstruction of an epidermis. Epidermal markers of proliferation, differentiation, and basement membrane components associated with a developing epidermis, including p63, collagen type IV, and cytokeratins 6, 10, and 14, were evaluated. Morphometric analysis of hematoxylin and eosin-stained cross sections revealed that HBO treatments significantly accelerated cornification of the stratum corneum compared with controls. Protein expression as determined by immunohistochemical analysis confirmed the accelerated epidermal maturation. In addition, early keratinocyte migration was enhanced by HBO. Thus, HBO treatments stimulate epidermal reconstruction in an HSE. These results further support the importance of oxygen during the process of wound healing and the potential role of HBO therapy in cutaneous wound healing.

Modelling the human epidermis in vitro: tools for basic and applied research

Culture models of tissues and organs are valuable tools developed by basic research that help investigation of the body functions. Modelling is aimed at simplifying experimental procedures in order to better understand biological phenomena, and consequently, when sufficiently characterized, culture models can also be utilized with high potential in applied research. In skin biology and pathology, the development of cultures of keratinocytes as monolayers has allowed the elucidation of most functional and structural characteristics of the cell type. Beside the multiple great successes that have been obtained with this type of culture, this review draws attention on several neglected characteristics of monolayer cultures. The more sophisticated models created in order to reconstruct the fully differentiated epidermis have followed the monolayers. The epidermal reconstruction produces all typical layers found in vivo and thus makes the model much less simple, but only this kind of model allows the study of full differentiation in keratinocyte and production of the cornified barrier. In addition to its interest in basic research, the reconstructed epidermis is currently gaining a lot of interest for applied research, particularly as an alternative to laboratory animals in the chemical and cosmetic industry. Today several commercial providers propose reconstructed skin or epidermis, but in vitro assays on these materials are still under development. In order to be beneficial at long term, the validation of assays must be performed on a material whose availability will not be interrupted. We warn here providers and customers that the longevity of in vitro assays will be guaranteed only if these assays are done with well-described models, prepared according to published procedures, and must consider having a minimum of two independent simultaneous producers of similar material.

Preparation of Cultured Skin for Transplantation Using Insulin-like Growth Factor I in Conjunction with Insulin-like Growth Factor Binding Protein 5, Epidermal Growth Factor, and Vitronectin

Cultured skin for transplantation is routinely prepared by growing patient keratinocytes in the presence of semidefined sources of growth factors including serum and feeder cells, but these materials require substantial risk remediation and can contribute to transplant rejection. We have therefore investigated the potential of a novel combination of recombinant and purified growth factors to replace serum and feeder cells in cultures of human keratinocytes suitable for clinical application. Our technique was investigated with respect to culture establishment, serial propagation, colony-forming efficiency, immunocytochemistry, epidermal reconstruction, and suitability to support transplantation by aerosolization. We demonstrate that insulin-like growth factor (IGF)-I--used in conjunction with epidermal growth factor (EGF), insulin-like growth factor binding protein (IGFBP)-5 and vitronectin--supports growth in the absence of serum. Moreover, a threefold greater number of cells are generated within 7 days compared to those grown under current best practice conditions using serum (P<0.05). The resulting test cultures are suitable for epidermal reconstruction and support the option for delivery in the form of an aerosolized cell suspension. Serial propagation, with the view to producing confluent sheets for extensive injuries, was achieved but with less consistency and this result correlated with a significant decline in colony-forming efficiency compared to controls. IGF-I used in conjunction with IGFBP-5, EGF, and vitronectin provides a superior alternative to serum for the rapid expansion and transplantation of cultured keratinocytes within the first week of treatment. Nevertheless, further optimization is required with respect to elimination of feeder cells and serial expansion of cultures for treatment of extensive injuries.

Reconstruction of epidermis by grafting of keratinocytes cultured on polymer support--clinical study.

Extensive wound coverage still represents a challenge for contemporary medicine. We demonstrate the results of a clinical trial of the grafting of cultured keratinocytes directly on a polymer cultivation support in the treatment of skin defects in seriously burned patients and in patients with trophic ulcers. Wound closure was evaluated clinically. The morphology and phenotypic pattern of the reconstructed epidermis, including the basal lamina, as well as the presence of Langerhans cells, were evaluated immunocytochemically using a panel of monoclonal antibodies. All layers of the reconstructed epidermis were normally differentiated (cytokeratin immunocytochemistry). The basal lamina contained collagen type IV and laminin. The reconstructed epidermis was extensively colonized by Langerhans cells. The results of the described technology are encouraging, especially in patients after a burn injury. The described procedure is suitable for the treatment of skin defects in clinical practice.

Fibroblasts and ascorbate regulate epidermalization in reconstructed human epidermis

Skin equivalent model provides a new investigating system to study the role of extracellular matrix and dermal factors such as collagen, basement membrane components and fibroblasts (Fb) which contribute to cell-cell and cell-matrix interactions. Although basement membrane factors is known to play an important role in epidermal differentiation and epidermal-matrix adhesion, comparative effects of these extracellular matrix and dermal factors on the reconstruction of epidermis are little known. In this study, we investigated effects of type I collagen (Coll I), type IV collagen plus laminin (LAM) coated Coll I (Coll IV+LAM), and human Fb enriched Coll I (Coll I+Fb) on epidermal reconstruction. When human keratinocytes were cultured on three different gels containing Coll I, Coll IV+LAM and Coll I+Fb, basal keratinocytes were cuboidal and perpendicular to the dermo-epidermal junction only in the gel containing Coll I+Fb. Proliferation marker expression was prominent and differentiation marker expression was similar with those of normal skin in the gel containing Coll I+Fb than in the other gel models. Since ascorbate is suspected to exert an effect as a modulator of proliferation and differentiation in keratinocytes, we tested the effects of ascorbate on human epidermis reconstruction. When 25 μg/ml ascorbate was added, disordered arrangement of epidermis was disappeared and differentiation marker expression was similar with its expression in normal skin. These data indicate that human Fb and a modulator of proliferation and differentiation such as ascorbate are essential for epidermalization in reconstructed epidermis.

Ex Vivo Study of Skin Phototypes

We studied skin phototypes ex vivo to validate a model of epidermal reconstruction with melanocytes. We made autologous epidermal reconstructs with keratinocytes and melanocytes of healthy donors of skin phototypes I to VI. Keratinocytes and melanocytes were seeded on a dead de-epidermized dermis (Pruniéras type) at a 1:20 melanocyte/keratinocyte ratio. Reconstructed epidermis was grown for 15 d at the air-liquid interface with or without ultraviolet B irradiation. A macroscopic, chromometric. histologic, and ultrastructural evaluation was performed. Reconstructs reproduced the initial phototype with few modifications. The intensity of melanin transfer correlated with the in vivo situation and was stimulated after ultraviolet B irradiation in reconstructs of all categories of skin phototypes.