Reconstructed human keloid models show heterogeneity within keloid scars.

Keloid scars are often described as having an actively growing peripheral margin with a regressing centre. The aim of this study was to examine the possible heterogeneity within keloids and the involvement of different regions within and around keloid scars in the pathogenesis, using an in vitro keloid scar model. In vitro skin models were constructed from keratinocytes and fibroblasts from normal skin and different regions within and around keloid scars: periphery, centre, and (adjacent) surrounding-normal-skin regions. Additionally, fibroblasts were isolated from the superficial-central and deep-central regions of the keloid and combined with central keratinocytes. All keloid regions showed increased contraction compared to normal skin models, particularly in central regions. Myofibroblasts were present in all keloid regions but were more abundant in models containing central-deep keloid fibroblasts. Secretion of anti-fibrotic HGF and extracellular matrix collagen IV gene expression was reduced in the central deep keloid compared to normal skin. No significant differences between peripheral and central regions within keloids were observed for inflammatory cytokine CCL20, CCL27, CXCL8, IL-6 and IL-18 secretion. Parameters for surrounding-normal-skin showed similarities to both non-lesional normal skin and keloids. In conclusion, a simple but elegant method of culturing keloid-derived keratinocytes and fibroblasts in an organotypic 3D scar model was developed, for the dual purpose of studying the underlying pathology and ultimately testing new therapeutics. In this study, these tissue engineered scar models show that the central keloid region shows a more aggressive keloid scar phenotype than the periphery and that the surrounding-normal-skin also shares certain abnormalities characteristic for keloids.

Characterization of In Vitro Reconstructed Human Normotrophic, Hypertrophic, and Keloid Scar Models.

To understand scar pathology, develop new drugs, and provide a platform for personalized medicine, physiologically relevant human scar models are required, which are characteristic of different scar pathologies. Hypertrophic scars and keloids are two types of abnormal scar resulting from unknown abnormalities in the wound healing process. While they display different clinical behavior, differentiation between the two can be difficult-which in turn means that it is difficult to develop optimal therapeutic strategies. The aim of this study was to develop in vitro reconstructed human hypertrophic and keloid scar models and compare these to normotrophic scar and normal skin models to identify distinguishing biomarkers. Keratinocytes and fibroblasts from normal skin and scar types (normotrophic, hypertrophic, keloid) were used to reconstruct skin models. All skin models showed a reconstructed differentiated epidermis on a fibroblast populated collagen-elastin matrix. Both abnormal scar types showed increased contraction, dermal thickness, and myofibroblast staining compared to normal skin and normotrophic scar. Notably, the expression of extracellular matrix associated genes showed distinguishing profiles between all scar types and normal skin (hyaluronan synthase-1, matrix-metalloprotease-3), between keloid and normal skin (collagen type IV), between normal scar and keloid (laminin α1), and between keloid and hypertrophic scar (matrix-metalloprotease-1, integrin α5). Also, inflammatory cytokine and growth factor secretion (CCL5, CXCL1, CXCL8, CCL27, IL-6, HGF) showed differential secretion between scar types. Our results strongly suggest that abnormal scars arise from different pathologies rather than simply being on different ends of the scarring spectrum. Furthermore, such normal skin and scar models together with biomarkers, which distinguish the different scar types, would provide an animal free, physiologically relevant scar diagnostic and drug testing platform for the future.

Comparison of advanced therapy medicinal product gingiva and skin substitutes and their in vitro wound healing potentials.

Skin and oral mucosa substitutes are a therapeutic option for closing hard-to-heal skin and oral wounds. Our aim was to develop bi-layered skin and gingiva substitutes, from 3 mm diameter biopsies, cultured under identical conditions, which are compliant with current European regulations for advanced therapy medicinal products. We present in vitro mode of action methods to (i) determine viability: epithelial expansion, proliferation (Ki-67), metabolic activity (MTT assay); (ii) characterize skin and gingiva substitutes: histology and immunohistochemistry; and (iii) determine potency: soluble wound healing mediator release (enzyme-linked immunosorbent assay). Both skin and gingiva substitutes consist of metabolically active autologous reconstructed differentiated epithelium expanding from the original biopsy sheet on a fibroblast populated connective tissue matrix (donor dermis). Gingival epithelium expanded 1.7-fold more than skin epithelium during the 3 week culture period. The percentage of proliferating Ki-67-positive cells located in the basal layer of the gingiva substitute was >1.5-fold higher than in the skin substitute. Keratins 16 and 17, which are upregulated during normal wound healing, were expressed in both the skin and gingiva substitutes. Notably, the gingiva substitute secreted higher amounts of key cytokines involved in mitogenesis, motogenesis and chemotaxis (interleukin-6 > 23-fold, CXCL8 > 2.5-fold) as well as higher amounts of the anti-fibrotic growth factor, hepatocyte growth factor (>7-fold), compared with the skin substitute. In conclusion, while addressing the viability, characterization and potency of the tissue substitutes, important intrinsic differences between skin and gingiva were discovered that may explain in part the superior quality of wound healing observed in the oral mucosa compared with skin.

Methods to study differences in cell mobility during skin wound healing in vitro.

Wound healing events which occur in humans are difficult to study in animals due to differences in skin physiology. Furthermore there are increasing restrictions in Europe for using animals for testing the therapeutic properties of new compounds. Therefore, in line with the 3Rs (reduction, refinement and replacement of test animals), a number of human in vitro models of different levels of complexity have been developed to investigate cell mobility during wound healing. Keratinocyte, melanocyte, fibroblast and endothelial cell mobility are described, since these are the residential cells which are responsible for restoring the main structural features of the skin. A monolayer scratch assay is used to study random fibroblast and endothelial cell migration in response to EGF and bFGF respectively and a chemotactic assay is used to study directional fibroblast migration towards CCL5. In order to study endothelial sprouting in response to bFGF or VEGF, which involves continuous degradation and resynthesis of a 3D matrix, a fibrin gel is used. Human physiologically relevant tissue-engineered skin models are used to investigate expansion of the stratified, differentiated epidermis (keratinocytes and melanocytes) over a fibroblast populated dermis and also to study migration and distribution of fibroblasts into the dermis. Together these skin models provide a platform for testing the mode of action of novel compounds for enhanced and scar free wound healing.

Differential response of human adipose tissue-derived mesenchymal stem cells, dermal fibroblasts, and keratinocytes to burn wound exudates: potential role of skin-specific chemokine CCL27.

Many cell-based regenerative medicine strategies toward tissue-engineered constructs are currently being explored. Cell-cell interactions and interactions with different biomaterials are extensively investigated, whereas very few studies address how cultured cells will interact with soluble wound-healing mediators that are present within the wound bed after transplantation. The aim of this study was to determine how adipose tissue-derived mesenchymal stem cells (ASC), dermal fibroblasts, and keratinocytes will react when they come in contact with the deep cutaneous burn wound bed. Burn wound exudates isolated from deep burn wounds were found to contain many cytokines, including chemokines and growth factors related to inflammation and wound healing. Seventeen mediators were identified by ELISA (concentration range 0.0006-9 ng/mg total protein), including the skin-specific chemokine CCL27. Burn wound exudates activated both ASC and dermal fibroblasts, but not keratinocytes, to increase secretion of CXCL1, CXCL8, CCL2, and CCL20. Notably, ASC but not fibroblasts or keratinocytes showed significant increased secretion of vascular endothelial growth factor (5-fold) and interleukin-6 (253-fold), although when the cells were incorporated in bi-layered skin substitute (SS) these differences were less pronounced. A similar discrepancy between ASC and dermal fibroblast mono-cultures was observed when recombinant human-CCL27 was used instead of burn wound exudates. Although CCL27 did not stimulate the secretion of any of the wound-healing mediators by keratinocytes, these cells, in contrast to ASC or dermal fibroblasts, showed increased proliferation and migration. Taken together, these results indicate that on transplantation, keratinocytes are primarily activated to promote wound closure. In contrast, dermal fibroblasts and, in particular, ASC respond vigorously to factors present in the wound bed, leading to increased secretion of angiogenesis/granulation tissue formation factors. Our findings have implications for the choice of cell type (ASC or dermal fibroblast) to be used in regenerative medicine strategies and indicate the importance of taking into account interactions with the wound bed when developing advanced therapies for difficult-to-close cutaneous wounds.

CCL5 and CCL20 mediate immigration of Langerhans cells into the epidermis of full thickness human skin equivalents.

Epidermal Langerhans cells (LC) play a key role in initiation and regulation of immune responses. Whereas LC migration out of the epidermis upon environmental assault is extensively studied, the mechanisms involved in the (re)population of the epidermis with LC are poorly understood. Here, we investigated the immigration of LC derived from the human MUTZ-3 cell line (MUTZ-LC) into the epidermis of a full thickness skin equivalent, comprising a fully differentiated epidermis on a fibroblast-populated dermis. MUTZ-LC were used to determine which epidermis-derived chemokines play a role in mediating LC trans-dermal migration into the epidermis. We found evidence for a role of keratinocyte-derived CCL5 and CCL20 in the chemo-attraction of MUTZ-LC. Neutralizing antibodies against CCL5 and CCL20 blocked LC migration towards keratinocytes. Secretion of these two chemokines was associated with incorporation of MUTZ-LC into the epidermis of full thickness skin equivalents. In conclusion, our findings suggest that epidermis derived CCL5 and CCL20 are pivotal mediators in recruitment of LC into the epidermis.

Irritant-induced migration of Langerhans cells coincides with an IL-10-dependent switch to a macrophage-like phenotype.

Langerhans cells (LCs) migrate after topical exposure of the skin to irritants, despite the supposed independence of irritant contact dermatitis from adaptive immunity. Whereas allergen-activated LCs are known to migrate to the draining lymph nodes (LNs), the fate of migrated LCs upon topical irritant exposure is unknown. Here, we identified a phenotypic switch of LCs after their migration into the dermis upon irritant exposure. With the aid of ex vivo intact human skin and epidermal sheets, we show that dermal fibroblasts are necessary for an IL-10-dependent postmigrational phenotypic switch of LCs into macrophage-like cells. Exposure of ex vivo skin to a panel of seven irritants resulted in a decrease in the number of CD1a(+) cells and an increase in CD14(+)/CD68(+) cells in the dermis. Neutralizing antibodies against IL-10 totally inhibited the phenotypic LC-to-macrophage transition, but did not influence the migration of CD1a(+) cells. Exposure of epidermal sheets to irritants resulted in a fibroblast-dependent LC-to-CD14(+)/CD68(+) switch coinciding with migration, which could be totally inhibited by neutralizing antibodies against either IL-10 or CCL2/CCL5 (two chemokines responsible for epidermal-to-dermal migration). We have thus identified an IL-10-dependent phenotypic switch of LCs into macrophage-like cells upon irritant exposure and emigration from the epidermis.

Use of a collagen-elastin matrix as transport carrier system to transfer proliferating epidermal cells to human dermis in vitro.

This in vitro study describes a novel cell culture, transport, and transfer protocol that may be highly suitable for delivering cultured proliferating keratinocytes and melanocytes to large open skin wounds (e.g., burns). We have taken into account previous limitations identified using other keratinocyte transfer techniques, such as regulatory issues, stability of keratinocytes during transport (single cell suspensions undergo terminal differentiation), ease of handling during application, and the degree of epidermal blistering resulting after transplantation (both related to transplanting keratinocyte sheets). Large numbers of proliferating epidermal cells (EC) (keratinocytes and melanocytes) were generated within 10-14 days and seeded onto a three-dimensional matrix composed of elastin and collagen types I, III, and V (Matriderm®), which enabled easy and stable transport of the EC for up to 24 h under ambient conditions. All culture conditions were in accordance with the regulations set by the Dutch Central Committee on Research Involving Human Subjects (CCMO). As an in vitro model system for clinical in vivo transfer, the EC were then transferred from Matriderm onto human acellular dermis during a period of 3 days. After transfer the EC maintained the ability to regenerate into a fully differentiated epidermis containing melanocytes on the human dermis. Proliferating keratinocytes were located in the basal layer and keratin-10 expression was located in differentiating suprabasal layers similar to that found in human epidermis. No blistering was observed (separation of the epidermis from the basement membrane). Keratin-6 expression was strongly upregulated in the regenerating epidermis similar to normal wound healing. In summary, we show that EC-Matriderm contains viable, metabolically active keratinocytes and melanocytes cultured in a manner that permits easy transportation and contains epidermal cells with the potential to form a pigmented reconstructed epidermis. This in vitro study has produced a robust protocol that is ready for clinical studies in the future.

Wound-healing factors secreted by epidermal keratinocytes and dermal fibroblasts in skin substitutes.

Full-skin substitutes, epidermal substitutes, and dermal substitutes are currently being used to heal deep burns and chronic ulcers. In this study, we investigated which wound-healing mediators are released from these constructs and whether keratinocyte-fibroblast interactions are involved. Autologous skin substitutes were constructed from human keratinocytes, fibroblasts, and acellular donor dermis. Full-thickness skin was used to represent an autograft. Secretion of wound-healing mediators was investigated by means of protein array, enzyme-linked immunosorbent assay, neutralizing antibodies, and conditioned culture supernatants. Full-skin substitutes and autografts produce high amounts of inflammatory/angiogenic mediators (IL-6, CCL2, CXCL1, CXCL8, and sST2). Epidermal and dermal substitutes produced less of these proteins. Epidermal-derived proinflammatory cytokines interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) were found to mediate synergistically the secretion of these wound-healing mediators (with the exception of sST2) from fibroblasts in dermal substitutes. The secretion of proinflammatory cytokines (IL-1alpha, TNF-alpha), chemokine/mitogen (CCL5) and angiogenic factor (vascular endothelial growth factor) by epidermal substitutes and tissue remodeling factors (tissue inhibitor of metalloproteinase-2, hepatocyte growth factor) by dermal substitutes was not influenced by keratinocyte-fibroblast interactions. The full-skin substitute has a greater potential to stimulate wound healing than epidermal or dermal substitutes. Both epidermal-derived IL-1alpha and TNF-alpha are required to trigger the release of dermal-derived inflammatory/angiogenic mediators from skin substitutes.

Culture of keratinocytes for transplantation without the need of feeder layer cells.

Patients with large burn wounds have a limited amount of healthy donor skin. An alternative for the autologous skin graft is transplantation with autologous keratinocytes. Conventionally, the keratinocytes are cultured with mouse feeder layer cells in medium containing fetal calf serum (FCS) to obtain sufficient numbers of cells. These xenobiotic materials can be a potential risk for the patient. The aim of the present study was to investigate if keratinocytes could be expanded in culture without the need of a feeder layer and FCS. Keratinocytes were cultured on tissue culture plastic with or without collagen type IV coating in medium containing Ultroser G (serum substitute) and keratinocyte growth factor (KGF). An in vitro skin equivalent model was used to examine the capacity of these cells to form an epidermis. Keratinocytes in different passages (P2, P4, and P6) and freshly isolated cells were studied. Keratinocytes grown on collagen type IV were able to form an epidermis at higher passage numbers than cells grown in the absence of collagen type IV (P4 and P2, respectively). In both cases the reconstructed epidermis showed an increased expression of Ki-67, SKALP, involucrin, and keratin 17 compared to normal skin. Only 50,000 keratinocytes grown on collagen type IV in P4 were needed to form 1 cm2 epidermis, whereas 150,000 of freshly isolated keratinocytes were necessary. Using this culture technique sufficient numbers of keratinocytes, isolated from 1 cm2 skin, were obtained to cover 400 cm2 of wound surface in 2 weeks. The results show that keratinocytes can be cultured without the need of a fibroblast feeder layer and FCS and that these cells are still able to create a fully differentiated epidermis. This culture technique can be a valuable tool for the treatment of burn wounds and further development of tissue engineered skin.

Culture of Keratinocytes for Transplantation without the Need of Feeder Layer Cells.

Patients with large burn wounds have a limited amount of healthy donor skin. An alternative for the autologous skin graft is transplantation with autologous keratinocytes. Conventionally, the keratinocytes are cultured with mouse feeder layer cells in medium containing fetal calf serum (FCS) to obtain sufficient numbers of cells. These xenobiotic materials can be a potential risk for the patient. The aim of the present study was to investigate if keratinocytes could be expanded in culture without the need of a feeder layer and FCS. Keratinocytes were cultured on tissue culture plastic with or without collagen type IV coating in medium containing Ultroser G (serum substitute) and keratinocyte growth factor (KGF). An in vitro skin equivalent model was used to examine the capacity of these cells to form an epidermis. Keratinocytes in different passages (P2, P4, and P6) and freshly isolated cells were studied. Keratinocytes grown on collagen type IV were able to form an epidermis at higher passage numbers than cells grown in the absence of collagen type IV (P4 and P2, respectively). In both cases the reconstructed epidermis showed an increased expression of Ki-67, SKALP, involucrin, and keratin 17 compared to normal skin. Only 50,000 keratinocytes grown on collagen type IV in P4 were needed to form 1 cm2 epidermis, whereas 150,000 of freshly isolated keratinocytes were necessary. Using this culture technique sufficient numbers of keratinocytes, isolated from 1 cm2 skin, were obtained to cover 400 cm2 of wound surface in 2 weeks. The results show that keratinocytes can be cultured without the need of a fibroblast feeder layer and FCS and that these cells are still able to create a fully differentiated epidermis. This culture technique can be a valuable tool for the treatment of burn wounds and further development of tissue engineered skin.