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PubMed2000

A new skin equivalent model: dermal substrate that combines de-epidermized dermis with fibroblast-populated collagen matrix.

Epidermis reconstructed on de-epidermized dermis (RE-DED) and on fibroblast-populated collagen matrix (Living Skin Equivalent) showed a histologic resemblance to native epidermis. However, some abnormalities have been found including different expression pattern of differentiation markers from native epidermis. In this study, to reconstruct an epidermis model resembling native epidermis more closely than previous skin equivalents, de-epidermized dermis (DED) was raised on fibroblast-populated collagen matrix and keratinocytes were cultured on top of the DED at the air-liquid interface. The new skin equivalent like RE-DED showed a similar morphology to that of native epidermis. Immunohistochemical studies revealed that differentiation markers such as involucrin, loricrin and filaggrin but not keratin 1 expressed similar pattern characteristics to native epidermis compared with those of RE-DED. In addition, the new model showed some fibroblasts in the DED as a result of migration from the fibroblast-populated collagen matrix, mimicking a living dermis in vivo. These results indicate that the new model seems to be a better skin equivalent model than previous models. Also, they provide additional evidence that the presence of fibroblasts improves epidermal differentiation.

Cells, Cultured↗
PubMed2002

Purification, characterization and cloning of tensilin, the collagen-fibril binding and tissue-stiffening factor from Cucumaria frondosa dermis.

The inner dermis of the sea cucumber, Cucumaria frondosa, is a mutable collagenous tissue characterized by rapid and reversible changes in its mechanical properties regulated by one or more protein effectors that are released from neurosecretory cells. One such effector, tensilin, is a collagen-fibril binding protein, named for its ability to induce dermis stiffening. Tensilin was purified using an affinity column constructed from C. frondosa collagen-fibrils. The protein migrates as a single band on SDS-PAGE (Mr approximately 33 kDa) and has an isoelectric point of 5.8. Equilibrium sedimentation experiments suggest a molecular mass of approximately 28.5-29.4 kDa. Carbohydrate analysis of tensilin revealed no measurable sugar content. The molar amount of tensilin was determined to be 0.38% that of collagen and 47% that of stiparin, a constitutive matrix glycoprotein. A full-length cDNA clone for tensilin was obtained from a C. frondosa inner dermis cDNA expression library. Predicted properties derived from the deduced peptide sequence were in agreement with those of the native protein. A noted feature of tensilin's deduced peptide sequence, particularly in its N-terminal domain, is its homology to tissue inhibitor of metalloproteinases. Tensilin's C-terminal tail has no known homology to other proteins but contains a putative collagen-fibril binding site.

Amino Acid Sequence↗
PubMed2002

Dermal nevus cells from congenital nevi cannot penetrate the dermis in skin reconstructs.

Congenital nevi are composed of pigment cells bearing common features with melanocytes but showing altered differentiation which leads to nesting and dermal involvement. Using a dead de-epidermized dermis seeded with a combination of keratinocytes and various sources of pigment cells (normal melanocytes, dermal nevus cells from congenital nevi, Bowes melanoma cells), we have studied the formation of nests and the dermal migration of pigment cells together with their secretion profiles of matrix metalloproteinases (MMP). Dermal fibroblasts were also used as control cells in epidermal reconstructs. Besides their morphologic features, the absence of pigment donation to keratinocytes was the major characteristic of dermal nevus cells. A positive correlation was established between the increasing percentage of seeded nevus cells and the patchy pigmentation of reconstructs, as well as the clustering of cells in junctional nests. However, the presence of nevus cells in the dermis of reconstructs was never detected, whereas melanoma cells and dermal fibroblasts could invade the dermis during the time span of the experiments. MMP9 was never expressed in congenital dermal nevus cells but pro-MMP2 was constitutively expressed by all strains of congenital nevus cells and dermal fibroblasts. Melanocytes produced comparable amounts of both pro-MMP2 and pro-MMP9, and Bowes melanoma cells secreted a marginal level of pro-MMP2. In view of their three-dimensional behaviour and secretion of MMPs, we propose that dermal congenital nevus cells correspond to an intermediate status of differentiation between normal melanocytes and melanoma cells. Activation of MMPs by a cofactor or the activation of another signalling pathway seems necessary to induce the dermal passage of nevus cells.

Adolescent↗
PubMed2003

Xanthophore migration from the dermis to the epidermis and dermal remodeling during Salamandra salamandra salamandra (L.) larval development.

During larval development of Salamandra salamandra salamandra chromatophores organize to form the definitive pigment pattern constituted by a black background with yellow patches that are characterized by epidermal xanthophores and dermal iridophores. Simultaneously the dermis undergoes remodeling from the larval stage to that typical of the adult. In the present study we ultrastucturally and immunocytochemically examined skin fragments of S. s. salamandra larvae and juveniles in order to investigate the modalities of xanthophore migration and differentiation in the context of dermal remodeling from the larval to adult stage. Semithin and thin sections showed that the dermis in newly born larvae consists of a compact connective tissue (basement lamella), to which fibroblasts and xanthophores adhere, and of a loose deep collagen layer. As larval development proceeds, fibroblasts and xanthophores invade the basement lamella, skin glands develop and the adult dermis forms. At metamorphosis, xanthophores reach the epidermis crossing through the basal lamina. We examined immunocytochemically the expression of signal molecules, such as fibronectin, vitronectin, beta1-integrin, chondroitin sulfate, E-cadherin, N-cadherin and plasminogen activator, which are known to be involved in regulating morphogenetic events. Their role in dermal remodeling and in pigment pattern formation is discussed.

Animals↗
PubMed1999

Microdialysis vs. suction blister technique for in vivo sampling of pharmacokinetics in the human dermis.

Our aim was to simultaneously investigate 2 techniques for in vivo sampling of peripheral compartment pharmacokinetics after systemic administration of acetylsalicylic acid. Ten volunteers were given 2 g acetylsalicylic acid orally. Blood samples and dialysates from 4 microdialysis probes inserted in the dermis of the forearm were collected for 5 h and suction blisters were raised 1-3 h after dosing. In microdialysates, both acetylsalicylic acid and the metabolite salicylic acid were measurable in the absence of hydrolysing enzymes. The mean Cmax (maximum concentration) of total, unbound salicylic acid was 9.5 microg/ml in microdialysates, 13.2 microg/ml in suction blister fluid and 56.5 microg/ml in plasma. Mean Tmax (time to Cmax) for salicylic acid was 188 and 161 min in plasma and microdialysates, respectively. The dermis-to-plasma Cmax ratio was 0.16+/-0.04 (mean +/- SD) by microdialysis sampling and 0.25+/-0.09 by the suction blister fluid method. Close correlations (p<0.01) were found between Cmax of salicylic acid in microdialysates and plasma, and between Cmax of salicylic acid in suction blister fluid and plasma. The 2 techniques were in excellent accordance with even closer correlation between maximum concentrations obtained by microdialysis and suction blister fluid sampling (p<0.001). However, comparing the tolerability of the sampling procedure, ease of analysis, and detail in chronology, microdialysis is superior for sampling in vivo pharmacokinetics in the dermis.

Adult↗
PubMed2000

Striated muscle: a normal component of the dermis and subcutis in many areas of the face.

Striated muscle is typically not mentioned in histology chapters of most dermatology and dermatopathology books. In others, authors only speak of its presence in the dermis or subcutis but do not provide any details about its quantity, distribution, or orientation. In the course of an investigation concerning regional histologic variations of the skin, the presence of striated muscle fibers (StrMFs) has been a noteworthy feature of many areas of the face. Striated muscle fibers are a normal constituent of the dermis of the eyebrow (few and deep fibers); eyelid and nose (excluding the nasal tip) (numerous and deep fibers); and lips and chin (numerous, vertical, and more superficial fibers). They are also found in the subcutis of all these areas with some differences in density and distribution among them. Striated muscle fibers form a compact layer beneath the subcutaneous fat on the forehead and cheek, but because of their deep location, they are rarely included in cutaneous biopsy specimens of the latter area. Mainly based on the presence of StrMFs in the dermis, an algorithm to accurately determine the location of biopsy specimens from the face has been elaborated. With the aid of other features such as the type of hair follicles and the presence and degree of solar elastosis, the sex and approximate age of the patient can be established in many cases. Some practical and scientific consequences of these findings are discussed.

Dermis↗
PubMed1998

Comparison of the effects of enucleation and orbital reconstruction using free-fat grafts, dermis grafts, and porous polyethylene implants in infant rabbits.

Twenty-nine infant rabbits from four litters were randomly assigned to three groups. No surgery was performed on left eyes. All right eyes were enucleated. Group 1, the control group, had no right-socket reconstruction. Group 2 had reconstruction using free-fat and dermis grafts. Group 3 had reconstruction using porous polyethylene implants. On attaining adult size, rabbits were killed, decapitated, and prepared for measurements. Orbital volume measurements were performed twice and orbital entrance areas were determined once. No significant differences were noted between first and second measurements of orbital volumes. Right orbital volumes were significantly smaller than left orbits in all groups except for the second measurement of the polyethylene implant group. Right orbital entrance areas were significantly smaller than left areas in the control and the free-fat and dermis graft groups. Right orbital entrance areas of the polyethylene group were not significantly smaller than the corresponding left orbital entrance areas. The use of free-fat and dermis grafts for orbital reconstruction in anophthalmic infant rabbits did not significantly stimulate orbital bone growth. In one trial, the polyethylene implant group displayed right orbital volumes and orbital entrance areas that were not significantly smaller than controls.

Adipose Tissue↗
PubMed2000

Facial autologous soft-tissue contouring by adjunction of tissue cocktail injection (micrograft and minigraft mixture of dermis, fascia, and fat).

Facial aging is both a physiologic and anatomic process characterized by changes in the skin and supporting tissues. The aging process produces an outer envelope that gradually expands while its contents gradually involute and the underlying structure weakens. This process results in an excess of skin that tends to create folds, grooves, and deepening furrows. Contour augmentation and filling depressions with autologous tissue or heterogeneous materials are widely used in face rejuvenation as an adjunctive procedure. There is unanimous agreement on the advantages of autogenous tissue grafts over alloplastic materials and heterogeneous transplants. It is also well known that the revascularization of a small graft (fat, dermis, and/or composite graft) is better than a large graft. For this reason, fat injections consisting of small particles have recently become popular. According to different authors, a graft take may vary from 30 to 50 percent. Nevertheless, it has been thoroughly documented that a graft consisting of dermis or fascia is superior to a fat graft in both the graft take rate and quality of the tissue. Strips of dermal graft have been used successfully for several years to fill lip contour and nasolabial folds. However, the main disadvantage of this technique is that utilization is restricted only to certain areas where there is a need for a small incision. To overcome this obstacle, the author developed a simple technique to obtain an injectable mass from a mixture of dermis, muscle strips, fat tissue, and fascia to use in body contouring (especially in the facial region) in large areas. The author describes the use of the technique in 450 patients. Follow-up in these patients from 6 months to 10 years showed that the application of the "tissue cocktail" procedure in select patients improved the author's results and created a marked increase in the number of satisfied patients with no complaints. The take and durability of this kind of graft were superior to the author's results using fat grafts. The tissue cocktail graft remained stable for several years, as effectively seen in the chin region. The sole complication from the procedure was some bruising, which was resolved in several days. No single infection or inclusion cyst was observed in this series.

Adipose Tissue↗
PubMed2002

Rapid alignment of collagen fibers in the dermis of undermined and not undermined skin stretched with a skin-stretching device.

A controlled, quantitative histochemical study was performed in five piglets to establish changes in undermined and not undermined stretched skin. The skin was stretched with a stretching device for 30 minutes to close a large skin defect. On each flank of the piglet, at a standard position, 9 x 9-cm wounds were created under general anesthesia. On one flank, the surrounding skin was undermined cranially and caudally over a 10-centimeter area. Sections of skin biopsies obtained during stretching were stained with picrosirius red and studied with routine light microscopy and polarized light microscopy in combination with image analysis. The length of collagen fibers was analyzed as a parameter of changes in the dermis resulting from skin stretching. This newly developed quantitative method appeared to be valid, specific, and reproducible, allowing for objective determination of changes in the length of the fibers in the plain of the sections. Changes in the orientation of collagen fibers in the dermis as a result of skin stretching were thereby determined. Epidermal thickness did not change significantly under the influence of stretching forces in both undermined and not undermined skin. However, the orientation of the collagen fibers changed significantly as a result of skin stretching. In undermined wounds, parallel alignment and elongation of the fibers in the plane of the sections was already observed after 15 minutes of stretching. The fibers became aligned in the direction of the stretching force, perpendicular to the wound margin. After 30 minutes of stretching, the mean major axes of the collagen fibers were longest in the plane of the sections (p < 0.001). This meant that elongation and parallel alignment of the collagen fibers had occurred. Stretching of not undermined skin for 15 minutes resulted in significantly stronger parallel alignment in the plane of the sections as compared with undermined skin. This was less well defined after 30 minutes of stretching in not undermined skin. It is concluded that skin stretching with a skin-stretching device for 30 minutes results in significant histomorphological changes of collagen fibers in the dermis of both undermined and not undermined skin. The fibers realign rapidly as a result of stretching forces and become aligned in the direction of the stretching force, perpendicular to the wound margin. These dynamic changes in collagen fibers explain the significantly decreased wound closing tension resulting from skin stretching and explain how skin stretches beyond its inherent extensibility.

Animals↗
PubMedDate not supplied

Amniotic membrane to the rescue of partially necrosed dermis fat graft.

A 25-year-old woman underwent dermis fat graft on the right side for persistent ocular pain with her polymethylmethacrylate (PMMA) spherical orbital implant. One week later she developed dermal necrosis of the dermis fat graft with potentially devastating fat atrophy if left uncovered. Amniotic membrane graft epithelial side up was sutured to the conjunctiva surrounding the fat. 2 weeks later the graft epithelialized with preservation of fat. Role of amniotic membrane graft as a routine lining over dermis fat grafts to promote conjunctival healing and prevent fat exposure needs to be studied in prospective studies.

Adipose Tissue↗
PubMed2006

Ultraviolet B radiation suppresses Langerhans cell migration in the dermis by down-regulation of alpha4 integrin.

BACKGROUND/PURPOSE: Ultraviolet B (UVB) radiation affects the migration and function of epidermal Langerhans cells (LC) and causes immunosuppression of contact hypersensitivity. It is known that LC leaves the epidermis after exposure to UVB. To know the behavior of LC in the dermis after UVB radiation, we studied the effect of UVB radiation on the expression of integrin families on freshly isolated or cultured murine LC. We also examined whether UVB radiation affects the migration of LC to secondary lymphoid tissue chemokine (SLC/6Ckine). METHODS: Integrin expressions of murine LC cultured in epidermal cell suspension were analyzed using flowcytometry. We used murine LC sorted flowcytometrically for binding assay to extracellular matrix and for migration assay to chemokine. Skin explant assay and immnohistochemical staining for 'cords formation' were performed as previously described. RESULTS: Twenty and 40 mJ/cm2 of UVB radiation down-regulated the expression of alpha4 integrin on 24 h-cultured LC, but not that of alpha6, beta1, or beta4 integrin. The number of cultured LC adhered to fibronectin, a ligand for alpha4 integrin, was decreased after UVB irradiation, while that to laminin, a ligand for alpha6 integrin, was not influenced. UVB radiation reduced the number of migrating LC to SLC. Furthermore, skin sheet explant experiments showed that UVB radiation inhibited the 'cords' formation in dermal vessels of the 48 h-cultured skin. CONCLUSIONS: These data suggest that UVB radiation may suppress the migration of LC from the dermis to lymphatic vessels. UVB radiation may downregulate the adherence of LC to dermal fibronectin and migration to SLC, and consequently suppress the migration of LC from the UVB-irradiated dermis to lymphatics.

Animals↗
PubMed2003

Measurement of protein metabolism in epidermis and dermis.

We found that, in the rabbit ear, the dermal protein contains 75.5% of cutaneous phenylalanine and 97.9% of cutaneous proline; the remaining 24.5% of phenylalanine and 2.1% of proline are in the epidermal protein. This finding led us to develop two novel models that use phenylalanine and proline tracers and the rabbit ear to quantify protein kinetics in the epidermis and dermis. The four-pool model calculates the absolute rates of protein kinetics and amino acid transport, and the two-pool model calculates the apparent rates of protein kinetics that are reflected in the blood. The results showed that both epidermis and dermis maintained their protein mass in the postabsorptive state. The rate of epidermal protein synthesis was 93.4 +/- 37.6 mg x 100 g(-1) x h(-1), which was 10-fold greater than that of the dermal protein (9.3 +/- 5.8 mg x 100 g(-1) x h(-1)). These synthetic rates were in agreement with those measured simultaneously by the tracer incorporation method. Comparison of the four-pool and two-pool models indicated that intracellular cycling of amino acids accounted for 75 and 90% of protein kinetics in the dermis and epidermis, respectively. We conclude that, in the skin, efficient reutilization of amino acids from proteolysis for synthesis enables the maintenance of protein mass in the postabsorptive state.

Amino Acids↗
PubMed2000

Dynamic mechanical characterization of a mutable collagenous tissue: response of sea cucumber dermis to cell lysis and dermal extracts.

The dermis of the holothurian Cucumaria frondosa is a mutable collagenous tissue (MCT). In this study, the inner and outer regions of the dermis were separated and used to make two different tissue extracts. These extracts were applied to intact pieces of dermis, one invoking a stiff mechanical state and the other invoking a compliant state. The extracts were effective on tissues incubated in artificial sea water (ASW) and in those incubated in Ca(2+)-chelated ASW. Furthermore, the extracts were effective on both fresh tissues and tissues in which the cells had been lysed by freeze-thawing, indicating that the sites of action are in the extracellular matrix. Dynamic oscillatory shear tests and analyses were used to measure both the dynamic shear stiffness (G*) and the relative damping (tan delta ) of the tissue. These two parameters proved to be inversely related to each other (i.e. when G* increased, tan delta decreased). A theoretical viscoelastic model is constructed to interpret the results of these tests. It is concluded that changes in the mechanical state of the tissue involve interactions between elastic elements within the tissue rather than an alteration of its viscous components.

Animals↗
PubMed1998

Characterization of the differentiated phenotype of an organotypic model of skin derived from human keratinocytes and dried porcine dermis.

A number of skin models have been developed, but a simple method for rapidly producing large quantities of differentiated epidermis has been missing. We show the differentiated phenotype of human keratinocytes in organotypic culture arising in vitro by air-exposure of keratinocytes cultured with feeders on dried pig dermis. Keratinocytes were seeded at low density on the dermis covered with irradiated NIH-3T3 feeder cells and after reaching confluence lifted to the air-medium interface. A well differentiated epidermis with distinct basal, spinous, granular and stratum corneum layers was formed within 1 week. In this way, 100 cm2 of the differentiated recombined human/pig skin (D-RHPS) can be obtained from 106 secondary keratinocytes in 14 days. The entire keratinocyte life cycle takes place on the dermal substrate - from single cells to stratified epidermis. The differentiation was characterized using a panel of monoclonal antibodies. Similarly as in the normal skin, keratin 14 was expressed in all cell layers, keratin 10 in suprabasal layers, beta1-integrin and epitopes to antibody LH8 in the basal layer, involucrin and transglutaminase in the granular and horny layer of the epidermis. Keratins 16 and 7/17, which are absent in the normal epidermis, but present suprabasally in the psoriatic one, were expressed strongly in all suprabasal layers and in a subpopulation of basal cells. The keratinocytes can be combined with two other cell types cultured either on the dermal side of the dermis and/or on the bottom of the dish. It appears that this simple skin model can be used in studies of epithelial/mesenchymal interactions and interactions between epidermal cells and infectious agents. It may be particularly useful for the study of human papilloma viruses.

3T3 Cells↗
PubMed1999

Application of acellular dermis and autograft on burns and scars.

The cases of two patients with burns treated with dermis allograft and of one patient for lip reconstructive aesthetic filling treated with less than one mm3 of radiosterilised acellular dermis are presented. This paper emphasizes the treatment with radiosterilised dermal grafts with a permanent character so far. Hospitals, therefore, can satisfy the demand for this kind of tissue in the case of disaster and patients with serious injuries. In the cases cited, histocompatibility analysis was not required, thus having the advantage of long-time storage of the radiosterilised dermis used on these patients. Neither inflammatory reaction nor acute phase re-absorption were observed. Moreover, shrink (contract) healing was diminished. After two years, the results are still satisfactory.

Adolescent↗
PubMed2000

[The application of meshed acellular dermis xenograft (pigskin) with split thickness skin autograft in 23 patients].

OBJECTIVE: An effective and inexpensive coverage, which can prevent scar formation and fill up scar tissue depression for full thickness skin burns in 23 patients was presented. METHODS: A deepithelized and acellular basement membranes of dermis from freshly harvested pigskin was obtained as meshed acellular dermis xenograft. The patients were treated in one or two stages. Two stages, 7 day postoperative the meshed acellular xenograft, a split thickness skin autograft was applied. One stage, the meshed acellular xenograft and split thickness skin autograft were applied at same time. RESULTS: The take rate of two staged treatment was (87.0 +/- 25.4)% and one stage was (60.3 +/- 3.4)%. All grafts showed in pink or near normal skin color and felt soft and smooth. CONCLUSIONS: The combination of meshed acellular dermis xenograft and split thickness skin autograft demonstrated an ideal way for the coverage of full thickness skin burn, which is effective and inexpensive.

Adolescent↗
PubMed2005

[Long-term result of repair of deeply burned hands with large sheet of split-thickness autoskin grafting with the preservation of denatured dermis].

OBJECTIVE: To evaluate the long-term results of repair of burn hands with large sheet of split-thickness autoskin grafting with the preservation of denatured dermis. METHODS: One hundred and fifty-two hands in 86 burn patients with deep partial thickness and full thickness burn were enrolled in the study. The burned hands were treated by tangential excision and grafted with large sheet of split-thickness autoskin with the preservation of denatured dermis. The patients were followed-up from 3 months to 3 years. The skin color, elasticity, degree of contracture and the functional grading of the operated hands were observed. RESULTS: Good function was found in one hundred and forty-one out of the 152 burn hands (92.8%). For the rest 11 hands, pigmentation was found in 4, poor appearance in 4, and 3 hands with both poor appearance and function. CONCLUSION: Large sheet of split-thickness autoskin grafting with the preservation of denatured dermis could be an optimal choice for the management of hands with deep partial thickness burn, and it could restore the appearance and function of the hands satisfactorily.

Adolescent↗
PubMed2006

The number and distribution of blood dendritic cells in the epidermis and dermis of healthy human subjects.

Human blood dendritic cells (BDC) can be divided into three subsets: plasmacytoid DC (PDC) and two myeloid subsets--MDC1 and MDC2. Several studies revealed the presence of both MDC and PDC in blood of healthy subjects, however no precise literature data exist on the number and distribution of BDC in the skin. The aim of our study was to assess the number and distribution of BDC and their subtypes in the healthy skin. The-study included 30 healthy volunteers (age 18-51). Punch biopsies were taken from the buttock skin from each subject, and immunofluorescent staining was performed using monoclonal mouse IgG1 antibodies directed against BDCA-1, BDCA-2, BDCA-3 and BDC-4. The BDC were present both in the epidermis and dermis. PDC were detected mainly in the dermis (mean 1.2 cells per field). Myeloid subtypes were observed mainly in the middle layers of the epidermis and in the upper part of the dermis (mean 1.8 cells per field). The detection of blood dendritic cells in the skin proves their role in immune cutaneous surveillance.

Adolescent↗