Elastin Expression in a Newly Developed Full-Thickness Skin Equivalent

El Ghalbzouri A., Ponec M.

Epithelial-mesenchymal interactions play an important role in controlling epidermal morphogenesis and homeostasis but little is known about the mechanisms of these interactions. To examine whether diffusible factors produced by fibroblasts and/or keratinocytes support epidermal morphogenesis and basement membrane formation, organotypic keratinocyte monocultures were established in media collected either from organotypic fibroblast or keratinocyte-monocultures or from keratinocyte-fibroblast cocultures, and the expression of keratin 10, 16, and 17 and basement membrane components (types IV and VII collagen, laminin 5, nidogen, BP 180, LAD-1) were examined. We found that diffusible factors released by keratinocytes were not sufficient to support the establishment of normalized epidermal phenotype and deposition of basement membrane components in contrast to fibroblast- or keratinocyte/fibroblast-derived factors. Keratinocytes appear to affect the spectrum of secreted soluble factors, as keratinocyte/fibroblast-derived factors were more effective to accomplish continuous linear deposition of laminin 5 and of nidogen. The finding that released amounts of keratinocyte growth factor and granulocyte macrophage colony stimulating factor were not sufficient to fully support epidermal morphogenesis and deposition of basement membrane components is suggestive for the involvement of other released diffusible factors. Generation of organotypic keratinocyte monocultures in the presence of fibroblast- or keratinocyte/fibroblast-derived soluble factors resulted in enhanced expression of keratins K16 and K17 and the absence of type IV collagen. This observation indicates that next to paracrine acting factors, epidermal homeostasis is controlled by mutual keratinocyte-fibroblast interaction.

Robert L., Fodil-Bourahla I., Bizbiz L., Robert A.M.

With increasing age elastic fibres in human skin are progressively lysed and skin elasticity is also decreasing. Still there is an age-dependent increase of elastic fibre surface density, mostly due to an alteration of the fibres. The present experiments were undertaken to explore if L-fucose and fucose-rich polysaccharides (FROP-s) could influence elastin biosynthesis. We show here, that topical application of a fucose-containing preparation to the skin of hairless rats increased after 4 weeks the elastic fibre surface density by about 40%, shown by quantitative morphology. Using human skin fibroblasts in explant cultures, the addition of L-fucose or of FROP-3 increased the biosynthesis of immunoprecipitable tropoelastin by about 40%. No increase was found however of desmosine-isodesmosine in skin explant cultures after 72 h of incubation. The effect of L-fucose and FROP-3 on the biosynthesis of collagen and non-collagen proteins excreted by the skin explant cultures was also investigated. L-fucose, but not FROP-3, decreased collagen biosynthesis but both increased non-collagen protein biosynthesis. These results show that L-fucose and FROP-3 stimulate tropoelastin biosynthesis in vitro, and elastic fibre formation in vivo. This stimulation concerns also several non-collagen proteins secreted by skin explant cultures. Elastic fibre formation necessitates the simultaneous synthesis of several microfibrillar glycoproteins as well as of tropoelastin. The increased elastic fibre density in the in vivo experiments suggests that this is indeed achieved by L-fucose and FROP-3, further demonstrating their efficiency in the control of age-dependent modifications of connective tissues in general and of skin in particular.

Noblesse E., Cenizo V., Bouez C., Borel A., Gleyzal C., Peyrol S., Jacob M., Sommer P., Damour O.

Elastic fiber formation involves the secretion of tropoelastin which is converted to insoluble elastin by cross-linking, initiated by the oxidative deamination of lysine residues by lysyl oxidase. Five lysyl oxidase genes have been discovered. This study deals with the expression of two isoforms, LOX and LOX-like (LOXL), in human foreskin and in a human skin-equivalent (SE) model that allows the formation of elastic fibers. In this model, keratinocytes are added to a dermal equivalent made of fibroblasts grown on a chitosan-cross-linked collagen-GAG matrix. LOX and LOXL were detected by immunohistochemistry in the dermis and the epidermis of both normal skin and in a SE. This expression was confirmed by in situ hybridization on the SE. LOX and LOXL expression patterns were confirmed in human skin. The ultrastructural localization of LOXL was indicative of its association with elastin-positive materials within the SE and human skin, though interaction with collagen could not be discarded. LOX was found on collagen fibers and could be associated with elastin-positive materials in the SE and human skin. LOXL and LOX were detected in keratinocytes where LOX was mainly expressed by differentiating keratinocytes, in contrast to LOXL that can be found in both proliferating and differentiating fibroblasts. These data favor a role for LOXL in elastic fiber formation, together with LOX, and within the epidermis where both enzymes should play a role in post-translational modification of yet unknown substrates.

Stark H., Willhauck M.J., Mirancea N., Boehnke K., Nord I., Breitkreutz D., Pavesio A., Boukamp P., Fusenig N.E.

Besides medical application as composite skin grafts, in vitro constructed skin equivalents (SEs) or organotypic co-cultures represent valuable tools for cutaneous biology. Major drawbacks of conventional models, employing collagen hydrogels as dermal equivalents (DEs), are a rather poor stability and limited life span, restricting studies to early phases of skin regeneration. Here we present an improved stabilised in vitro model actually providing the basis for skin-like homeostasis. Keratinocytes were grown on dermal equivalents (DEs) reinforced by modified hyaluronic acid fibres (Hyalograft-3D) and colonised with skin fibroblasts, producing genuine dermis-type matrix. These SEs developed a superior epidermal architecture with regular differentiation and ultrastructure, which occurred also faster than in SEs based on collagen-DEs. Critical aspects of differentiation, still unbalanced in early stages, were perfectly re-normalised, most strikingly the co-expression of keratins K1/K10 and downregulation of regeneration-associated keratins such as K16. The restriction of integrin and K15 distribution as well as keratinocyte proliferation to the basal layer underlined the restored tissue polarity, while the drop of growth rates towards physiological levels implied finally accomplishment of homeostasis. This correlated to faster basement membrane (BM) formation and ultrastructurally defined dermo-epidermal junction including abundant anchoring fibrils for strong tissue connection. Whereas the fibroblasts in the scaffold initially secreted a typical provisional regenerative matrix (fibronectin, tenascin), with time collagens of mature dermis (type I and III) were accumulating giving rise to an in vivo-like matrix with regularly organised bundles of striated collagen fibrils. In contrast to the more catabolic state in conventional DEs, the de novo reconstruction of genuine dermal tissue seemed to be a key element for maintaining prolonged normal keratinocyte proliferation (followed up to 8 wks), fulfilling the criteria of tissue-homeostasis, and possibly providing a stem cell niche.

Pouliot R., Larouche D., Auger F.A., Juhasz J., Xu W., Li H., Germain L.

The best alternative to a split-thickness graft for the wound coverage of patients with extensive burns should be in vitro reconstructed autologous skin made of both dermis and epidermis and devoid of exogenous extracellular matrix proteins and synthetic material. We have designed such a reconstructed human skin (rHS) and present here its first in vivo grafting on athymic mice.The rHS was made by culturing newborn or adult keratinocytes on superimposed fibrous sheets obtained after culturing human fibroblasts with ascorbic acid. Ten days after keratinocyte seeding, reconstructed skins were either cultured at the air-liquid interface or grafted on athymic mice. We present the macroscopic, histologic, and phenotypic properties of such tissues in vitro and in vivo after grafting on nude mice.After maturation in vitro, the reconstructed skin exhibited a well-developed human epidermis that expressed differentiated markers and basement membrane proteins. Four days after grafting, a complete take of all grafts was obtained. Histological analysis revealed that the newly generated epidermis of newborn rHS was thicker than that of adult rHS after 4 days but similar 21 days after grafting. The basement membrane components (bullous pemphigoid antigens, laminin, and type IV and VII collagens) were detected at the dermo-epidermal junction, showing a continuous line 4 days after grafting. Ultrastructural studies revealed that the basement membrane was continuous and well organized 21 days after transplantation. The macroscopic aspect of the reconstructed skin revealed a resistant, supple, and elastic tissue. Elastin staining and elastic fibers were detected as a complex network in the rHS that contributes to the good elasticity of this new reconstructed tissue.This new rHS model gives supple and easy to handle skins while demonstrating an adequate wound healing on mice. These results are promising for the development of this skin substitute for permanent coverage of burn wounds.

Sahota P.S., Burn J.L., Heaton M., Freedlander E., Suvarna S.K., Brown N.J., Mac Neil S.

We have previously shown that reconstructed human skin engineered from autologous keratinocytes, fibroblasts, and sterilized donor allodermis stimulates angiogenesis within 5–7 days when placed on well-vascularized wound beds in nude mice. When this reconstructed skin was used clinically in more demanding wound beds, some grafts were lost, possibly due to delayed vascularization. As this reconstructed skin lacks any endothelial cells, our aim in this study was to develop an angiogenic reconstructed skin model in which to explore strategies to improve angiogenesis both in vitro and in vivo. We report that culture of small-vessel human dermal microvascular endothelial cells (HuDMECs) was achieved using magnetic beads coated with an antibody to platelet cell adhesion molecule as a means of purifying the culture. Keratinocytes, fibroblasts, and HuDMECs could be cultured from the same skin biopsy. Initial studies culturing HuDMECs and other sources of endothelial cells with the tissue-engineered skin showed that these cells were capable of slowly entering the dermis under standard culture conditions in vitro. In conclusion, this provides us with a model in which to explore strategies for improving angiogenesis in vitro and also establishes the culture methodologies for the production of reconstructed skin containing autologous keratinocytes, fibroblasts, and endothelial cells. (WOUND REP REG 2003;11:275–284)

Mao J., Zhao L., de Yao K., Shang Q., Yang G., Cao Y.

A novel absorbable scaffold composed of chitosan and gelatin was fabricated by freezing and lyophilizing methods, resulting in an asymmetric structure. This bilaminar texture is suitable for preparing a bilayer skin substitute. The methods employed to confirm the applicability of this chitosan-gelatin scaffold as an ideal skin substitute were a water uptake ability test, in vitro fibroblast proliferation, and scaffold tests in which fibroblasts were co-cultured with keratinocytes. The chitosan-gelatin scaffolds were more wettable and adsorbed more water than did chitosan alone. In static cell culture the thinner scaffold is better than the thicker one, and because of diffusion limitations in the scaffold, culture time must be within 3 weeks before transplantation to living tissues. Keratinocytes were co-cultured with fibroblasts in chitosan-gelatin scaffolds to construct an artificial bilayer skin in vitro. The artificial skin obtained was flexible and had good mechanical properties. Moreover, there was no contraction observed in the in vitro cell culture tests. The data from this study suggest that chitosan-gelatin scaffolds are suitable for skin tissue engineering goals.

Guerret S., Govignon E., Hartmann D.J., Ronfard V.

Type I collagen is a clinically approved biomaterial largely used in tissue engineering. It acts as a regenerative template in which the implanted collagen is progressively degraded and replaced by new cell-synthesized tissue. Apligraf, a bioengineered living skin, is composed of a bovine collagen lattice containing living human fibroblasts overlaid with a fully differentiated epithelium made of human keratinocytes. To investigate its progressive remodeling, athymic mice were grafted and the cellular and the extracellular matrix components were studied from 0 to 365 days after grafting. Biopsies were analyzed using immunohistochemistry with species-specific antibodies and electron microscopy techniques. We observed that this bioengineered tissue provided living and bioactive cells to the wound site up to 1 year after grafting. The graft was rapidly incorporated within the host tissue and the bovine collagen present in the graft was progressively replaced by human and mouse collagens. A normal healing process was observed, i.e., type III collagen appeared transiently with type I collagen, the major collagen isoform present at later stages. New molecules, such as elastin, were produced by the living human cells contained within the graft. This animal model combined with species-specific immunohistochemistry tools is thus very useful for studying long-term tissue remodeling of bioengineered living tissues.

Hinterhuber G., Marquardt Y., Diem E., Rappersberger K., Wolff K., Foedinger D.

Organotypic human skin equivalents of keratinocytes and fibroblasts embedded in collagen matrix have been the subject of studies dealing with various culture conditions. Development of standardized living skin equivalents using defined culture media containing respective supplements can provide important instruments of investigation in skin biology. In addition, tissue engineering has created human skin substitutes for treatment of acute and chronic wounds. In our study, we generate a modified organotypic human skin equivalent using normal human serum instead of fetal calf serum (FCS). This living skin equivalent shows regular stratification of the epidermis and the dermal-epidermal junction zone at the light and electron microscopic level after 1 and 3 weeks of coculture. Indirect immunofluorescence reveals regular expression of differentiation antigens and the major structural proteins collagen IV, laminin 5 and the integrin chains alpha 6 and beta 4 at the dermo-epidermal junction zone. Immunoelectron microscopy demonstrates expression of collagen IV, alpha 6 and beta 4 integrin after 1 and 3 weeks of coculture. This organotypic skin model could be the basis for autologous skin grafting for acute or chronic wounds using autologous serum as well as patients' keratinocytes and fibroblasts, thus minimizing the risk of transmitting infectious agents.

Berthod F., Germain L., Li H., Xu W., Damour O., Auger F.A.

Wound healing of deep and extensive burns can induce hypertrophic scar formation, which is a detrimental outcome for skin functionality. These scars are characterized by an impaired collagen fibril organization with fibril bundles oriented parallel to each other, in contrast with a basket weave pattern arrangement in normal skin. We prepared a reconstructed skin made of a collagen sponge seeded with human fibroblasts and keratinocytes and grown in vitro for 20 days. We transplanted it on the back of nude mice to assess whether this reconstructed skin could prevent scar formation. After transplantation, murine blood vessels had revascularized one-third of the sponge thickness on the fifth day and were observed underneath the epidermis at day 15. The reconstructed skin extracellular matrix was mostly made of human collagen I, organized in loosely packed fibrils 5 days after transplantation, with a mean diameter of 45 nm. After 40-90 days, fibril bundles were arranged in a basket weave pattern while their mean diameter increased to 56 nm, therefore exactly matching mouse skin papillary dermis organization. Interestingly, we showed that an elastic system remodeling was started off in this model. Indeed, human elastin deposits were organized in thin fibrils oriented perpendicular to epidermis at day 90 whereas elastic system usually took years to be re-established in human scars. Our reconstructed skin model promoted in only 90 days the remodeling of an extracellular matrix nearly similar to normal dermis (i.e. collagen fibril diameter and arrangement, and the partial reconstruction of the elastic system).

LAPLANTE A.F., GERMAIN L., AUGER F.A., MOULIN V.

Wound closure of epithelial tissues must occur efficiently to restore rapidly their barrier function. We have developed a tissue-engineered wound-healing model composed of human skin keratinocytes and fibroblasts to better understand the mechanisms of reepithelialization. It allowed us to quantify the reepithelialization rate, which was significantly accelerated in the presence of fibrin or platelet-rich plasma. The reepithelialization of these 6 mm excisional wounds required the contribution of keratinocyte proliferation, migration, stratification, and differentiation. The epidermis regenerated progressively from the surrounding wound margins. After 3 days, the neoepidermis showed a complete spectrum of changes. Near the wound margin, the differentiation of the neoepidermis (keratins 1/10, filaggrin, and loricrin) and regeneration of the dermoepidermal junction (laminin 5 and collagen IV) were more advanced than toward the wound center, where the proliferative index was significantly increased. The spatial distribution of keratinocytes distinguished by particular features suggests two complementary mechanisms of reepithelialization: 1) the passive displacement of the superficial layers near the wound margin that would rapidly regenerate a barrier function and 2) the crawling of keratinocytes over each other at the tip of the progressing neoepidermis. Therefore, this study brings a new perspective to long-standing questions concerning wound reepithelialization.

Schlotmann K., Kaeten M., Black A.F., Damour O., Waldmann-Laue M., Förster T.

A tissue engineered human skin equivalent is successfully used for the testing of raw materials and cosmetic formulations. This reconstructed skin is supported by a collagen-glycosaminoglycan-chitosan biopolymer in which human keratinocytes and dermal fibroblasts were co-cultured to form a tissue that closely reproduces the in vivo architecture of normal human skin and takes into account the complex interactions between epidermis and dermis. On the other hand, dermal and epidermal responses can be assessed separately in the dermal or skin equivalent. The three-dimensional model has important advantages compared to monolayer cell cultures and epidermis models in efficacy testing: (i) the possibility of long-term cultivation with repeated application of cream formulations containing bioactives and (ii) the similarity to human skin concerning the interaction between dermis and epidermis. These similarities include the expression of keratinocyte differentiation markers such as cytokeratin 10, filaggrin and transglutaminase, as well as proteins of the basal lamina (laminin, collagen type IV) and extracellular matrix proteins such as elastin. The efficacy of selected bioactives was determined using different endpoints, for example, stimulation of collagen synthesis in the dermal and skin equivalents was shown in comparison to vitamin C as a positive control. On skin equivalents using immunofluorescence techniques we also demonstrated stimulation of the differentiation marker filaggrin, which is important for skin moisturization. The results could be used for claim substantiation, e.g. for the treatment of dry and aged skin.

Wang C.K., Nelson C.F., Brinkman A.M., Miller A.C., Hoeffler W.K.

We show that an inherent ability of two distinct cell types, keratinocytes and fibroblasts, can be relied upon to accurately reconstitute full-thickness human skin including the dermal-epidermal junction by a cell-sorting mechanism. A cell slurry containing both cell types added to silicone chambers implanted on the backs of severe combined immunodeficient mice sorts out to reconstitute a clearly defined dermis and stratified epidermis within 2 wk, forming a cell-sorted skin equivalent. Immunostaining of the cell-sorted skin equivalent with human cell markers showed patterns similar to those of normal full-thickness skin. We compared the cell-sorted skin equivalent model with a composite skin model also made on severe combined immunodeficient mice. The composite grafts were constructed from partially differentiated keratinocyte sheets placed on top of a dermal equivalent constructed of devitalized dermis. Electron microscopy revealed that both models formed ample numbers of normal appearing hemidesmosomes. The cell-sorted skin equivalent model, however, had greater numbers of keratin intermediate filaments within the basal keratinocytes that connected to hemidesmosomes, and on the dermal side both collagen filaments and anchoring fibril connections to the lamina densa were more numerous compared with the composite model. Our results may provide some insight into why, in clinical applications for treating burns and other wounds, composite grafts may exhibit surface instability and blistering for up to a year following grafting, and suggest the possible usefulness of the cell-sorted skin equivalent in future grafting applications.

Duplan-Perrat F., Damour O., Montrocher C., Peyrol S., Grenier G., Jacob M., Braye F.

Elastic fibers form a complex network that contributes to the elasticity of connective tissues. Alterations in the elastic fiber network are involved in several disease affecting organs in which compliance of the connective tissue is essential: skin, main vasculature, lung, joints, muscle, and ligament. The aim of our work was to study the deposition, maturation, and organization of elastic fiber components in a dermal equivalent model consisting of collagen-GAG-chitosan seeded with fibroblasts. The influence of keratinocytes was studied in parallel, thus constituting a skin equivalent model. These models were examined by transmission electron microscopy (TEM) and by immunohistochemistry to determine the staining patterns of fibrillin-1 and elastin proteins representative of the microfibrillar framework and of the elastic fibers, respectively. After 2 mo of fibroblast culture in the dermal equivalent, elastin was undetectable, whereas fibrillin-1 staining was weak and microfibrils were infrequently observed by TEM. In the skin equivalent, fibrillin-1 and elastin were detected by immunostaining 15 d after epidermization and TEM revealed the typical structure and organization of the elastic network in the dermis, with elastin deposition on the microfibrillar scaffold. This in vitro skin equivalent model is to our knowledge the first in which elastic fibers have been detected, thus demonstrating the influence of keratinocytes on the maturation and organization of the elastic network.

van Dorp A.G., Verhoeven M.C., Koerten H.K., van Blitterswijk C.A., Ponec M.

The purpose of this study was to find an optimal polymer matrix and to optimize the culture conditions for human keratinocytes and fibroblasts for the development of a human skin substitute. For this purpose porous, dense bilayers made of a block copolymer of poly(ethylene glycol terephthalate) (PEGT) and poly(butylene terephthalate) (PBT; Polyactivetrade mark) with a PEGT/PBT weight ratio of 55/45 and a PEG molecular weight (MW) of 300, 600, 1000, or 4000 Da were used. The best performance was achieved with PEGT/PBT copolymer with MW of PEG 300 D (300PEG55PBT45). When fibroblasts were seeded into the porous underlayer and cultured for 3 weeks in medium supplemented with 100 microg/mL ascorbic acid, all pores were filled with fibroblasts and with extracellular matrix, which was judged from the presence of collagen types I, III, and IV, and laminin. When seeded onto the dense top layer of the bilayered (cell free or fibroblast populated) copolymer matrix, human keratinocytes grew out into confluent sheets. After subsequent lifting to the air-liquid interface, a multilayered epithelium with a morphology corresponding to that of the native epidermis was formed. Some differences could still be observed: the expression and localization of some differentiation specific proteins was different and close to that seen in hyperproliferative epidermis; a basal lamina and anchoring zone were absent.

Böttcher P., Steinmeyer L., Stark H., Breitkreutz J., Mewes K.R.

The MUTZ-3 cell line is a surrogate for Langerhans cells (LCs) employed in New Approach Methodologies for assessing the skin sensitizing potential of chemicals. However, MUTZ-3 cells must first be differentiated to achieve the LC-typical phenotype. As all protocols use high fetal calf serum (FCS) concentrations, we aimed at reducing, or even replacing FCS, while maintaining MUTZ-LC characteristics. Additionally, we assessed the impact of the poorly defined 5637-conditioned medium (5637CM) on MUTZ-LC differentiation. With reducing the FCS content by 75 %, the desired differentiation status was achieved after 7 instead of 14 days, identified by elevated CD207 and CD1a expression. Culture with Ultroser G, a synthetic surrogate for FCS, resulted in an insufficient number of MUTZ-LCs. 5 % FCS-differentiated MUTZ-LCs could be activated with DNCB, an extreme sensitizer, as demonstrated by increased CD83 expression. 5637CM did not affect MUTZ-LC differentiation and is therefore not needed as a supplement. For their intended role in an immunocompetent skin model to assess the sensitizing potential of chemicals, MUTZ-LCs were successfully integrated into the Phenion® Full-Thickness skin model, as demonstrated by CD1a expression. These results are important steps towards medium standardization and the generation of an immunocompetent skin model according to the 3R principles.

Hölken J.M., Wurz A., Friedrich K., Böttcher P., Asskali D., Stark H., Breitkreutz J., Buhl T., Vierkotten L., Mewes K.R., Teusch N.

AbstractIn the past decades studies investigating the dendritic cell (DC) activation have been conducted almost exclusively in animal models. However, due to species-specific differences in the DC subsets, there is an urgent need for alternative in vitro models allowing the investigation of Langerhans cell (LC) and dermal dendritic cell (DDC) activation in human tissue. We have engineered a full-thickness (FT) human skin tissue equivalent with incorporated LC surrogates derived from the human myeloid leukemia-derived cell line Mutz-3, and DDC surrogates generated from the human leukemia monocytic cell line THP-1. Topical treatment of the skin models encompassing Mutz-LCs only with nickel sulfate (NiSO4) or 1-chloro-2,4-dinitrobenzene (DNCB) for 24 h resulted in significant higher numbers of CD1a positive cells in the dermal compartment, suggesting a sensitizer-induced migration of LCs. Remarkably, exposure of the skin models encompassing both, LC and DDC surrogates, revealed an early sensitizer-induced response reflected by increased numbers of CD1a positive cells in the epidermis and dermis after 8 h of treatment. Our human skin tissue equivalent encompassing incorporated LC and DDC surrogates allows the investigation of DC activation, subsequent sensitizer identification and drug discovery according to the principles of 3R.

Meloni M., de Rooij B., Janssen F.W., Rescigno F., Lombardi B.

Backgrounds/Objectives: Skin wound healing is a physiological process orchestrated by epithelial and mesenchymal cells able to restore tissue continuity by re-organizing themselves and the ECM. This research study aimed to develop an optimized in vitro experimental model of full-thickness skin, to address molecular and morphological modifications occurring in the re-epithelization and wound healing process. Methods: Wound healing starting events were investigated within an experimental window of 8 days at the molecular level by gene expression and immunofluorescence of key epidermal and dermal biomarkers. To mirror the behavior of infected wounds, the established wound healing model was then colonized with S. aureus, and the efficacy of a novel antibacterial agent, XZ.700, was investigated. Viable counts (CFU/tissue), IF, and ultrastructural analysis (SEM) were performed to evaluate S. aureus colonization inside and around the wound bed in an experimental window of 3 h of colonization and 24 h of treatment. Results: Endolysin showed an efficacy in counteracting bacterial growth and invasion within the wound bed, reducing the S. aureus load compared to its placebo, thanks to its selective antimicrobial activity interfering with biofilm formation. Conclusions: The preclinical in vitro infected wound model on FT-kin showed interesting applications to assess the repair efficacy of dermo-pharmaceutical and cosmetic formulations.

Raabe H.A., Costin G., Allen D.G., Lowit A., Corvaro M., O’Dell L., Breeden-Alemi J., Page K., Perron M., Flint Silva T., Westerink W., Baker E., Sullivan K.

Qiao N., Dumas V., Bergheau A., Ouillon L., Laroche N., Privet-Thieulin C., Perrot J., Zahouani H.

The skin, the outermost organ of the human body, is vital for sensing and responding to stimuli through mechanotransduction. It is constantly exposed to mechanical stress. Consequently, various mechanical therapies, including compression, massage, and microneedling, have become routine practices for skin healing and regeneration. However, these traditional methods require direct skin contact, restricting their applicability. To address this constraint, we developed shear wave stimulation (SWS), a contactless mechanical stimulation technique. The effectiveness of SWS was compared with that of a commercial compression bioreactor used on reconstructed skin at various stages of maturity. Despite the distinct stimulus conditions applied by the two methods, SWS yielded remarkable outcomes, similar to the effects of the compression bioreactor. It significantly increased the shear modulus of tissue-engineered skin, heightened the density of collagen and elastin fibers, and resulted in an augmentation of fibroblasts in terms of their number and length. Notably, SWS exhibited diverse effects in the low- and high-frequency modes, highlighting the importance of fine-tuning the stimulus intensity. These results unequivocally demonstrated the capability of SWS to enhance the mechanical functions of the skin in vitro, making it a promising option for addressing wound healing and stretch mark recovery.

Girard F., Lajoye C., Camman M., Tissot N., Berthelot Pedurand F., Tandon B., Moedder D., Liashenko I., Salameh S., Dalton P.D., Rielland M.

AbstractIn vitro skin models are validated methods for screening cosmetics and pharmaceuticals, but still have limitations. The bilayer poly(ε‐caprolactone) scaffold/membrane model described here overcomes some of these deficits by integrating a solution electrospun (SES) membrane at the dermoepidermal interface and a melt electrowritten (MEW) scaffold that provides an optimal open‐pore environment for the dermis. To the knowledge, this scaffold/membrane model is the only one capable of creating a properly differentiated, full thickness skin model with neosynthesized extracellular matrix (ECM) in only 18 days. Both the wavy and straight fiber scaffold designs create a well‐organized dermis, but dermal collagen organization differs between designs. Adding cells and vitamin C to the scaffolds improves the mechanical properties to more closely mimic native human skin. These findings establish bicomponent scaffolds as a promising advancement for rapidly creating different skin models with varied properties. The versatility and adaptability of the described model can be used for studying how the biological and physical microenvironment impact skin, and testing dermo‐cosmetics and pharmaceutical treatments on different ages of skin. Furthermore, it can be an excellent new tool for studying wound healing and development into its use as a graft or wound dressing is ongoing.

Page K., Westerink W., Sullivan K., McDonald T., Roper C.

The skin is a potential route of exposure to antimicrobial cleaning products (ACP). Skin irritation, reversible damage to the skin, is an endpoint for protecting consumers and operators accidently exposed to these complex mixtures. To assess skin irritation of 24 ACP formulations, a new protocol was developed and adapted from OECD Test Guideline No. 439 with EpiDerm™ (epidermis model) replaced by Phenion® FT (full thickness tissue, including epidermis and dermis) as the test system. A full thickness tissue was utilized to provide a more human in vivo-like model. Formulations were applied to Phenion® FT and cell viability measured by MTT reduction after a 15-min exposure and 42 h post exposure period. A prediction model was applied, and results compared with in vivo rabbit skin irritation data. Concordance between in vivo and in vitro was demonstrated to be suitable (i.e., sensitivity 78%, specificity 83%, and accuracy 79%) using this modified OECD Test Guideline No. 439 method with a 70% cell viability selected as the most reasonable cut off for discriminating non-irritants (EPA Class IV). These results were considered suitable to develop a draft IATA i.e., with any ACP formulation identified as EPA Category IV in this test. The method will be further refined to distinguish irritant categories.

Hoerst A., Hermann M., Mewes K.R., Steinmeyer L., Buchholzer M.

For years, the strive for in vitro methods for toxicological assessment suitable to replace animal studies gained progressive importance. OECD Test Guideline (TG) 431 was implemented in 2004, allowing to circumvent animal testing according to OECD TG 404 while reliably predicting skin corrosion potential of many substances and products. However, non-animal assays often show protocol-dependent limitations, that complicate or even prevent the testing of several groups of substances. In this study, the suitability of the OECD TG 431 for assessment of the skin corrosion potential of known acidic, thus often skin corrosive or irritating acrylic and methacrylic acid-based adhesives and monomers, was investigated. The commercially available Phenion® Open Source Reconstructed Epidermis (OS-REp) model, developed at Henkel & Co. KGaA, was used. The EpiDerm™ prediction model was considered most applicable to the Phenion® OS-REp model. All Proficiency Substances listed in OECD TG 431, amongst them six acids, were correctly classified and subcategorized as Skin Corr. 1 A or 1B/C corrosives. The OS-REp model was shown to be suitable for the assessment of skin corrosion potential in accordance with OECD TG 431. However, our results also indicate that acrylic and methacrylic monomer-based adhesives might fall outside the applicability domain of this guideline.

Roy B., Pekec T., Yuan L., Shivashankar G.V.

AbstractCell‐based therapies are essential for tissue regeneration and wound healing during aging. Autologous transplantation of aging cells is ineffective due to their increased senescence and reduced tissue remodeling capabilities. Alternatively, implanting reprogrammed aged cells provides unique opportunities. In this paper, we demonstrate the implantation of partially reprogrammed aged human dermal fibroblasts into in vitro aged skin models for tissue regeneration and wound healing. The partially reprogrammed cells were obtained using our previously reported, highly efficient mechanical approach. Implanted cells showed enhanced expression of extracellular matrix proteins in the large area of aged tissue. In addition, the implanted cells at wound sites showed increased extracellular matrix protein synthesis and matrix alignment. Transcriptome analysis, combined with chromatin biomarkers, revealed these implanted cells upregulated tissue regeneration and wound healing pathways. Collectively our results provide a novel, nongenetic, partial reprogramming of aged cells for cell‐based therapies in regenerative medicine.

Hölken J.M., Friedrich K., Merkel M., Blasius N., Engels U., Buhl T., Mewes K.R., Vierkotten L., Teusch N.E.

We have integrated dermal dendritic cell surrogates originally generated from the cell line THP-1 as central mediators of the immune reaction in a human full-thickness skin model. Accordingly, sensitizer treatment of THP-1-derived CD14-, CD11c+ immature dendritic cells (iDCs) resulted in the phosphorylation of p38 MAPK in the presence of 1-chloro-2,4-dinitrobenzene (DNCB) (2.6-fold) as well as in degradation of the inhibitor protein kappa B alpha (IκBα) upon incubation with NiSO4 (1.6-fold). Furthermore, NiSO4 led to an increase in mRNA levels of IL-6 (2.4-fold), TNF-α (2-fold) and of IL-8 (15-fold). These results were confirmed on the protein level, with even stronger effects on cytokine release in the presence of NiSO4: Cytokine secretion was significantly increased for IL-8 (147-fold), IL-6 (11.8-fold) and IL-1β (28.8-fold). Notably, DNCB treatment revealed an increase for IL-8 (28.6-fold) and IL-1β (5.6-fold). Importantly, NiSO4 treatment of isolated iDCs as well as of iDCs integrated as dermal dendritic cell surrogates into our full-thickness skin model (SM) induced the upregulation of the adhesion molecule clusters of differentiation (CD)54 (iDCs: 1.2-fold; SM: 1.3-fold) and the co-stimulatory molecule and DC maturation marker CD86 (iDCs ~1.4-fold; SM:~1.5-fold) surface marker expression. Noteworthy, the expression of CD54 and CD86 could be suppressed by dexamethasone treatment on isolated iDCs (CD54: 1.3-fold; CD86: 2.1-fold) as well as on the tissue-integrated iDCs (CD54: 1.4-fold; CD86: 1.6-fold). In conclusion, we were able to integrate THP-1-derived iDCs as functional dermal dendritic cell surrogates allowing the qualitative identification of potential sensitizers on the one hand, and drug candidates that potentially suppress sensitization on the other hand in a 3D human skin model corresponding to the 3R principles (“replace”, “reduce” and “refine”).

Zidarič T., Kleinschek K.S., Maver U., Maver T.

The skin can self-renew, thanks to the presence of stem cells in the hypodermisHypodermis. However, when the skin is damaged in the deeper layers, as in second or third-degree burnsBurns, the normal wound healingWound healing responses are impeded, resulting in a chronic injury.

Jennen D.G., van Herwijnen M., Jetten M., Vandebriel R.J., Keizers P., Geertsma R.E., de Jong W.H., Kleinjans J.C.

Usage of injectable dermal fillers applied for aesthetic purposes has extensively increased over the years. As such, the number of related adverse reactions has increased, including patients showing severe complications such as product migration, topical swelling and inflammatory reactions of the skin. In order to understand the underlying molecular events of these adverse reactions we performed a genome-wide gene expression study on the multi-cell type human Phenion® Full-Thickness Skin Model exposed to five experimental hyaluronic acid (HA) preparations with increasing cross-linking degree, four commercial fillers from Perfectha®, and non-resorbable filler Bio-Alcamid®. In addition, we evaluated whether cross-linking degree or particle size of the HA-based fillers could be associated with the occurrence of adverse effects. In all cases, exposure to different HA fillers resulted in a clearly elevated gene expression of cytokines and chemokines related to acute inflammation as part of the foreign body response. Furthermore, for one experimental filler genes of OXPHOS complexes I-V were significantly down-regulated (adjusted p-value < 0.05), resulting in mitochondrial dysfunction which can be linked to over-expression of pro-inflammatory cytokines TNFα and IL-1β and chemokine CCL2. Our hypothesis that cross-linking degree or particle size of the HA-based fillers is related to the biological responses induced by these fillers could only partially be confirmed for particle size. In conclusion, our innovative approach resulted in gene expression changes from a human 3D skin model exposed to dermal fillers that mechanistically substantiate aforementioned adverse reactions, and thereby adds to the weight of evidence that these fillers may induce inflammatory and fibrotic responses.

Holzknecht J., Dubrac S., Hedtrich S., Galgóczy L., Marx F.

Candida albicans represents one of the most prevalent opportunistic fungal pathogens, causing superficial skin and mucosal infections in humans with certain predisposing health conditions and life-threatening systemic infections in immunosuppressed patients. The emerging drug resistance of this human-pathogenic yeast and the limited number of antifungal drugs for prevention and treatment of infections urgently demands the identification of new antifungal compounds with novel mechanisms of action.

Motter Catarino C., Kaiser K., Baltazar T., Motter Catarino L., Brewer J.R., Karande P.

A variety of human skin models have been developed for applications in regenerative medicine and efficacy studies. Typically, these employ matrix molecules that are derived from non-human sources along with human cells. Key limitations of such models include a lack of cellular and tissue microenvironment that is representative of human physiology for efficacy studies, as well as the potential for adverse immune responses to animal products for regenerative medicine applications. The use of recombinant extracellular matrix proteins to fabricate tissues can overcome these limitations. We evaluated animal- and non-animal-derived scaffold proteins and glycosaminoglycans for the design of biomaterials for skin reconstruction in vitro. Screening of proteins from the dermal-epidermal junction (collagen IV, laminin 5, and fibronectin) demonstrated that certain protein combinations when used as substrates increase the proliferation and migration of keratinocytes compared to the control (no protein). In the investigation of the effect of components from the dermal layer (collagen types I and III, elastin, hyaluronic acid, and dermatan sulfate), the primary influence on the viability of fibroblasts was attributed to the source of type I collagen (rat tail, human, or bovine) used as scaffold. Furthermore, incorporation of dermatan sulfate in the dermal layer led to a reduction in the contraction of tissues compared to the control where the dermal scaffold was composed primarily of collagen type I. This work highlights the influence of the composition of biomaterials on the development of complex reconstructed skin models that are suitable for clinical translation and in vitro safety assessment.

Dai M., Belaïdi J., Fleury G., Garanger E., Rielland M., Schultze X., Lecommandoux S.

Three-dimensional (3D) bioprinting offers a great alternative to traditional techniques in tissue reconstruction, based on seeding cells manually into a scaffold, to better reproduce organs' complexity. When a suitable bioink is engineered with appropriate physicochemical properties, such a process can advantageously provide a spatial control of the patterning that improves tissue reconstruction. The design of an adequate bioink must fulfill a long list of criteria including biocompatibility, printability, and stability. In this context, we have developed a bioink containing a precisely controlled recombinant biopolymer, namely, elastin-like polypeptide (ELP). This material was further chemoselectively modified with cross-linkable moieties to provide a 3D network through photopolymerization. ELP chains were additionally either functionalized with a peptide sequence Gly-Arg-Gly-Asp-Ser (GRGDS) or combined with collagen I to enable cell adhesion. Our ELP-based bioinks were found to be printable, while providing excellent mechanical properties such as stiffness and elasticity in their cross-linked form. Besides, they were demonstrated to be biocompatible, showing viability and adhesion of dermal normal human fibroblasts (NHF). Expressions of specific extracellular matrix (ECM) protein markers as pro-collagen I, elastin, fibrillin, and fibronectin were revealed within the 3D network containing cells after only 18 days of culture, showing the great potential of ELP-based bioinks for tissue engineering.