Reconstitution of structure and cell function in human skin grafts derived from cryopreserved allogeneic dermis and autologous cultured keratinocytes

Grafts of allogeneic dermis plus autologous epidermal cell cultures were used to replace extensively burned skin. Cryopreserved split-thickness cadaveric skin was grafted onto debrided burn wound, and autologous keratinocytes were cultured from uninjured donor sites. Several weeks later, allograft epidermis was abraded and replaced with the keratinocyte cultures. The final grafts were thus composites of autologous cultured epidermis and allogeneic dermis. In a case with 28 months follow-up, reconstitution of the dermal-epidermal (BMZ.1) and microvascular (BMZ.2) basement membrane zones was studied immunohistochemically and ultrastructurally. Immediately before grafting, thawed cryopreserved skin reacted with antibodies against laminin and type IV collagen in normal patterns. Twenty-nine days after grafting, BMZ.1 reacted weakly with both antibodies, and anticollagen type IV reactivity was absent from BMZ.2. Antilaminin reactivity of BMZ.2, however, was moderately intense, consistent with recent neovascularization. On day 29, the allograft epidermis was replaced with autologous keratinocyte cultures. Twenty-five days later (54 d after allografting), staining of both BMZs was intense with both antibodies. Ultrastructurally, at day 76 (47 d after culture placement) BMZ.1 revealed only small hemidesmosomes, few incipient anchoring fibrils, and a discontinuous lamina densa. BMZ.2, however, was fully reconstituted. By 124 d, both BMZs appeared normal. Observations in the dermis at 76 d included the presence of lymphocytes, organellar debris, and hyperactive collagen fibrillogenesis, all indicative of dermal remodelling. The microvasculature was well differentiated, but no elastic fibers or nerves were found. In the epidermis, melanocytes and evidence of melanosome transfer were seen at 5, 47, and 95 d after grafting of keratinocyte cultures. We conclude that the composite procedure reconstitutes skin with excellent textural and histologic qualities.     

Immunohistochemical studies on regeneration in cultured epidermal autografts used to treat full-thickness burn wounds

The normal pattern of healing following culture grafting is not well-established. The present study describes the immunohistochemcal expression of differentiation markers during various stages of wound healing after resurfacing full-thickness burn wounds with cultured epidermal autografts. For this purpose, biopsy specimens were obtained from six patients 6 days to 4-5 years after transplantation. A panel of monoclonal antibodies was used against various differentiation-specific protein markers, including cytokeratins, involucrin, transgluta-minase and filaggrin. Findings from early and late biopsies were compared with site-matched normal skin and cultured epidermal sheets. The persistence of cytokeratins 6 and 16, and to a lesser extent, the abnormal distribution of involucrin up to 4-5 years after resurfacing burns with cultured autografts was observed. These findings confirm previous studies–that the newly formed epidermis after culture grafting remains in a hyperproliferative state for a long time, which may be caused by the absence of a modulating dermal factor. Our results suggest that keratinocyte maturation following culture autografting does not return to normal for at least 4–5 years after healing of full-thickness burn wounds.     

Interleukin 6 indirectly induces keratinocyte migration

IL-6-deficient transgenic mice (IL-6 KO) display significantly delayed cutaneous wound healing. To further elucidate the role of IL-6 in skin wound healing, epidermal keratinocyte and dermal fibroblast cells were isolated from neonatal IL-6 KO mice and treated with rmIL-6. It was found that rmIL-6 alone did not significantly modulate the proliferation or migration of cultured IL-6 KO keratinocytes. rmIL-6, however, significantly induced the migration of IL-6 KO keratinocytes (up to 5-fold) when co-cultured with dermal fibroblasts. Culture supernatants from IL-6-treated fibroblasts were also found to induce the migration of keratinocytes to a similar degree. Genomics analysis of treated fibroblasts indicated that rmIL-6 does not induce any known soluble keratinocyte migratory factors. rmIL-6 treatment of fibroblast, however, induced a rapid and sustained phosphorylation of STAT3 protein. These data indicate that IL-6 could influence wound healing by inducing keratinocyte migration through the production of a soluble fibroblast-derived factor, and its activity may be associated with STAT3 activation.     

Liposome-encapsulated ursolic acid increases ceramides and collagen in human skin cells

Skin wrinkling and xerosis associated with aging result from decreases in dermal collagen and stratum corneum ceramide content. This study demonstrated that ursolic acid incorporated into liposomes (URA liposomes) increases both the ceramide content of cultured normal human epidermal keratinocytes (NHEK), and the collagen content of cultured normal human dermal fibroblasts. In addition, URA liposomes increased the ceramide content of the skin of human subjects, with increases in hydroxy ceramides occurring after only 3 days of treatment. Both URA liposomes and retinoic acid decreased markers of keratinocyte differentiation (keratin 1, keratin 10 and involucrin) in cultured NHEK. Thus, URA liposomes have effects on keratinocyte differentiation and dermal fibroblast collagen synthesis similar to those of retinoids. However, this study showed that URA liposomes increase ceramides in NHEK, in contrast to the decreases previously shown to be caused by retinoids. URA liposomes have the potential to be used alone or in combination with other agents to restore or maintain skin ceramide and collagen content.     

Treatment of leg ulcers with cryopreserved allogeneic cultured epithelium. A multicenter study.

BACKGROUND--In the past few years, several authors have described the usefulness of cultured allogeneic epidermal sheets in promoting wound healing of burns, leg ulcers, and donor sites. This study reports clinical results obtained by different departments in the treatment of chronic leg ulcers by cryopreserved cultured allogeneic epithelium. The freezing procedure and the assessment of viability of the cryopreserved epithelium are also described. A total of 30 ulcers were treated using 138 cryopreserved allografts. OBSERVATIONS--Twenty ulcers (66.6%) healed completely within 12 weeks. Four ulcers showed a 30% to 84.4% reduction in size by 3 weeks but did not heal completely; the remaining six ulcers did not show any improvement. A strong stimulation of granulation tissue formation and of reepithelialization from the wound edge were observed. RESULTS--The results indicate that frozen cultured epidermis, stored in a skin bank, is a valid and generally applicable alternative therapy for the treatment of chronic ulcers.     

Suprabasal induction of ornithine decarboxylase in adult mouse skin is sufficient to activate keratinocytes

To study the effects of de novo induction of ornithine decarboxylase (ODC) activity in adult, quiescent skin, we generated transgenic mice in which the suprabasal expression of an inducible form of the ODC protein fused to a modified estrogen receptor ligand-binding domain (ODCER) is driven by an involucrin promoter. After topical treatment with the inducing agent 4-hydroxytamoxifen (4OHT), ODC activity and putrescine levels were dramatically increased in the epidermis but not in the dermis of transgenic mice. 4OHT treatment stimulated both proliferation as measured by bromodeoxyuridine incorporation in basal epidermal cells and differentiation shown by increased expression of differentiation markers. Furthermore, induction of ODC activity did not rescue primary epidermal keratinocyte cultures isolated from ODCER2 mice from a calcium-triggered DNA synthesis block, as measured by [3H]thymidine incorporation. In vivo induction of epidermal ODC enzyme activity significantly stimulated the vascularization of ODCER transgenic skin. Increased expression of interleukin-1beta and keratin 6, markers of keratinocyte activation seen in wound healing, was also observed in 4OHT-treated transgenic skin. These results suggest that de novo suprabasal induction of ODC activity in adult mouse skin activates keratinocytes and stimulates vascularization in the dermal layer in a manner similar to skin undergoing wound healing.     

Human hair follicle dermal sheath and papilla cells support keratinocyte growth in monolayer coculture

Traditional skin grafting techniques are effective but limited methods of skin replacement. Autologous transplantation of rapidly cultured keratinocytes is successful for epidermal regeneration, but the current gold‐standard technique requires mouse fibroblast feeders and serum‐rich media, with serum‐free systems and dermal fibroblast (DF) feeders performing relatively poorly. Here, we investigated the capacity of human hair follicle dermal cells to act as alternative supports for keratinocyte growth. Dermal papilla (DP) dermal sheath (DS), DF and 3T3 cells were used as inactivated feeder cells for human keratinocyte coculture. Under conditions favouring dermal cells, proliferation of keratinocytes in the presence of either DS or DP cells was significantly enhanced compared with DF cells, at levels comparable to keratinocytes cultured under gold‐standard conditions. Secreted protein acidic and rich in cysteine (SPARC) expression increased DS and DP cells relative to DFs; however, further experiments did not demonstrate a role in keratinocyte support.     

Fetuin‐A promotes primary keratinocyte migration: independent of epidermal growth factor receptor signalling

Please cite this paper as: Fetuin‐A promotes primary keratinocyte migration: independent of epidermal growth factor receptor signalling. Experimental Dermatology 2010; 19 : e289–e292. Abstract: Previously, we reported that fetuin‐A is a major component of ovine foetal skin and significantly enhances ‘wound closure’ in primary keratinocyte cultures. In this study, we found that in human newborn foreskin, a high level of fetuin‐A protein is detected throughout the dermis. However, in adult skin a low level of fetuin‐A is observed throughout the epidermal and dermal layers, except at regions surrounding hair follicles and at the epidermal‐dermal junction where the level of fetuin‐A is relatively high. Fetuin‐A significantly induces actin‐rich protrusions in human primary keratinocytes. Interestingly, blockade of epidermal growth factor (EGF) receptor signalling has a limited effect on fetuin‐A promoted ‘wound closure’ on primary human keratinocytes, but significantly inhibits fetuin‐A’s effect on HaCaT cells. These results indicate that high levels of fetuin‐A may partially contribute to less scar formation in newborn foreskin and that the effect of fetuin‐A on primary keratinocyte migration is independent of EGF receptor signalling.     

Cutaneous wound healing: an update.

Our understanding of wound-healing mechanisms has progressed over the past decade. Wound healing is traditionally divided into three phases--the inflammatory phase, the proliferation phase, and the remodeling phase--and involves a well-orchestrated interaction among blood vessels (platelets, macrophages, neutrophils, endothelial cells, and smooth muscle cells), epidermis (keratinocytes, melanocytes, and Langerhans cells), adnexal structures (outer root sheath cells and hair dermal papilla cells), dermis (fibroblasts and myofibroblasts), nervous system (neurons), and subcutaneous fatty layers (adipocytes). We review recent discoveries of basic and clinical aspects of wound healing including several revolutions that occurred in wound management: occlusive dressing therapy, use of living skin equivalents, and topical administration of growth factors. As we previously proposed, the use of tissue substitutes and autologous epidermal sheets led to a new concept of skin grafting through the keratinocyte activation phase in the graft healing mechanism. In this review, we also discuss a representative patient who presented with plantar wounds caused by calcaneal osteomyelitis and healed by the coverage of epidermal grafting.     

Keratinocyte grafts for wound healing

Attempts by early investigators to culture human keratinocytes in vitro were hampered by fibroblast overgrowth in culture. In addition, under the culture conditions at the time, the keratinocytes rapidly differentiated and did not actively propagate.¹ In the 1960s tissue expiant cultures were used in attempts to obtain improved epidermal expansion. In this technique, a skin sample was placed with the dermal surface in contact with the culture dish to allow attachment and then incubated in culture medium. The epidermis grew down the side of the explant, attached to the dish, and then migrated outward to produce epithelial layers that could be used as allografts in animal models²; however, proliferation of epidermal cells using this method was insufficient to produce the large amounts of epidermis required for grafting.^3–5 In 1975 Rheinwald and Green described a significant advance in keratinocyte culture techniques that allowed serial subcultivation of human keratinocytes. Whole skin was trypsinized to produced a single-cell suspension of keratinocytes. These were then cultivated on a feeder layer of lethally irradiated 3T3 fibroblasts. The technique has been refined and modified so that it is now possible, starting with a skin sample of 1 cm², to expand the amount of cultured epithelium 10,000-fold to a size comparable to that of the adult body surface. Using the enzyme dispase, the epidermal sheet can be detached from the culture dish (Fig 1). A nonadherent dressing or vaseline gauze is then placed on the outer surface as a backing to facilitate handling of the graft^6–10 (Fig 2).An important advance in this methodology was the introduction of epidermal growth factor, which was a potent stimulator of keratinocyte yield in culture.¹⁰ In addition, many new keratinocyte culture techniques using a chemically more defined medium have been described by multiple groups.^11–18 From grafting experiments on nude mice, it was found that cultured epidermal cells regenerate a fully differentiated epidermis with normal architecture and a relatively complete pattern of keratinization including synthesis of high-molecular-weight keratins.¹⁹     

Epidermal cell confluence and implications for a two-step mechanism of wound closure

In contrast to freshly isolated cells, some cultured keratinocytes have the ability to adhere and spread in protein-free media. Reported here are experiments testing the hypothesis that the social history of keratinocytes influences their ability to spread in defined media. The experiments indicate that confluent cells lack the ability to spread in defined media while subconfluent cells have this property. The inability of dissociated confluent cells to spread in protein-free media is referred to phenomenologically as a "confluent block." The confluent block is acquired rapidly (1-3 days) and lost slowly (5-7 days). The ability of subconfluent cells to spread in the absence of media protein is sensitive to cycloheximide. Aortic endothelial cells and dermal fibroblasts do not demonstrate a confluent block. These observations are consonant with a two-step mechanism of epidermal wound repair: the first occurs immediately after wounding during which the cells require substratum-active proteins, and the second occurs 5-7 days later when the cells are able to synthesize their own substratum.     

Modulation of bullous pemphigoid antigens by gamma interferon in cultured human keratinocytes

We report the effects of human recombinant gamma interferon (gamma-IFN) on the expression of bullous pemphigoid (BP) antigens by human cultured keratinocytes. Secondary epidermal cell cultures were grown on 3T3 mouse fibroblasts; when confluent, some cultures were maintained in control medium while others were exposed to various concentrations of gamma-IFN (100, 200, 400 U/ml) for 14 days. The expression of BP antigens was analyzed by indirect immunofluorescence on epithelial sheets and immunoblotting of Tris, SDS, beta-mercaptoethanol culture extracts using different BP sera. Our results show that gamma-IFN alters the expression of BP antigens in a way varying according to the skin donor: we observed results ranging from complete loss and decreased expression to unchanged reactivity patterns. Thus, gamma-IFN modifies BP antigen expression; this behavior has been previously shown for other adhesion molecules such as fibronectin and thrombospondin. However, the observed variability of the expression of BP antigens according to the skin donor suggests an unexpected variability in keratinocyte sensitivity to gamma interferon, which remains to be explored both in vitro and in vivo.     

构建生长因子缓释功能的复合型组织工程皮肤替代物

目的设计制备具有缓释表皮生长因子(EGF)功能的复合型组织工程皮肤替代物并进行相关性能检测。方法在组织工程脱细胞真皮的研究基础上,利用天然材料制备凝胶状类似表皮结构的生物膜作为覆盖,并加载表皮生长因子缓释系统,构建具有促表皮愈合功能的复合型组织工程皮肤替代物。并对此皮肤替代物的形态学、体外释放性能以及实际疗效做以考察。结果缓释EGF的复合型组织工程皮肤替代物具有良好的形态学性能,生物相容性好,EGF在体外的释放可达14d以上。动物实验表明实验组创面愈合率在术后各时间点均高于对照组(P<0.05);组织学观察显示愈合创面的表皮愈合程度明显优于对照组。结论具有EGF缓释功能的复合型组织工程皮肤替代物能有效促进猪全层皮肤缺损的创面愈合,可作为具有应用前景的覆盖材料进一步研究。     

Recent advances in epidermal cell cultures

Summary Among the many skin culture systems, three have been selected in this short review because of their specific potentials in Dermatological research. H. Green cultures newborn human forsekin keratinocytes on a mouse 3T3 feeder layer. Keratinocytes grow and keratinize. The feeder cells release factor (s) which allows serial propagation of keratinocytes to be achieved. The cell yield is further increased by adding epidermal growth factor. This system has already proved to be a potent tool for the study of keratinization at the molecular level. A. Freeman has described a system in which explants of adult human skin are cultured on the dermal aspect of dead split-thickness pig skin. Keratinocytes can be passaged several times. Their differentiation is remarkable: it includes the production of keratohyaline, membrane coating granules, pemphigus as well as pemphigoid antigens. This system is interesting in the study of epidermal morphogenesis and may be applicable to the treatment of burns.The culture of epidermal cells from adult guinea pig ear in comparison with that of dermal fibroblasts is being used to study the specificity of action of pharmacological compounds on growth and keratinization of epidermal cells. Furthermore, the isolation (and culture) of pure populations of basal cells appears as a promising approach to the study of the mechanisms which moderate epidermal cell proliferation.     

A model system to analyse the ability of human keratinocytes to form hair follicles

Earlier studies showed that dermal cells lose trichogenic capacity with passage, but studies on the effect of keratinocyte passage on human hair follicle neogenesis and graft quality have been hampered by the lack of a suitable model system. We recently documented human hair follicle neogenesis in grafted dermal‐epidermal composites, and in the present study, we determined the effects of keratinocyte passage on hair follicle neogenesis. Dermal equivalents were made with cultured human dermal papilla cells and were overlaid with either primary or passaged human keratinocytes to form dermal‐epidermal composites; these were then grafted onto immunodeficient mice. Superior hair follicle neogenesis was observed using early keratinocyte cultures. Characteristics such as formation of hair shafts and sebaceous glands, presence of hair follicles with features of anagen or telogen follicles, and reproducible hair and skin function parameters make this model a tool to study human hair follicle neogenesis and development.     

Toxic epidermal necrolysis treated with intravenous high-dose immunoglobulins: our experience

BACKGROUND: Toxic epidermal necrolysis (TEN) is a rare severe acute exfoliative drug-induced skin disorder which has recently been ascribed to alterations in the control of keratinocyte apoptosis, mediated by an interaction between the cell surface death receptor Fas and its respective ligand. A therapeutic approach with intravenous immunoglobulins (IVIG) associated with pulse methylprednisolone, based on the inhibition of Fas-mediated keratinocyte death by naturally occurring Fas-blocking antibodies included in human immunoglobulin preparations, has produced good preliminary results. OBJECTIVE: To analyse the efficacy of IVIG in the treatment of TEN. Patients: Nine patients with erythematous body surface area ranging from 38 to 85% and dermo-epidermal detachment from 4 to 37% were treated. RESULTS: Eight patients were healed and 1 died of septic shock and multiple organ failure. Interruption of further epidermal detachment occurred after an average of 4.8 days from the onset of IVIG therapy. Complete wound healing occurred after an average of 12 days. Concerning complications, 3 out of 8 surviving patients had acute respiratory failure requiring mechanical ventilation and 1 acute renal failure was treated with dialysis. Late sequelae were limited to dyschromia and nail dystrophies. No hypertrophic scars were observed. CONCLUSION: IVIG therapy represents a safe and valid approach for TEN.     

Allogeneic cultured epidermal grafts heal chronic ulcers although they do not remain as proved by DNA analysis

To investigate whether allogeneic cultured keratinocytes are rejected or not, and to find out how beneficial their effect on wound healing could be, patients with chronic ulcers were grafted with allogeneic cultured human keratinocytes. In order to examine the epidermal origin of the healed wound, DNA analysis was performed and compared to donor and recipient blood-cell DNA. Healing was observed in 84% of the grafted ulcers by granulation tissue stimulation and would edge effect. In little time 60% of the grafted chronic ulcers healed completely. Although no rejection was observed, DNA analysis revealed that the grafted allogeneic keratinocytes were finally replaced by the patient's own epidermis. This study confirmed that cultured allogeneic keratinocytes that have been grafted on ulcers, play an important role in the wound healing process.     

Epidermal cells delivered for cutaneous wound healing

Re-epithelialization is the first and most important step in cutaneous wound healing. The vital role of epidermal cells, or keratinocytes, in accelerating wound healing has long been established. The technique of delivering the cultured and uncultured epidermal cells to the wound bed takes a variety of forms including cultured epithelial autografts (CEAs), tissue-engineered skin equivalent, epidermal suspension and microbead-loaded composite. These techniques, together with the keratinocyte culturing method and scaling up equipment, are still the ongoing research. Application of these techniques also bears direct impact on the outcome of the wounded patients. Best understanding of the delivery technique and its relationship with the culturing method and delivery vehicle could benefit not only the wounded patient but also the development of tissue-engineered skin equivalent.     

Tissue-Engineered Skin

Tissue-engineered skin is a significant advance in the field of wound healing and was developed due to limitations associated with the use of autografts. These limitations include the creation of a donor site which is at risk of developing pain, scarring, infection and/or slow healing. A number of products are commercially available and many others are in development. Cultured epidermal autografts can provide permanent coverage of large area from a skin biopsy. However, 3 weeks are needed for graft cultivation. Cultured epidermal allografts are available immediately and no biopsy is necessary. They can be cryopreserved and banked, but are not currently commercially available. A nonliving allogeneic acellular dermal matrix with intact basement membrane complex (Alloderm®) is immunologically inert. It prepares the wound bed for grafting allowing improved cultured allograft ‘take’ and provides an intact basement membrane. A nonliving extracellular matrix of collagen and chondroitin-6-sulfate with silicone backing (Integra®) serves to generate neodermis. A collagen and glycosaminoglycan dermal matrix inoculated with autologous fibroblasts and keratinocytes has been investigated but is not commercially available. It requires 3 to 4 weeks for cultivation. Dermagraft® consists of living allogeneic dermal fibroblasts grown on degradable scaffold. It has good resistance to tearing. An extracellular matrix generated by allogeneic human dermal fibroblasts (TransCyte?) serves as a matrix for neodermis generation. Apligraf® is a living allogeneic bilayered construct containing keratinocytes, fibroblasts and bovine type I collagen. It can be used on an outpatient basis and avoids the need for a donor site wound. Another living skin equivalent, composite cultured skin (OrCel?), consists of allogeneic fibroblasts and keratinocytes seeded on opposite sides of bilayered matrix of bovine collagen. There are limited clinical data available for this product, but large clinical trials are ongoing. Limited data are also available for 2 types of dressing material derived from pigs: porcine small intestinal submucosa acellular collagen matrix (Oasis?) and an acellular xenogeneic collagen matrix (E-Z-Derm?). Both products have a long shelf life. Other novel skin substitutes are being investigated. The potential risks and benefits of using tissue-engineered skin need to be further evaluated in clinical trials but it is obvious that they offer a new option for the treatment of wounds.     

A graft model for hair development

Abstract:  Follicular cell implantation (FCI) is an experimental cell therapy for the treatment of hair loss that uses cultured hair follicle cells to induce new hair formation. This treatment is based on the demonstration that adult dermal papilla cells (DPC) retain the hair inductive capacity they acquired during hair morphogenesis in the embryo. For FCI, hair inductive cells are isolated from scalp biopsies and then propagated in culture in order to provide enough cells to generate many new follicles from a few donor follicles. Following expansion in culture, the cells are implanted into the scalp where they induce the formation of new follicles. Because the process relies on the ability to retain the potential for hair induction during the expansion of DPC in culture, we sought a consistent, reliable and easily performed in vivo assay in which to test hair induction. In this study, we describe a simple graft model that supports hair morphogenesis. The assay combines dermal cells with embryonic mouse epidermis that provides the keratinocyte component of induced follicles. The grafts are placed under a protective skin flap in the host athymic mouse where the cells will form a skin graft with hair if the dermal cells are hair inductive DPC. Using the assay, freshly isolated and cultured mouse embryo dermal cells as well as cultured dermal papilla cells from other species all induced hair formation. The induced hairs were aesthetically indistinguishable from those of the epidermal donor in length, thickness, and pigmentation, and they were histologically normal.