De-epidermized Dermis Research Articles

Cutaneous Cell Therapy Manufacturing Timeframe Rationalization: Allogeneic Off-the-Freezer Fibroblasts for Dermo-Epidermal Combined Preparations (DE-FE002-SK2) in Burn Care.

Autologous cell therapy manufacturing timeframes constitute bottlenecks in clinical management pathways of severe burn patients. While effective temporary wound coverings exist for high-TBSA burns, any means to shorten the time-to-treatment with cytotherapeutic skin grafts could provide substantial therapeutic benefits. This study aimed to establish proofs-of-concept for a novel combinational cytotherapeutic construct (autologous/allogeneic DE-FE002-SK2 full dermo-epidermal graft) designed for significant cutaneous cell therapy manufacturing timeframe rationalization. Process development was based on several decades (four for autologous protocols, three for allogeneic protocols) of in-house clinical experience in cutaneous cytotherapies. Clinical grade dermal progenitor fibroblasts (standardized FE002-SK2 cell source) were used as off-the-freezer substrates in novel autologous/allogeneic dermo-epidermal bilayer sheets. Under vitamin C stimulation, FE002-SK2 primary progenitor fibroblasts rapidly produced robust allogeneic dermal templates, allowing patient keratinocyte attachment in co-culture. Notably, FE002-SK2 primary progenitor fibroblasts significantly outperformed patient fibroblasts for collagen deposition. An ex vivo de-epidermalized dermis model was used to demonstrate the efficient DE-FE002-SK2 construct bio-adhesion properties. Importantly, the presented DE-FE002-SK2 manufacturing process decreased clinical lot production timeframes from 6-8 weeks (standard autologous combined cytotherapies) to 2-3 weeks. Overall, these findings bear the potential to significantly optimize burn patient clinical pathways (for rapid wound closure and enhanced tissue healing quality) by combining extensively clinically proven cutaneous cell-based technologies.

Full Skin Equivalent Models for Simulation of Burn Wound Healing, Exploring Skin Regeneration and Cytokine Response.

Healing of burn injury is a complex process that often leads to the development of functional and aesthetic complications. To study skin regeneration in more detail, organotypic skin models, such as full skin equivalents (FSEs) generated from dermal matrices, can be used. Here, FSEs were generated using de-epidermalized dermis (DED) and collagen matrices MatriDerm® and Mucomaix®. Our aim was to validate the MatriDerm- and Mucomaix-based FSEs for the use as in vitro models of wound healing. Therefore, we first characterized the FSEs in terms of skin development and cell proliferation. Proper dermal and epidermal morphogenesis was established in all FSEs and was comparable to ex vivo human skin models. Extension of culture time improved the organization of the epidermal layers and the basement membrane in MatriDerm-based FSE but resulted in rapid degradation of the Mucomaix-based FSE. After applying a standardized burn injury to the models, re-epithelization occurred in the DED- and MatriDerm-based FSEs at 2 weeks after injury, similar to ex vivo human skin. High levels of pro-inflammatory cytokines were present in the culture media of all models, but no significant differences were observed between models. We anticipate that these animal-free in vitro models can facilitate research on skin regeneration and can be used to test therapeutic interventions in a preclinical setting to improve wound healing.

Plasma-polymerized pericyte patches improve healing of murine wounds through increased angiogenesis and reduced inflammation.

Pericytes have the potential to be developed as a cell therapy for the treatment of wounds; however, the efficacy of any cell therapy relies on the successful delivery of intact and functioning cells. Here, the effect of delivering pericytes on wound repair was assessed alongside the development of a surface-functionalized pericyte patch. Plasma polymerization (PP) was used to functionalize the surface of silicone patches with heptylamine (HA) or acrylic acid (AA) monomers. Human pericytes were subsequently delivered to murine excisional wounds by intradermal injection or using the pericyte-laden patches and the comparative effects on wound healing, inflammation and revascularization determined. The AA surface provided the superior transfer of the cells to de-epidermized dermis. Excisional murine wounds treated either with pericytes injected directly into the wound or with the pericyte-laden AA patches showed improved healing with decreased neutrophil infiltration and reduced numbers of macrophages in the wounds. Pericyte delivery also enhanced angiogenesis through a mechanism independent of VEGF signalling. Pericytes, when delivered to wounds, improved healing responses by dampening inflammation and promoting angiogenesis. Delivery of pericytes using PP-AA-functionalized patches was equally as effective as direct injection of pericytes into wounds. Pericyte-functionalized dressings may therefore be a clinically relevant approach for the treatment of wounds.

Suitability of biological acellular dermal matrices as a skin replacement

Tissue defects are associated with loss of epidermal and dermal components of the skin. For full-thickness tissue defects, dermal equivalents are useful to enable rapid wound closure. Split-thickness skin grafts are associated with loss of tissue elasticity resulting in scar contractures that can impair joint mobility. Synthetic collagen matrices and allogeneic acellular dermal matrices (ADM) are commercially available and could serve as skin tissue replacement. The aim of this study was to investigate whether ADM of different dermal layers or bioartificial matrices can serve as cutaneous replacement. For this purpose, cellular migration, differentiation and the inflammatory reaction were studied in an established ex vivo skin organ model. Human split-thickness skin grafts were transplanted onto ADM (Epiflex, DIZG, Berlin, Germany), de-epidermized dermis (DED) or an artificial collagen-elastin matrix (Matriderm, Dr. Suwelack, Billerbeck, Germany). Epithelial migration was studied using an established skin culture model at the air-liquid interface. In addition, the effect of tissue from different dermal compartments, e. g. papillar and reticular dermis, on epithelial migration was compared. Epithelial resurfacing and differentiation of matrices as well as the inflammatory reaction were studied using histological, immunohistochemical and biochemical analyses. Significantly more epithelial outgrowth area was found on DED (2.54 mm ± 0.43 mm, mean ± SEM) compared to papillary ADM (1.32 mm ± 0.44 mm, p = 0.039), to reticular ADM (no horizontal growth, p < 0.0001) and collagen-elastin matrix (0.78 mm ± 0.11 mm, p = 0.0056) measured by fluorescence microscopy over 10 days presumably due to the presence of pro-migratory basement membrane residues on DED. Reepithelialization was significantly higher (p < 0.002) on papillary dermis compared to ADM of reticular origin. In contrast to the biological matrices, a complete horizontal penetration was found in the macroporous collagen-elastin matrix. Pro-inflammatory mediators varied depending on the human skin donor and matrix. In summary, the biochemical structure of the matrix' surface and its origin influenced the epithelial behaviour with regard to migration, differentiation and inflammatory response.

Identification of Malassezia furfur Secreted Aspartyl Protease 1 (MfSAP1) and Its Role in Extracellular Matrix Degradation.

Malassezia is the most abundant eukaryotic microbial genus on human skin. Similar to many human-residing fungi, Malassezia has high metabolic potential and secretes a plethora of hydrolytic enzymes that can potentially modify and structure the external skin environment. Here we show that the dominant secreted Malassezia protease isolated from cultured Malassezia furfur is an aspartyl protease that is secreted and active at all phases of culture growth. We observed that this protease, herein named as MfSAP1 (M. furfur secreted aspartyl protease 1) has a broader substrate cleavage profile and higher catalytic efficiency than the previously reported protease homolog in Malassezia globosa. We demonstrate that MfSAP1 is capable of degrading a wide range of human skin associated extracellular matrix (ECM) proteins and ECM isolated directly from keratinocytes and fibroblasts. Using a 3-D wound model with primary keratinocytes grown on human de-epidermized dermis, we show that MfSAP1 protease can potentially interfere with wound re-epithelization in an acute wound model. Taken together, our work demonstrates that Malassezia proteases have host-associated substrates and play important roles in cutaneous wound healing.

Use of a Rho kinase inhibitor to increase human tonsil keratinocyte longevity for three-dimensional, tissue engineered tonsil epithelium equivalents.

The generation of tissue-engineered epithelial models is often hampered by the limited proliferative capacity of primary epithelial cells. This study aimed to isolate normal tonsillar keratinocytes (NTK) from human tonsils, increase the lifespan of these cells using the Rho kinase inhibitor Y-27632 and to develop tissue-engineered equivalents of healthy and infected tonsil epithelium. The proliferation rate of isolated NTK and expression of c-MYC and p16INK4A were measured in the absence or presence of the inhibitor. Y-27632-treated NTK were used to generate tissue-engineered tonsil epithelium equivalents using de-epidermised dermis that were then incubated with Streptococcus pyogenes to model bacterial tonsillitis, and the expression of pro-inflammatory cytokines was measured by cytokine array and ELISA. NTK cultured in the absence of Y-27632 rapidly senesced whereas cells cultured in the presence of this inhibitor proliferated for over 30 population doublings without changing their phenotype. Y-27632-treated NTK produced a multi-layered differentiated epithelium that histologically resembled normal tonsillar surface epithelium and responded to S. pyogenes infection by increased expression of pro-inflammatory cytokines including CXCL5 and IL-6. NTK can be isolated and successfully cultured in vitro with Y-27632 leading to a markedly prolonged lifespan without any deleterious consequences to cell morphology. This functional tissue-engineered equivalent of tonsil epithelium will provide a valuable tool for studying tonsil biology and host-pathogen interactions in a more physiologically relevant manner.

Quantitative comparison of the spreading and invasion of radial growth phase and metastatic melanoma cells in a three-dimensional human skin equivalent model.

BackgroundStandard two-dimensional (2D) cell migration assays do not provide information about vertical invasion processes, which are critical for melanoma progression. We provide information about three-dimensional (3D) melanoma cell migration, proliferation and invasion in a 3D melanoma skin equivalent (MSE) model. In particular, we pay careful attention to compare the structure of the tissues in the MSE with similarly-prepared 3D human skin equivalent (HSE) models. The HSE model is identically prepared to the MSE model except that melanoma cells are omitted. Using the MSE model, we examine melanoma migration, proliferation and invasion from two different human melanoma cell lines. One cell line, WM35, is associated with the early phase of the disease where spreading is thought to be confined to the epidermis. The other cell line, SK-MEL-28, is associated with the later phase of the disease where spreading into the dermis is expected.Methods3D MSE and HSE models are constructed using human de-epidermised dermis (DED) prepared from skin tissue. Primary fibroblasts and primary keratinocytes are used in the MSE and HSE models to ensure the formation of a stratified epidermis, with a well-defined basement membrane. Radial spreading of cells across the surface of the HSE and MSE models is observed. Vertical invasion of melanoma cells downward through the skin is observed and measured using immunohistochemistry. All measurements of invasion are made at day 0, 9, 15 and 20, providing detailed time course data.ResultsBoth HSE and MSE models are similar to native skin in vivo, with a well-defined stratification of the epidermis that is separated from the dermis by a basement membrane. In the HSE and MSE we find fibroblast cells confined to the dermis, and differentiated keratinocytes in the epidermis. In the MSE, melanoma cells form colonies in the epidermis during the early part of the experiment. In the later stage of the experiment, the melanoma cells in the MSE invade deeper into the tissues. Interestingly, both the WM35 and SK-MEL-28 melanoma cells lead to a breakdown of the basement membrane and eventually enter the dermis. However, these two cell lines invade at different rates, with the SK-MEL-28 melanoma cells invading faster than the WM35 cells.DiscussionThe MSE and HSE models are a reliable platform for studying melanoma invasion in a 3D tissue that is similar to native human skin. Interestingly, we find that the WM35 cell line, that is thought to be associated with radial spreading only, is able to invade into the dermis. The vertical invasion of melanoma cells into the dermal region appears to be associated with a localised disruption of the basement membrane. Presenting our results in terms of time course data, along with images and quantitative measurements of the depth of invasion extends previous 3D work that has often been reported without these details.

Construction of tissue-engineered skin by culture of human amniotic epithelial cells on human de-epidermized dermis for the repair of skin defects in nude mice: an experimental study

Objective To evaluate the feasibility of repair of full-thickness skin defects in nude mice with tissue-engineered skin which was constructed by culture of human amniotic epithelial cells (hAECs) and fibroblasts on human de-epidermized dermis (DED) . Methods Healthy human amniotic tissues were treated with trypsin at a low concentration in multi-steps to prepare hAECs, and a two-step collagenase digestion was used to treat healthy children′s prepuce tissues to prepare fibroblast suspensions. When fibroblasts were cultured in vitro up to passage 3-5 and hAECs up to passage 2, they were seeded on the reticular dermal surface and basement membrane surface of the DED respectively to construct the tissue-engineered skin. A total of 20 healthy male nude mice aged 3-4 weeks were enrolled into this experiment, and full-thickness skin defects were made on the middle of the back of mice. Then, these mice were randomly divided into 2 groups by using a lottery method, and reconstructed full-thickness tissue-engineered skin grafts and vaseline oil gauze were used to cover the wounds in the tissue-engineered skin group and control group respectively. The whole body and transplantation sites of the nude mice were observed on day 7, 14, 21 and 28 after transplantation, the wound healing time and rate were compared between the above two groups, and skin tissues at the transplantation site were harvested at 4 weeks after transplantation and subjected to histological examination. Results HAECs had stem-cell characteristics and expressed octamer-binding protein-4 (OCT-4) and embryonic marker stage-specific embryonic antigen-4 (SSEA-4) . After 2-week organ culture, the in vitro reconstructed tissue-engineered skin showed 4-9 continuous layers of stratified epithelium, and the histological structure of the epidermis was similar to that of the normal human skin. Compared with the control group, the tissue-engineered skin group showed significantly higher wound healing rates on day 7, 14 and 21 after transplantation (57.49% ± 6.11% vs. 22.93% ± 4.26%, 92.80% ± 3.10% vs. 54.57% ± 7.94%, 98.83% ± 0.25% vs. 91.16% ± 4.79%, respectively; n= 10, t= 27.36, 32.23, 11.80, respectively, all P < 0.001) , shorter wound healing time[ (21.51 ± 1.51) d vs. (28.80 ± 1.14) d, n= 10, t= 42.23, P < 0.001], with the color of skin grafts closer to that of autologous skin on day 28 after transplantation. Histological examination revealed distinct stratification of the epithelium, obvious keratinization and favorable growth of cells in the dermis in the tissue-engineered skin group, but thin epithelium with some defects, indistinct stratification of the dermis, and inflammatory cell infiltration in the control group. Conclusion Tissue-engineered skin constructed by the culture of hAECs and fibroblasts on human DED can survive in nude mice after transplantation, resulting in a more favorable healing of wounds, and is expected to serve as a kind of ideal tissue-engineered skin. Key words: Tissue engineering; Epithelial cells; Dermis; Skin transplantation; Wound healing; De-epidermized dermis; Human amniotic epithelial cells

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

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

Modeling and validating three dimensional human normal cervix and cervical cancer tissues in vitro.

The use of three dimensional in vitro systems in cancer research is a promising path for developing effective anticancer therapies. The aim of this study was to engineer a functional 3-D in vitro model of normal and cancerous cervical tissue.Normal epithelial and immortalized cervical epithelial carcinoma cell lines were used to construct 3-D artificial normal cervical and cervical cancerous tissues. De-epidermised dermis (DED) was used as a scaffold for both models. Morphological analyses were conducted by using hematoxylin and eosin staining and characteristics of the models were studied by analyzing the expression of different structural cytokeratins and differential protein marker Mad1 using immunohistochemical technique.Haematoxylin and eosin staining results showed that normal cervical tissue had multi epithelial layers while cancerous cervical tissue showed dysplastic changes. Immunohistochemistry staining results revealed that for normal cervix model cytokeratin 10 was expressed in the upper stratified layer of epithelium while cytokeratin 5 was expressed mainly in the middle and basal layer. Cytokeratin 19 was weakly expressed in a few basal cells. Cervical cancer model showed cytokeratin 19 expression in different epithelial layers and weak or no expression for cytokeratin 5 and cytokeratin 10. Mad1 expression was detected in some suprabasal cells.The 3-D in vitro models showed stratified epithelial layers and expressed the same types and patterns of differentiation marker proteins as seen in corresponding in vivo tissue in either normal cervical or cervical cancerous tissue. Findings imply that they can serve as functional normal and cervical cancer models.

Initial application of autologous de-epidermized dermis tissue in immediate breast reconstruction after modified radical mastectomy

Initial application of autologous de-epidermized dermis tissue in immediate breast reconstruction after modified radical mastectomy

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation.

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

Reconstruction of tissue-engineered skin using keratinocyte serum-free medium

Objective To reconstruct tissue-engineered skin by co-culture of human fibroblasts, keratinocytes and melanocytes on human de-epidermized dermis with keratinocyte serum-free medium (K-SFM) . Methods Healthy children's prepuce tissues were treated with pancreatin and collagenase to prepare epidermal and dermal cell suspensions respectively. After keratinocytes and melanocytes were cultured separately up to passage 3 and fibroblasts up to passage 5, the density of these cells was adjusted to 2.5 × 105/ml for the following experiment. Human de-epidermized dermis containing some components of the basement membrane was prepared. Firstly, fibroblast suspensions were seeded at the bottom of 6-well plates followed by 24-hour culture. Subsequently, the prepared de-epidermized dermis was added into the 6-well plates, then, keratinocyte and melanocyte suspensions were seeded on the surface of the de-epidermized dermis with the melanocyte ∶ keratinocyte ∶ fibroblast ratio being 1∶4∶1. After 4 hours of culture, the cell mixtures were divided into two groups: an experimental group cultured with K-SFM, a control group cultured with a mixed medium containing DMEM with 10% fetal bovine serum, K-SFM and M254 at a ratio of 1∶1∶1. De-epidermized dermis cultured with K-SFM or the mixed medium alone served as blank control groups. After submerged cultivation for 3 days, the tissue cultures were maintained at an air-liquid interface for another 11 days with the culture medium changed every 3 days. Finally, these cultures were subjected to hematoxylin and eosin staining, periodic acid-Schiff (PAS) staining and immunohistochemical staining for Melan-A, S-100, HMB45, cytokeratin-Pan, P63, K5, K6, K14, Ki67, vimentin, collagen Ⅳ and laminin. Results After 14-day submerged and air-liquid interface culture, a well-structured epidermis developed in both the experimental group and control group with the formation of the stratum corneum. The immunohistochemical study showed positive staining for cytokeratin-Pan, P63, K5, K6, K14, Ki67, laminin in both the experimental group and control group, but positive staining for Melan-A, S-100, HMB45 and collagen Ⅳ in only the experimental group. Conclusion Tissue-engineered skin can be constructed using keratinocytes, melanocytes and fibroblasts co-cultured on de-epidermized dermis with K-SFM in vitro. Key words: Skin; Tissue engineering; Culture media, serum-free; Fibroblasts; Melanocytes; Keratinocytes

Using human epithelial amnion cells in human de-epidermized dermis for skin regeneration

BackgroundHuman amniotic epithelial cells (hAECs) is a desirable reserve of stem cells. Human de-epidermized dermis (DED) retains basic tissue structure and parts of the basement membrane (BM) components at the acelluIar dermal surface, and provides a potential tool for skin regeneration. ObjectiveTo evaluate the potential role of hAECs in skin regeneration, we used DED to perform organotypic culture of hAECs to develop organotypic skin. MethodsHAECs were isolated and cultured. Biological characteristics of hAECs were determined by immunocytochemistry and flow cytometry. To prepare DED, the epidermis was removed and then repeated freeze-thaw cycles. HAECs and fibroblast were seeded onto DED to perform the submerged culture for 3 days and then to be maintained at the air-liquid interface for 14 days to form organotypic culture. To identify whether the obtained DED retain the BM structure and components, the histological characteristics of DED and the BM were detected by immunohistochemistry. To evaluate whether the organotypic skin has similar histological characteristics with normal human skin, the marks of epidermal proliferation and differentiation and basement membrane component were detected by immunohistochemistry. Moreover, cell ultrastructure, cell–cell contact and ultrastructure of BM were examined under the transmission electron microscopy. ResultsHAECs has stem-cell characteristics with strong pluripotent Oct-4 and embryonic marker SSEA-4 expression. DED has effectively cleansed the cell components and continuous distributions of laminin and collagen IV. The histological appearance of tissue-engineered skin in vitro has 4 to 9 continuous layers of stratified epithelium and is similar to normal human skin in morphology. Immunohistochemical studies revealed that proliferation and differentiation markers such as Ki67, CK19, CK14, CK10, filaggrin but not CK18 expressed similar pattern characteristics to normal human epidermis. In addition, Periodic acid-Schiff stain showed that a uniform red staining strip located at the epidermal-dermal junction. BM component proteins (type IV collagen and laminin) and cell adhesion protein (desmoglein) were detected by immunohistochemistry in organotypic skin. Ultrastructurally, desmosomes, hemidesmosomes and BM zone (BMZ) were observed in organotypic skin. ConclusionsOur studies indicate that the hAECs is a promising stem cell source for tissue-engineered skin, and DED with hAECs is a potential application prospects in regenerative medicine.

In-vitro melanoma models: invasive growth is determined by dermal matrix and basement membrane.

A critical first step in the metastatic progression of cutaneous melanoma, invasive growth into the dermal compartment, would ideally be studied in the proper three-dimensional tissue microenvironment. In this study, we compared the growth and behavior of four melanoma cell lines originating from primary and metastatic human cutaneous melanomas (AN, RU, M14, and WK) in in-vitro human skin equivalents (HSEs) generated with four different dermal matrices: human fibroblast-seeded rat tail collagen, human fibroblast-derived matrix (FDM), noncellular human de-epidermized dermis (DED), and a novel fully cellular human DED with an intact pre-existent basement membrane. Melanoma cells showed proliferation in all HSEs, indicating that the microenvironment formed in all HSEs studied here allows the growth of melanoma cells in concert with epidermal keratinocytes for multiple weeks in vitro. Melanoma cells did not affect epidermal proliferation and terminal differentiation. Growth of melanoma cells in the dermal compartment, as a measure of invasive potential, differs markedly between the four types of in-vitro human melanoma models. Notably, the growth of melanoma cells in the dermal matrix was observed in all HSEs cultured with cell lines originating from metastatic melanoma, except for cDED-based HSEs, and the growth of melanoma cells of nonmetastatic origin was observed in the dermal compartment of FDM-based HSEs. Our results show that the type of dermal equivalent and the presence of an intact basement membrane should be taken into consideration when studying melanoma invasion using in-vitro HSEs.

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

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

The use of allodermis prepared from Euro skin bank to prepare autologous tissue engineered skin for clinical use

Over the past two decades a range of 3D models for human skin have been described. Some include native collagen and intrinsic basement membrane proteins and fibroblasts, others are based on xenogeneic collagen or synthetic supports often without fibroblasts. The aim of this study was to look at the influence of media calcium, basement membrane and fibroblasts on the quality of 3D tissue engineered skin produced using human de-epidermized acellular dermis. In this study we deliberately used Euro skin as the source of the donor dermis to examine to what extent this could provide an effective dermal substrate for producing 3D skin for clinical use. Keratinocytes were cultured in the presence and absence of fibroblasts and both with and without basement membrane on decellularized dermis at calcium concentrations ranging from 250μM to 1.6mM over a period of 14 days. Results showed the formation of a well attached epithelium with many of the features of normal skin in the presence of a basement membrane. This was largely independent of the presence of fibroblasts and not greatly influenced by the concentration of calcium in the media. However there was a clear requirement for physiological levels of calcium in the formation of a stratified epithelium in the absence of a basement membrane.

Exposure to acute electromagnetic radiation of mobile phone exposure range alters transiently skin homeostasis of a model of pigmented reconstructed epidermis

Exposure to electromagnetic radiations (EMR) produced by mobile phone concerns half the world's population and raises the problem of their impact on human health. In this study, we looked at the effects of mobile phone exposure (GSM basic, 900MHz, SAR 2mWg(-1) , 6h) on a model of pigmented skin. We have analysed the expression and localization of various markers of keratinocyte and melanocyte differentiation 2, 6, 18 and 24h after EMR exposure of reconstructed epidermis containing either only keratinocytes or a combination of keratinocytes and melanocytes grown on dead de-epidermized dermis, using histology, immunohistochemistry and Western blot. No changes were found in epidermal architecture, localization of epidermal markers, presence of apoptotic cells and the induction of p53 in both types of epidermis (with or without melanocytes) after exposure to EMR. In pigmented reconstructs, no change in the location and dendricity of melanocytes and in melanin transfer to neighbouring keratinocytes was detected after EMR exposure. Loricrin, cytokeratin 14 were significantly decreased at 6h. The level of all markers increased at 24h as compared to 6h post-EMR exposure, associated with a significant decrease of the 20S proteasome activity. Our data indicate that exposure to 900MHz frequency induces a transient alteration of epidermal homoeostasis, which may alter the protective capacity of the skin against external factors. Presence or absence of melanocytes did not modify the behaviour of reconstructs after EMR exposure.

Architectural characterization of organotypic cultures of H400 and primary rat keratinocytes

Organotypic epithelial structures can be cultured using primary or immortalized keratinocytes. However, there has been little detailed quantitative histological characterization of such cultures in comparison with normal mucosal architecture. The aim of this study is to identify morphological markers of tissue architecture that can be used to monitor tissue structure, maturation, and differentiation and to enable quantitative comparison of organotypic cultures (OCs) with normal oral mucosa. OCs of oral keratinocytes [immortalized H400 or primary rat keratinocytes (PRKs)] were generated using the three scaffolds of de-epidermalized dermis (DED), polyethylene terephthalate (PET), and collagen gels for up to 14 days. Cultures and normal epithelium were analyzed immunohistochemically and by using the semi-quantitative reverse transcriptase polymerase chain reaction (sq-RT-PCR) for E-cadherin, desmoglein-3, plakophilin, involucrin, cytokeratins-1, -5, -6, -10, -13, and Ki67. The epithelial thickness of OCs was measured in stained sections using image processing. Histological analysis revealed that air-liquid interface (ALI) cultures generated stratified organotypic epithelial structures by 14-days. The final thickness of these cultures as well as the degree of maturation/stratification (including stratum corneum formation) varied significantly depending on the scaffold used. For certain scaffolds, the immunohistochemical profiles obtained recapitulated those of normal oral epithelium indicating comparable in vitro differentiation and proliferation. In conclusion, quantitative microscopy approaches enabled unbiased architectural characterization of OCs. The scaffold materials used in the present study (DED, collagen type-I and PET) differentially influenced cell behavior in OCs of oral epithelia. H400 and PRK OCs on DED at the ALI demonstrated similar characteristics in terms of gene expression and protein distribution to the normal tissue architecture.