A new model for preclinical testing of dermal substitutes for human skin reconstruction.

BACKGROUND: Currently, acellular dermal substitutes used for skin reconstruction are usually covered with split-thickness skin grafts. The goal of this study was to develop an animal model in which such dermal substitutes can be tested under standardized conditions using a bioengineered dermo-epidermal skin graft for coverage. METHODS: Bioengineered grafts consisting of collagen type I hydrogels with incorporated human fibroblasts and human keratinocytes seeded on these gels were produced. Two different dermal substitutes, namely Matriderm(®), and an acellular collagen type I hydrogel, were applied onto full-thickness skin wounds created on the back of immuno-incompetent rats. As control, no dermal substitute was used. As coverage for the dermal substitutes either the bioengineered grafts were used, or, as controls, human split-thickness skin or neonatal rat epidermis were used. Grafts were excised 21 days post-transplantation. Histology and immunofluorescence was performed to investigate survival, epidermis formation, and vascularization of the grafts. RESULTS: The bioengineered grafts survived on all tested dermal substitutes. Epidermis formation and vascularization were comparable to the controls. CONCLUSION: We could successfully use human bioengineered grafts to test different dermal substitutes. This novel model can be used to investigate newly designed dermal substitutes in detail and in a standardized way.

Cryptic epitopes induce high-titer humoral immune response in patients with cancer.

In search of novel markers for diagnosis, prognosis, and therapy of cancer, screening of rcDNA expression libraries with patient's sera has been established as a valuable tool for identification of cancer-specific Ags. Interestingly, besides the expected humoral responses to annotated proteins, patients with cancer were frequently found to have serum Abs that bind to peptides without homology to known proteins. So far, the nature of these unconventional epitopes and their possible significance in tumor immunology have never been thoroughly investigated. In our study, we specifically analyzed humoral immune response toward such peptides in patients with pancreatic or breast cancer using yeast-displayed cDNA expression libraries derived from tumor tissue. A detailed analysis of the identified peptides revealed that they originated from translation of sequences outside annotated open reading frames and may derive from the use of alternative start codons or from DNA indel mutations. In several cases, the corresponding mRNA templates have a known association with cancer. In a final analysis, we were able to detect one of these tumor Ags in cancer tissue arrays by a selected Fab-Ab. We conclude that cryptic epitopes may elicit specific humoral immune responses in patients with cancer and thus play a role in immunologic surveillance. Due to the high prevalence of immune responses against some of the peptides, they may also be valuable markers for cancer diagnosis, prognosis, or therapy monitoring.

Skingineering I: engineering porcine dermo-epidermal skin analogues for autologous transplantation in a large animal model.

BACKGROUND: Extended full thickness skin defects still represent a considerable therapeutic challenge as ideal strategies for definitive autologous coverage are still not available. Tissue engineering of whole skin represents an equally attractive and ambitious novel approach. We have recently shown that laboratory-grown human skin analogues with near normal skin anatomy can be successfully transplanted on immuno-incompetent rats. The goal of the present study was to engineer autologous porcine skin grafts for transplantation in a large animal model (pig study = intended preclinical study). MATERIALS AND METHODS: Skin biopsies were taken from the pig's abdomen. Epidermal keratinocytes and dermal fibroblasts were isolated and then expanded on culture dishes. Subsequently, highly concentrated collagen hydrogels and collagen/fibrin hydrogels respectively, both containing dermal fibroblasts, were prepared. Fibroblast survival, proliferation, and morphology were monitored using fluorescent labelling and laser scanning confocal microscopy. Finally, keratinocytes were seeded onto this dermal construct and allowed to proliferate. The resulting in vitro generated porcine skin substitutes were analysed by H&E staining and immunofluorescence. RESULTS: Dermal fibroblast proliferation and survival in pure collagen hydrogels was poor. Also, the cells were mainly round-shaped and they did not develop 3D-networks. In collagen/fibrin hydrogels, dermal fibroblast survival was significantly higher. The cells proliferated well, were spindle-shaped, and formed 3D-networks. When these latter dermal constructs were seeded with keratinocytes, a multilayered and partly stratified epidermis readily developed. CONCLUSION: This study provides compelling evidence that pig cell-derived skin analogues with near normal skin anatomy can be engineered in vitro. These tissue-engineered skin substitutes are needed to develop a large animal model to establish standardized autologous transplantation procedures for those studies that must be conducted before "skingineering" can eventually be clinically applied.

Skingineering II: transplantation of large-scale laboratory-grown skin analogues in a new pig model.

BACKGROUND: Tissue engineering of skin with near-normal anatomy is an intriguing novel strategy to attack the still unsolved problem of how to ideally cover massive full-thickness skin defects. After successful production of large, pig cell-derived skin analogues, we now aim at developing an appropriate large animal model for transplantation studies. MATERIALS AND METHODS: In four adult Swiss pigs, full-thickness skin defects, measuring 7.5 × 7.5 cm, were surgically created and then shielded against the surrounding skin by a new, self-designed silicone chamber. In two animals each, Integra dermal regeneration templates or cultured autologous skin analogues, respectively, were applied onto the wound bed. A sophisticated shock-absorbing dressing was applied for the ensuing 3 weeks. Results were documented photographically and histologically. RESULTS: All animals survived uneventfully. Integra healed in perfectly, while the dermo-epidermal skin analogues showed complete take of the dermal compartment but spots of missing epidermis. The chamber proved effective in precluding ("false positive") healing from the wound edges and the special dressing efficiently kept the operation site intact and clean for the planned 3 weeks. CONCLUSION: We present a novel and valid pig model permitting both transplantation of large autologous, laboratory-engineered skin analogues and also keeping the site of intervention undisturbed for at least three postoperative weeks. Hence, the model will be used for experiments testing whether such large skin analogues can restore near-normal skin, particularly in the long term. If so, clinical application can be envisioned.

The tumor suppressor cybL, a component of the respiratory chain, mediates apoptosis induction.

A genetic screen was established to clone apoptosis-inducing genes in a high-throughput format. It led to the isolation of several proapoptotic genes whose proteins are localized to mitochondria. One of the isolated genes is cytochrome bL (cybL also known as SDHC, CII-3, or QPs-1), a component of the respiratory chain complex II. It was further investigated because both cybL and another component of complex II, cybS, have recently been identified as tumor suppressor proteins, some of which act by controlling apoptosis. Our studies reveal that cell death induction by cybL expression is concomitant with a transient inhibition of complex II and the generation of reactive oxygen species. Importantly, cells that are constitutively deficient in cybL are resistant to a variety of proapoptotic cytostatic drugs and to the effects of the Fas receptor. Our results therefore identify complex II as a sensor for apoptosis induction and could explain the unexpected observation that complex II is inactivated in tumors.

Collagen hydrogels strengthened by biodegradable meshes are a basis for dermo-epidermal skin grafts intended to reconstitute human skin in a one-step surgical intervention.

Extensive full-thickness skin loss, associated with deep burns or other traumata, represents a major clinical problem that is far from being solved. A promising approach to treat large skin defects is the use of tissue-engineered full-thickness skin analogues with nearly normal anatomy and function. In addition to excellent biological properties, such skin substitutes should exhibit optimal structural and mechanical features. This study aimed to test novel dermo-epidermal skin substitutes based on collagen type I hydrogels, physically strengthened by two types of polymeric net-like meshes. One mesh has already been used in clinical trials for treating inguinal hernia; the second one is new but consists of a FDA-approved polymer. Both meshes were integrated into collagen type I hydrogels and dermo-epidermal skin substitutes were generated. Skin substitutes were transplanted onto immuno-incompetent rats and analyzed after distinct time periods. The skin substitutes homogeneously developed into a well-stratified epidermis over the entire surface of the grafts. The epidermis deposited a continuous basement membrane and dermo-epidermal junction, displayed a well-defined basal cell layer, about 10 suprabasal strata and a stratum corneum. Additionally, the dermal component of the grafts was well vascularized.

De novo epidermal regeneration using human eccrine sweat gland cells: higher competence of secretory over absorptive cells.

In our previous work, we showed that human sweat gland-derived epithelial cells represent an alternative source of keratinocytes to grow a near normal autologous epidermis. The role of subtypes of sweat gland cells in epidermal regeneration and maintenance remained unclear. In this study, we compare the regenerative potential of both secretory and absorptive sweat gland cell subpopulations. We demonstrate the superiority of secretory over absorptive cells in forming a new epidermis on two levels: first, the proliferative and colony-forming efficiencies in vitro are significantly higher for secretory cells (SCs), and second, SCs show a higher frequency of successful epidermis formation as well as an increase in the thickness of the formed epidermis in the in vitro and in vivo functional analyses using a 3D dermo-epidermal skin model. However, the ability of forming functional skin substitutes is not limited to SCs, which supports the hypothesis that multiple subtypes of sweat gland epithelial cells hold regenerative properties, while the existence and exact localization of a keratinocyte stem cell population in the human eccrine sweat gland remain elusive.

Human eccrine sweat gland cells turn into melanin-uptaking keratinocytes in dermo-epidermal skin substitutes.

Recently, Biedermann et al. (2010) have demonstrated that human eccrine sweat gland cells can develop a multilayered epidermis. The question still remains whether these cells can fulfill exclusive and very specific functional properties of epidermal keratinocytes, such as the incorporation of melanin, a feature absent in sweat gland cells. We added human melanocytes to eccrine sweat gland cells to let them develop into an epidermal analog in vivo. The interaction between melanocytes and sweat gland-derived keratinocytes was investigated. The following results were gained: (1) macroscopically, a pigmentation of the substitutes was seen 2-3 weeks after transplantation; (2) we confirmed the development of a multilayered, stratified epidermis with melanocytes distributed evenly throughout the basal layer; (3) melanocytic dendrites projected to suprabasal layers; and (4) melanin was observed to be integrated into former eccrine sweat gland cells. These skin substitutes were similar or equal to skin substitutes cultured from human epidermal keratinocytes. The only differences observed were a delay in pigmentation and less melanin uptake. These data suggest that eccrine sweat gland cells can form a functional epidermal melanin unit, thereby providing striking evidence that they can assume one of the most characteristic keratinocyte properties.

Modified plastic compression of collagen hydrogels provides an ideal matrix for clinically applicable skin substitutes.

Tissue engineering of clinically applicable dermo-epidermal skin substitutes is crucially dependent on the three-dimensional extracellular matrix, supporting the biological function of epidermal and dermal cells. This matrix essentially determines the mechanical stability of these substitutes to allow for safe and convenient surgical handling. Collagen type I hydrogels yield excellent biological functionality, but their mechanical weakness and their tendency to contract and degrade does not allow producing clinically applicable transplants of larger sizes. We show here that plastically compressed collagen type I hydrogels can be produced in clinically relevant sizes (7×7 cm), and can be safely and conveniently handled by the surgeon. Most importantly, these dermo-epidermal skin substitutes mature into a near normal skin that can successfully reconstitute full-thickness skin defects in an animal model.

Human eccrine sweat gland cells can reconstitute a stratified epidermis.

Eccrine sweat glands are generally considered to be a possible epidermal stem cell source. Here we compared the multilayered epithelia formed by epidermal keratinocytes and those formed by eccrine sweat gland cells. We demonstrated both in vitro and in vivo the capability of human eccrine sweat gland cells to form a stratified interfollicular epidermis substitute on collagen hydrogels. This is substantiated by the following findings: (1) a stratified epidermis consisting of 10-12 cell layers is formed by sweat gland cells; (2) a distinct stratum corneum develops and is maintained after transplantation onto immuno-incompetent rats; (3) proteins such as filaggrin, loricrin, involucrin, envoplakin, periplakin, and transglutaminases I and III match with the pattern of the normal human skin; (4) junctional complexes and hemidesmosomes are readily and regularly established; (5) cell proliferation in the basal layer reaches homeostatic levels; (6) the sweat gland-derived epidermis is anchored by hemidesmosomes within a well-developed basal lamina; and (7) palmo-plantar or mucosal markers are not expressed in the sweat gland-derived epidermis. These data suggest that human eccrine sweat glands are an additional source of keratinocytes that can generate a stratified epidermis. Our findings raise the question of the extent to which the human skin is repaired and/or permanently renewed by eccrine sweat gland cells.

Formation of human capillaries in vitro: the engineering of prevascularized matrices.

Initial take, development, and function of transplanted engineered tissue substitutes are crucially dependent on rapid and adequate blood perfusion. Therefore, the development of rapidly and efficiently vascularized tissue grafts is vital for tissue engineering and regenerative medicine. Here we report on the construction of a network of highly organotypic capillaries in engineered tissue substitutes. We employed a three-dimensional culture system consisting of human microvascular endothelial cells. These were reproducibly expanded at high purity and subsequently seeded into biodegradable, fibrin-based hydrogels. The process of capillary formation in vitro followed the principles of both angiogenesis and postnatal vasculogenesis and a distinct sequence of other developmental steps that closely resemble embryonic neovascularization. Capillary lumen formation in vitro was initiated by the deposition of a basement membrane and intensive pinocytosis, followed by the generation of intracellular vacuoles, successive fusion of these vacuoles, and finally the formation of a long, continuous lumen. After transplantation the vascular structures were stabilized by mural cells of the recipient animal. Our findings suggest that the in vitro engineering of prevascularized matrices is within reach.

Markers to evaluate the quality and self-renewing potential of engineered human skin substitutes in vitro and after transplantation.

We screened a series of antibodies for their exclusive binding to the human hair follicle bulge. In a second step these antibodies were to be used to identify basal keratinocytes and potential epithelial stem cells in the human epidermis and in engineered skin substitutes. Of all the antibodies screened, we identified only one, designated C8/144B, that exclusively recognized the hair follicle bulge. However, C8/144B-binding cells were never detected in the human epidermal stratum basale. In the bulge C8/144B-binding cells gave rise to cytokeratin 19-positive cells, which were also tracked in the outer root sheath between bulge and the hair follicle matrix. Remarkably, cytokeratin 19-expressing cells were never detected in the hair follicle infundibulum. Yet, cytokeratin 19-expressing keratinocytes were found in the epidermal stratum basale of normal skin as a subpopulation of cytokeratin 15-positive (not C8/144B-positive) basal keratinocytes. Cytokeratin 19/cytokeratin 15-positive keratinocytes decreased significantly with age. We suggest that cytokeratin 19-expressing cells represent a subpopulation of basal keratinocytes in neonates and young children (up to 1.5 years) that is particularly adapted to the lateral expansion of growing skin. Our data show that cytokeratin 19 in combination with cytokeratin 15 is an important marker to routinely monitor epidermal homeostasis and (at least indirectly) the self-renewing potential of engineered skin.

Type II keratin cDNAs from the rainbow trout: implications for keratin evolution.

From a teleost fish, the rainbow trout Oncorhynchus mykiss, we have cloned and sequenced cDNAs encoding five different type II keratins. The corresponding protein spots, as separated by 2D-PAGE of trout cytoskeletal preparations, have been identified by peptide mass mapping using MALDI mass spectrometry. Three of the sequenced keratins are expressed in the epidermis (subtype IIe), and two in simple epithelia and mesenchymal cells (subtype IIs). The IIs keratins are both orthologs of human K8. This leaves unsequenced only the trace component S3 of the biochemically established trout keratin catalog. A phylogenetic tree has been constructed from a multiple alignment of the rod domains of the new keratin sequences together with type II sequences from other vertebrates such as shark, zebrafish, and human; lamprey K8 (recently sequenced in our laboratory) has been used as outgroup. This tree suggests, in a highly bootstrap-supported manner, that the teleost IIe keratins diversified independently from the mammalian IIe keratins. In contrast, all the species investigated express K8-like keratins, suggesting that the different IIe branches evolved from K8-like progenitors. The tree also indicates that the published zebrafish sequences represent IIe keratins and that the biochemically identified K8 ortholog in zebrafish has not yet been sequenced.