Influence of subcutaneous adipose tissue mass on dermal elasticity and sagging severity in lower cheek

Background: Subcutaneous adipose tissue lies just beneath the dermal layer, but the interaction between the two is not well understood. Recently, we showed that the subcutaneous adipose layer affects dermal properties in an obese rodent model. Objective: To explore the influence of the subcutaneous adipose layer on dermal properties and morphology in humans. Methods: Subcutaneous adipose mass was measured by ultrasound using a Prosound alpha 5^®, dermal elasticity was measured using an in vivo suction skin elasticity meter (Cutometer MPA 580^®), and sagging severity were evaluated morphologically using photograph‐based grading criteria at the lower cheek in 70 Japanese female volunteers (age; 31–59, BMI; 17.1–36.2). The correlations among the results were examined. Results: Thickness of the subcutaneous adipose layer was significantly and negatively correlated with dermal elasticity parameters, i.e., elastic deformation, elastic deformation recovery, extensibility, total deformation recovery, ratio of viscoelasticity to elastic distention and overall elasticity, including creep and creep recovery. Furthermore, we investigated their influence on facial morphology, such as sagging. The subcutaneous adipose mass was significantly and positively correlated with sagging severity at the lower cheek (R=0.442, P<0.001), and there was a significant negative correlation between dermal elasticity and sagging severity (R=?0.358, P<0.01). Conclusion: These results indicate that increment of subcutaneous adipose mass impairs dermal elasticity, which in turn exacerbates sagging severity.     

High‐fat diet exacerbates imiquimod‐induced psoriasis‐like dermatitis in mice

Psoriasis, a chronic inflammatory skin disease, is closely related to systemic metabolism. An elevated body mass index (BMI) is a risk factor for psoriasis; inflammasomes are activated by adipose tissue macrophages in obese subjects. We hypothesized that hyperlipidaemia is involved in the pathogenesis of psoriasis and examined the role of a high‐fat diet (HFD) in the development of psoriasis in imiquimod (IMQ)‐treated mice. The body weight and serum level of cholesterol were significantly higher in mice fed an HFD than in a regular diet (RD). HFD mice had higher psoriasis skin scores, and the number of neutrophils infiltrating into the lesional skin was elevated. IL‐17A mRNA expression was significantly increased in the skin of IMQ‐treated HFD mice; the expression of IL‐22, IL‐23 and TNF‐α mRNA was not enhanced. Caspase‐1 and IL‐1β were activated in the skin of IMQ‐treated HFD mice, and their serum level of IL‐17A, TNF‐α and IL‐1β was significantly upregulated. Our findings strongly suggest that hyperlipidaemia is involved in the development and progression of psoriasis via systemic inflammation and inflammasome activation.     

Increment of subcutaneous adipose tissue is associated with decrease of elastic fibres in the dermal layer

Obesity is a significant risk factor for various skin disorders, including pressure ulcer and delayed wound healing. We previously showed that increment of subcutaneous adipose tissue contributes to poor skin condition by decreasing dermal elasticity. Here, we examined the mechanism involved. Histologic observation of abdominal skin from middle‐aged females with a wide range of body mass index (BMI), an indicator of subcutaneous fat mass, showed that dermal elastic fibre abundance was significantly decreased with increment of BMI. Concomitantly, adipocytes were significantly enlarged. Adipocyte enlargement was significantly negatively correlated with dermal elastic fibre abundance. We hypothesized that enlarged adipocytes negatively influence dermal elastic fibres, so we investigated elastic fibre‐degrading factors in in vitro‐cultured enlarged adipocytes. MMP9 gene expression and secretion were significantly increased; further, these changes were blocked by extracellular signal‐regulated kinase (ERK) inhibitor. Nuclear translocation (activation) of AP‐1, a downstream ERK signalling molecule, was also observed in enlarged adipocytes. MMP9 abundance was significantly increased in skin of subjects with high BMI and enlarged adipocytes. These results suggest that increment of subcutaneous adipose tissue leads to adipocyte enlargement together with increased degradation of dermal elastic fibres, mediated at least in part by an ERK signalling‐mediated increase of MMP9 in enlarged adipocytes.     

Defining dermal adipose tissue

Here, we explore the evolution and development of skin‐associated adipose tissue with the goal of establishing nomenclature for this tissue. Underlying the reticular dermis, a thick layer of adipocytes exists that encases mature hair follicles in rodents and humans. The association of lipid‐filled cells with the skin is found in many invertebrate and vertebrate species. Historically, this layer of adipocytes has been termed subcutaneous adipose, hypodermis and subcutis. Recent data have revealed a common precursor for dermal fibroblasts and intradermal adipocytes during development. Furthermore, the development of adipocytes in the skin is independent from that of subcutaneous adipose tissue development. Finally, the role of adipocytes has been shown to be relevant for epidermal homoeostasis during hair follicle regeneration and wound healing. Thus, we propose a refined nomenclature for the cells and adipose tissue underlying the reticular dermis as intradermal adipocytes and dermal white adipose tissue, respectively.     

Ovariectomy is sufficient to accelerate spontaneous skin ageing and to stimulate ultraviolet irradiation-induced photoageing of murine skin

Background: Wrinkling and sagging of the skin during photoageing is physiologically associated with diminished elasticity, which can be attributed to increased fibroblast-derived elastase activity. This degrades the dermal elastic fibres needed to maintain the three-dimensional structure of the skin. We previously reported that ovariectomy accelerates ultraviolet (UV)B-induced wrinkle formation in rat hind limb skin by altering the three-dimensional structure of elastic fibres. OBJECTIVES: In this study, we used hairless mice to assess the effects of ovariectomy with or without chronic UVA or UVB radiation on sagging and wrinkling of skin, on the elasticity of skin, as well as on matrix metalloproteinase activities in the skin. METHODS: Ovariectomies or sham operations were performed on 6-week-old female ICR/HR hairless mice. RESULTS: Even in the ovariectomy group without UV irradiation, the skin elasticity was significantly decreased during the 3-13 weeks after ovariectomy, which was accompanied by a significant increase in elastase activity in the skin. After UVA or UVB irradiation, skin elasticity was significantly decreased to a greater extent in the ovariectomy group than in the sham operation group, and this was accompanied by a reciprocal increase in elastase activity but not in the activities of collagenases I or IV in the skin. Consistent with the decreased skin elasticity, UVA irradiation for 12 weeks elicited more marked sagging in the ovariectomy group than in the sham operation group. UVB irradiation for 12 weeks also induced more marked wrinkle formation in the ovariectomy group than in the sham operation group. CONCLUSIONS: These results suggest that ovariectomy alone is sufficient to accelerate skin ageing and to increase UV sensitivity, which results in the further deterioration of the skin and photoageing, and may account for the accelerated skin ageing seen in postmenopausal women.     

Skin fragility in obese diabetic mice: possible involvement of elevated oxidative stress and upregulation of matrix metalloproteinases

The purpose of this study was to test the hypothesis that obese diabetic mice exhibit marked skin fragility, which is caused by increased oxidative stress and increased matrix metalloproteinase (MMP) gene expression in the subcutaneous adipose tissue. Scanning electron microscopy of skin samples from Tsumura-Suzuki obese diabetic (TSOD) mice revealed thinner collagen bundles, and decreased density and convolution of the collagen fibres. Furthermore, skin tensile strength measurements confirmed that the dorsal skin of TSOD mice was more fragile to tensile force than that of non-obese mice. The mRNA expressions of heme oxygenase 1 (Hmox1), a marker of oxidative stress, Mmp2 and Mmp14 were increased in the adipose tissue of TSOD mice. Antioxidant experiments were subsequently performed to determine whether the changes in collagen fibres and skin fragility were caused by oxidative stress. Strikingly, oral administration of the antioxidant dl-α-tocopherol acetate (vitamin E) decreased Hmox1, Mmp2 and Mmp14 mRNA expressions, and improved the skin tensile strength and structure of collagen fibres in TSOD mice. These findings suggest that the skin fragility in TSOD mice is associated with dermal collagen damage and weakened tensile strength, and that oxidative stress and MMP overexpression in the subcutaneous adipose tissue may, at least in part, affect dermal fragility via a paracrine pathway. These observations may contribute to novel clinical interventions, such as dietary supplementation with antioxidants or application of skin cream containing antioxidants, which may overcome skin fragility in obese patients with diabetes.     

Focal dermal hypoplasia syndrome

An 11-month-old female with focal dermal hypoplasia syndrome is described. Historically, the skin lesions were characterized by a marked decrease in dermal connective tissue. Ultrastructurally, there were a number of fine filamentous structures measuring 5 to 70 mn within and between the bundles of collagen; most of the dermal fibroblasts of the lesions possessed no remarkably developed rough endoplasmic reticulum and Golgi complex. Multilocular fat cells which are regarded as young fat cells were often seen. It is suggested that this disease may be characterized by a decreased rate of collagen synthesis by fibroblasts and an overgrowth of adipose tissue.     

Negative regulation of dermal fibroblasts by enlarged adipocytes through release of free fatty acids

Subcutaneous adipose tissue lies just beneath the dermal layer, but the interaction between the two types of tissue remains obscure. Recently, we reported that obesity is associated with decreased dermal elasticity. To investigate the mechanism of the adipose tissue/dermal interaction, fibroblasts were cocultured with small or enlarged adipocytes, using a membrane insert to prevent direct contact. Enlarged adipocytes reduced 3T3-L1 fibroblast proliferation and gene expression of collagen (I)-α1 (col (I)-α1) and elastin while increasing gene expression of matrix metalloproteinase 13 (MMP13). In contrast, small adipocytes had no such effects. These results indicate that factors secreted by enlarged adipocytes influence dermal condition. As enlarged adipocytes are known to release free fatty acids (FFAs), the effects of these acids on 3T3-L1 fibroblasts were examined. Palmitic acid decreased fibroblast proliferation, reduced gene expressions of col (I)-α1 and elastin, and increased MMP13. Similar effects were observed in human dermal fibroblasts. The influence of palmitic acid on fibroblasts was inhibited by eicosapentaenoic acid (EPA), an inhibitor of Toll-like receptors (TLRs). Furthermore, EPA inhibited the effects of enlarged adipocytes on fibroblasts in the coculture system. These data indicate that enlarged adipocytes negatively control the function of dermal fibroblasts through the activation of TLRs by secreted FFAs.     

Impaired contact hypersensitivity in diet-induced obese mice

BACKGROUND: Epidemiological studies suggest that obesity is associated with the impairment of immunity. However, there is no experimental evidence that obesity prejudices immune responses. OBJECTIVE: This study was designed to determine the effects of obesity on contact hypersensitivity (CHS) response using a diet-induced obese (DIO) mouse model. METHODS: The effect of high fat diet (HFD) on CHS response to trinitrochlorobenzene (TNCB) was assessed by ear swelling, cytokine production, functional analysis of epidermal Langerhans cells, and adoptive transfer of immune cells. Immune response to ovalbumin was also analyzed in DIO mice. RESULTS: C57BL/6 mice but not BALB/c mice that fed with HFD for 4 weeks or more became obese and showed impaired CHS response, although both strain of mice showed enhanced irritant response to TNCB. CHS response was slightly impaired when C57BL/6 mice fed with HFD for 1 or 2 weeks. This suggests that diet-induced obesity or the HFD itself impairs the CHS response in the susceptible mice. The adoptive transfer of immune cells from DIO mice sensitized with TNCB to na?ve mice failed to show vigorous CHS, which suggests dysfunction of an afferent phase of CHS in DIO mice. However, the number and allo-stimulating ability of epidermal Langerhans cells were comparable between DIO mice and lean mice. In addition, the immune response to ovalbumin (delayed type hypersensitivity, and antigen-dependent production of antibodies and cytokine) was preserved in DIO mice. CONCLUSION: These results suggest that the diet-induced obesity or the HFD only partially impairs immunity in the susceptible mice.     

The hairless mouse model for assaying the atrophogenicity of topical corticosteroids.

The daily application of corticosteroids for 18 days to the dorsal skin of hairless mice resulted in loss of volume of all the cutaneous compartments. The epidermis thinned, sebaceous glands regressed, dermal thickness was reduced, horn-filled cysts shrunk, subcutaneous fat disappeared, and regression of the muscular layer occurred. The magnitude of these changes correlated strongly with the accepted potency ranking of these agents by clinical efficacy. Thus, atrophogenicity predicts anti-inflammatory activity. This model furnishes a simple screening method for assaying corticosteroid activity and for optimizing proprietary formulations.     

Dietary deficiencies of unsaturated fatty acids and starch cause atopic dermatitis‐like pruritus in hairless mice

Hairless mice fed with a special diet (named HR‐AD) show atopic dermatitis (AD)‐like pruritic skin inflammation that is almost completely resolved with the supplementation of an unsaturated fatty acid (UFA), the linoleic acid (LA). This suggests that the dietary deficiency of LA is the key cause of this dermatitis. However, because there is no appropriate control diet for HR‐AD, the involvement of other dietary ingredients cannot be ruled out. Furthermore, it has not yet been tested whether only UFA deficiency can cause such AD‐like pruritus. In this study, using semi‐purified custom diets, we attempted to reproduce this syndrome. Four‐week‐old hairless mice were maintained on a widely used standard diet American Institute of Nutrition‐76A (AIN‐76A), its modifications, or HR‐AD. Several modifications of fat and carbohydrate components revealed that dietary deficiency of both UFAs and cornstarch was required to induce severe skin barrier dysfunction as typically occurred in HR‐AD‐fed mice. An UFA‐ and cornstarch‐deficient diet caused severe AD‐like pruritus comparable to HR‐AD, despite weak Th2 immune responses and absence of immunoglobulin E production. On the other hand, a diet lacking UFAs but containing cornstarch significantly alleviated the development of pruritic dermatitis. Furthermore, the supplementation of wheat starch similarly improved skin barrier function. In conclusion, this study showed that a lack of certain starches might also be the cause of diet‐induced AD. Our findings could help to reproduce the diet‐induced AD itch model and also provide evidence that certain starches can have protective and ameliorative effects on AD‐like pruritus.     

Human adipose tissue-derived cells delay re-epithelialization in comparison with skin fibroblasts in organotypic skin culture

BACKGROUND: Wound healing of deep and extensive burns can induce hypertrophic scar formation. During the early steps of wound healing fibroblasts migrate into the wounded area. Fibroblastic cells present in tissues other than dermis may also migrate into the wounded area and participate in the wound healing process. OBJECTIVES: To examine the influence of human fibroblastic cells derived from subcutaneous fat or dermis on epidermal morphogenesis in vitro. METHODS: We prepared human skin equivalents (HSEs) made of a collagen type I matrix populated either with dermal fibroblasts or adipose tissue-derived cells (ADCs), on top of which keratinocytes were seeded and subsequently grown at the air-liquid interface. RESULTS: A fully differentiated epidermis was formed on matrices populated with ADCs. However, the HSE formed differed in a number of features from HSE generated with dermal fibroblasts. The major differences included: marked contraction of the dermal matrix, low lateral migration of keratinocytes, high keratin 17 expression indicating increased keratinocyte activation, delayed deposition of collagen IV at the epidermal/matrix junction, accumulation of alpha-smooth muscle actin-positive cells only underneath the epidermal compartment and positioning of these cells in a direction parallel to the epidermal compartment. The latter two phenomena have also been found in scar tissue. CONCLUSIONS: The possibility of generating HSEs with different cell types represents an attractive approach for in vitro studies focusing on the mechanism of wound healing.     

Sagging of the cheek is related to skin elasticity, fat mass and mimetic muscle function

Background/purpose: Facial sagging is associated with aging, although the mechanism remains unclear. The aim of this study was to investigate the mechanism of facial sagging by examining the relationship of sagging severity to changes of skin elasticity, fat mass and facial muscle function at the cheek.
Methods: Faces of 108 healthy Japanese female volunteers, aged 20–60 years were photographed at an angle of 45°. Standard scores of sagging severity were established by analyzing the photographs. We examined the correlations of scored sagging levels with skin elasticity measured with a Cutometer MPA 580^®, fat content estimated by bioelectrical impedance analysis and facial muscle function (lip sealing force and occlusal force) in middle-aged female volunteers (30–40 years) with a wide range of sagging scores.
Results: Because the upper, lower and lateral areas in the cheek may show severe sagging, a six-grade score of sagging severity was separately established for each area. Each score was significantly correlated positively with age (20–60 years). In middle-aged volunteers, the sagging scores in all three areas of the cheek were significantly and negatively associated with skin elasticity. Body fat percentage was significantly and positively correlated with the sagging scores in the lower and lateral areas, although the correlation was only weakly positive in the upper area. Mimetic muscle function, measured in terms of lip sealing pressure, was significantly and negatively correlated with the sagging score only at the upper area of the cheek, but masticatory muscle function, measured in terms of occlusal force pressure, was not associated with the sagging score.
Conclusions: Sagging may be associated with the reduction of skin elasticity and mimetic muscle function and increase of fat mass, but the relationships are different in different areas of the cheek.     

Comparison of sagging at the cheek and lower eyelid between male and female faces

Background: Facial sagging is a well‐known morphological feature associated with aging and reduced dermal elasticity. Its morphological characteristics and mechanism have been studied in females, but it is unclear whether or not there is a gender difference. Aims: The aim of this study was to clarify the morphological characteristics of sagging and the mechanism of sagging formation in male faces as compared with female faces, focusing on changes in dermal elasticity. Methods: Faces of 98 healthy Japanese male volunteers, in their 20s–60s, were photographed at an angle of 45°. Upper and lower cheek sagging severity was evaluated by using photograph‐based grading criteria. In addition, new photograph‐based grading criteria of sagging severity at the lower eyelid were established and used. Dermal elasticity was measured using a non‐invasive, in vivo suction skin elasticity meter, Cutometer^®. Furthermore, photographs of 108 healthy Japanese female volunteers in their 20s–60s were used to compare the difference in the morphological characteristics of sagging between males and females. Results: Male facial sagging was prominent at the lower eyelid, upper cheek and lower cheek. Sagging severity in the upper and lower cheek was almost the same between males and females at all ages, whereas sagging at the lower eyelid in males was significantly more severe than that in females after middle age. Although dermal extensibility (U_f) was not related to age, total deformation recovery (U_a), ?(amount of deformation) ?(U_f?U_a), overall elasticity of the skin including creep and creep recovery (U_a/U_f), net elasticity excluding viscoelastic creep (U_r/U_e), ratio of elastic recovery to total deformation (U_r/U_f) and ?(ratio of viscoelastic to elastic distention) ?(U_v/U_e) were all significantly negatively related to age in both men and women. Furthermore, as in female faces, male facial sagging was significantly negatively related to dermal elasticity parameters, such as ?(U_f?U_a), U_a/U_f, U_r/U_e and U_r/U_f. Conclusion: These results indicate that the morphology and areas of sagging in male faces are similar to those in females in the cheek, but sagging at the lower eyelid is more severe in males after middle age. Furthermore, the dermal elasticity of male facial skin decreased with age similar to that of females, and may therefore be associated with the sagging formation in male faces.     

Protective effect of topically applied conjugated hexadienes against ultraviolet radiation-induced chronic skin damage in the hairless mouse

Albino hairless mice (SkH:HR-1) exposed chronically to suberythemal doses of ultraviolet B (UVB) radiation display visible skin wrinkling and tumors. Topical treatment of mice with solutions of conjugated dienes (2,4-hexadien-1-ol and derivatives of it) prior to each UVB radiation exposure reduces significantly the severity of these visible alterations. Chronic suberythemal doses of ultraviolet A radiation induce skin sagging, a distinctly different visible skin alteration. The severity of skin sagging is not reduced by topical application of the conjugated dienes tested here.     

Bleomycin inhibits adipogenesis and accelerates fibrosis in the subcutaneous adipose layer through TGF‐β1

Systemic sclerosis [scleroderma (SSc)]‐associated skin fibrosis is characterized by increased fibrosis in the dermis and a reduction in the thickness of the subcutaneous adipose tissue layer. Although many studies have examined fibrosis in SSc, only a few studies have focused on the associated reduction in the thickness of the subcutaneous adipose tissue layer. In this study, we investigated the effects of SSc‐induced fibrosis on adipose tissue. We found that bleomycin suppresses adipogenesis in adipose‐derived stem cells (ASCs) and stimulates ASCs to express transforming growth factor β1 (TGF‐β1), which suppresses adipogenesis and promotes fibrosis. Furthermore, we found that adipocyte‐conditioned medium suppressed collagen synthesis by fibroblasts in fibrosis‐like conditions. We concluded that in the skin affected by bleomycin‐induced fibrosis, increased TGF‐β1 expression suppresses adipogenesis and promotes adipocyte fibrosis. It was also suggested that adipocytes have an inhibitory effect on the progression of fibrosis.     

Zellulitis

Cellulite is a condition affecting the dermal and subdermal compartment and developing on thighs and buttocks of almost every woman. Macroscopically, cellulite is characterized by dimpling, visible either spontaneously or after provoking maneuvers. While published concepts on the pathophysiology of cellulite are in parts inconclusive or contradictory, there are at least some recent studies describing or confirming structural and anatomic changes on dermal and subdermal tissues: (1) A gender specific dimorphism with subdermal septae oriented orthogonally towards the skin surface as well as protrusion of fat tissue into the dermis. (2) These intradermal fat protrusions additionally correlate with the presence of cellulite. (3) There is also a correlation between the thickness of the subdermal fat layer and the presence of cellulite. (4) Cellulite also correlates with focal hypertrophic subcutaneous septae and a reduced density of septae in general. Treatment of cellulite aims at (1) the reduction of the subcutaneous fat layer, (2) increase in dermal thickness and elasticity and (3) dissection of hypertrophic connective tissue septae, responsible for the most pronounced dermal indentions. A variety of treatment options have evoleved ranging from topical retinol to interstitial laser     

Dietary glucosylceramide improves skin barrier function in hairless mice

BACKGROUND: Sphingolipids are known to play an important role in both water retention and epidermal permeability barrier function in mammalian stratum corneum. However, little is known about the effects on epidermal function of orally administered sphingolipids. OBJECTIVE: We examined the effect of dietary glucosylceramide (GluCer) on the maintenance and recovery of epidermal barrier function. METHODS: Hairless mice were fed a particular diet (HR-AD) for 4 weeks to induce chronic skin perturbation. Subsequently, a normal diet supplemented with GluCer (from rice bran and germ) was provided for the next 4 weeks. Transepidermal water loss (TEWL) and stratum corneum flexibility were measured throughout this recovery phase. Additional hairless mice were fed a diet with or without a maize-extracted GluCer supplement for 5 weeks, then their skin was acutely perturbed with repeated tape-stripping, and the TEWL was measured. RESULTS: Although skin functions were generally lower following chronic perturbation, in GluCer-fed mice the TEWL was significantly reduced at 2 weeks and the stratum corneum flexibility was increased at 3 weeks compared to controls. Following acute barrier perturbation by tape-stripping, mice an HR-AD fed a GluCer diet exhibited enhanced recovery compared with the control diet group. CONCLUSION: These results demonstrate that in hairless mice skin barrier functions impaired by chronic or acute perturbations were improved by dietary GluCer. The oral administration of GluCer may be useful for the preservation and recovery of epidermal barrier functions an HR-AD.     

Ionizing radiation induces cutaneous lipid remolding and skin adipocytes confer protection against radiation-induced skin injury

BackgroundRadiation-induced skin injury is a serious concern during radiotherapy and radiation accidents. Skin fat represents the dominant architectural component of the human skin. However, the interplay between skin fat and the progression of radiation-induced skin injury remains largely unexplored.ObjectiveThis study aims to elucidate the interplay between skin fat and the progression of radiation-induced skin injury.MethodsSD rats were irradiated with an electron beam. mRNA profiles were determined by RNA-Seq. The skin lipid mass was monitored by magnetic resonance imaging (MRI) and lipid profiles were measured by liquid chromatography-mass spectrometry (LC–MS). Human mature adipocytes isolated from dermal and subcutaneous white adipose tissues (WATs) were co-cultured with human keratinocytes (HaCaT) and skin fibroblasts (WS1) in the transwell culture system. Cell migration ability was measured by migration assay.ResultsRadiation modulated cutaneous lipid metabolism by downregulating multiple pathways. Moreover, radiation decreased skin fat mass with altered lipid metabolite profiles. The rats fed with a high-fat diet showed resistance to radiogenic skin injury compared with that with a control diet, indicating that skin lipid plays a radioprotective role. Mature adipocytes promoted the migration but not the proliferation of co-cultured skin keratinocytes and fibroblasts. Palmitic acid, the most abundant fatty acid in skin tissues, facilitated the migration of WS1 cells. Moreover, fatty acid-binding protein 4 (FABP4) could be incorporated into skin cells and promote DNA damage repair in irradiated skin fibroblasts.ConclusionRadiation induces cutaneous lipid remolding, and skin adipocytes confer a protective role against radiation-induced skin injury.     

The suitability of cells from different tissues for use in tissue-engineered skin substitutes

Tissue-engineered skin substitutes may be a future remedy for burn wounds and chronic wounds, as wound contraction and scar formation cannot be prevented with the current standard treatment. The aim of this study therefore was to identify readily available sources of fibroblasts suitable for dermal substitution. Three different tissues were studied: dermal tissue from split-skin graft, subcutaneous fat tissue and eschar tissue obtained through debridement of burn wounds. We determined the cellular profile and the cell numbers immediately after isolation and after 2 and 14 days of fibroblast culture using flow cytometry and cell counting with a cytometer. In addition, parts of the isolated cell suspensions were seeded directly into a porous collagen dermal substitute to investigate contraction over time. Various cell types were isolated from the three different tissues, but after 14 days of culturing predominantly fibroblasts (>90%) were detected. Keratinocytes, granulocytes and macrophages, if present, disappeared within 14 days. In the cell populations derived from dermal tissue, the percentage of myofibroblasts had decreased significantly by day 14 (from 8% to 3%, P=0.028). In contrast, this percentage had increased in the cell populations derived from fat and eschar (from 23% to 40% and from 20% to 38%, respectively). The fibroblast yield from dermal tissue after 2 weeks of culturing (50 x 10(6) cells/g of tissue) was significantly higher than the yield from fat and eschar tissue (2 x 10(6) cells/g of each tissue, P=0.029). Immunohistochemistry of collagen matrices seeded and cultured with fat- and eschar-derived cells revealed a high prevalence of myofibroblasts, whereas hardly any myofibroblasts were detected in the matrices seeded with dermal cells. The contraction of the eschar matrices was highest (74+/-6% remaining area), whereas dermal matrices contracted significantly less (92+/-7% remaining area, P=0.029) with intermediate contraction for fat matrices. We conclude that fibroblast cultures can be established from dermal tissue, fat tissue and eschar tissue. Dermis is the best fibroblast source for use in skin substitutes as it yields the highest numbers of fibroblasts with minimal numbers of myofibroblasts.