Superficial Dermal Fibroblasts Research Articles

Dermal fibroblast from superficial layers of pig skin exhibits more proliferative capacity than that from deep layers

ObjectiveTo further examine the feasibility of using pigs as an animal model for the study of dermal fibroblast heterogeneity and to explore the proliferative capacity of dermal fibroblasts from different layers of pig skin in vitro and in vivo. Material and methodsCultured superficial and deep dermal fibroblasts were subjected to cell growth assay, cell cycle analysis, immunocytochemical staining and western blotting for proliferating cell nuclear antigens. Moreover, skin samples autografted with superficial/deep dermal fibroblasts were subjected to immunohistochemical staining and western blotting for proliferating cell nuclear antigen. ResultsThe cell growth assay showed that the growth curve of the superficial dermal fibroblast was progressively higher than that of the deep layer. The cell cycle analysis showed that the (G2+S) percentage of the superficial dermal fibroblasts was significantly higher than that of the deep layer fibroblasts. The immunocytochemical staining and western blotting showed that the expression of proliferating cell nuclear antigen in the cultured superficial dermal fibroblast was significantly higher than that of the deep layer cells. The immunohistochemical staining showed that the positive rate of proliferating cell nuclear antigen in the skin samples autografted with the superficial dermal fibroblast was significantly higher than that of the deep layer. ConclusionsThis study has demonstrated that similar to human dermal fibroblasts, dermal fibroblasts from different layers of pig skin exhibit distinct proliferative capacity, which increases the feasibility of using pigs as an animal model for future studies on the heterogeneity of dermal fibroblasts.

Improved Dermal Regeneration Using a Combination of Dermal Substitutes and Dermal Fibroblast Optimization: A Hypothesis

In human adults, the repair of cutaneous wounds usually leads to scar formation rather than regeneration. Dermal substitutes have been used as a regenerative template for reducing scar formation and improving the extent of dermal regeneration. However, achievement of complete regeneration is still a long way off. Dermal substitutes are characterized by unusual regenerative activity, appearing to function by acting as temporary configurational guides for cell infiltration and synthesis of new stroma. Fibroblasts are important cells with many vital functions in wound-healing processes. They are heterogeneous with distinct characteristics according to their source location, such as subcutaneous tissue, superficial-layer dermis, and deep-layer dermis. Many studies have shown that superficial dermal fibroblasts possess the potential to form dermis-like tissue. Fibroblasts in deep-layer dermis and subcutaneous tissue may play a critical role in the formation of hypertrophic scars. Fibroblast phenotype affects the newly formed dermal architecture and influences the dermal regeneration effect induced by dermal substitutes. It is hypothesized that better regeneration of the dermis can be achieved using dermal substitutes along with dermal fibroblast optimization.

Advances in Skin Substitutes-Potential of Tissue Engineered Skin for Facilitating Anti-Fibrotic Healing.

Skin protects the body from exogenous substances and functions as a barrier to fluid loss and trauma. The skin comprises of epidermal, dermal and hypodermal layers, which mainly contain keratinocytes, fibroblasts and adipocytes, respectively, typically embedded on extracellular matrix made up of glycosaminoglycans and fibrous proteins. When the integrity of skin is compromised due to injury as in burns the coverage of skin has to be restored to facilitate repair and regeneration. Skin substitutes are preferred for wound coverage when the loss of skin is extensive especially in the case of second or third degree burns. Different kinds of skin substitutes with different features are commercially available; they can be classified into acellular skin substitutes, those with cultured epidermal cells and no dermal components, those with only dermal components, and tissue engineered substitutes that contain both epidermal and dermal components. Typically, adult wounds heal by fibrosis. Most organs are affected by fibrosis, with chronic fibrotic diseases estimated to be a leading cause of morbidity and mortality. In the skin, fibroproliferative disorders such as hypertrophic scars and keloid formation cause cosmetic and functional problems. Dermal fibroblasts are understood to be heterogeneous; this may have implications on post-burn wound healing since studies have shown that superficial and deep dermal fibroblasts are anti-fibrotic and pro-fibrotic, respectively. Selective use of superficial dermal fibroblasts rather than the conventional heterogeneous dermal fibroblasts may prove beneficial for post-burn wound healing.

Superficial Dermal Fibroblasts Enhance Basement Membrane and Epidermal Barrier Formation in Tissue-Engineered Skin: Implications for Treatment of Skin Basement Membrane Disorders

Basement membrane is a highly specialized structure that binds the dermis and the epidermis of the skin, and is mainly composed of laminins, nidogen, collagen types IV and VII, and the proteoglycans, collagen type XVIII and perlecan, all of which play critical roles in the function and resilience of skin. Both dermal fibroblasts and epidermal keratinocytes contribute to the development of the basement membrane, and in turn the basement membrane and underlying dermis influence the development and function of the epidermal barrier. Disruption of the basement membrane results in skin fragility, extensive painful blistering, and severe recurring wounds as seen in skin basement membrane disorders such as epidermolysis bullosa, a family of life-threatening congenital skin disorders. Currently, there are no successful strategies for treatment of these disorders; we propose the use of tissue-engineered skin as a promising approach for effective wound coverage and to enhance healing. Fibroblasts and keratinocytes isolated from superficial and deep dermis and epidermis, respectively, of tissue from abdominoplasty patients were independently cocultured on collagen-glycosaminoglycan matrices, and the resulting tissue-engineered skin was assessed for functional differences based on the underlying specific dermal fibroblast subpopulation. Tissue-engineered skin with superficial fibroblasts and keratinocytes formed a continuous epidermis with increased epidermal barrier function and expressed higher levels of epidermal proteins, keratin-5, and E-cadherin, compared to that with deep fibroblasts and keratinocytes, which had an intermittent epidermis. Further, tissue-engineered skin with superficial fibroblasts and keratinocytes formed better basement membrane, and produced more laminin-5, nidogen, collagen type VII, compared to that with deep fibroblasts and keratinocytes. Overall, our results demonstrate that tissue-engineered skin with superficial fibroblasts and keratinocytes forms significantly better basement membrane with higher expression of dermo-epidermal adhesive and anchoring proteins, and superior epidermis with enhanced barrier function compared to that with deep fibroblasts and keratinocytes, or with superficial fibroblasts, deep fibroblasts, and keratinocytes. The specific use of superficial fibroblasts in tissue-engineered skin may thus be more beneficial to promote adhesion of newly formed skin and wound healing, and is therefore promising for the treatment of patients with basement membrane disorders and other skin blistering diseases.

Deep dermal fibroblast profibrotic characteristics are enhanced by bone marrow–derived mesenchymal stem cells

Hypertrophic scars are a significant fibroproliferative disorder complicating deep injuries to the skin. We hypothesize that activated deep dermal fibroblasts are subject to regulation by bone marrow-derived mesenchymal stem cells (BM-MSCs), which leads to the development of excessive fibrosis following deep dermal injury. We found that the expression of fibrotic factors was higher in deep burn wounds compared with superficial burn wounds collected from burn patients with varying depth of skin injury. We characterized deep and superficial dermal fibroblasts, which were cultured from the deep and superficial dermal layers of normal uninjured skin obtained from abdominoplasty patients, and examined the paracrine effects of BM-MSCs on the fibrotic activities of the cells. In vitro, deep dermal fibroblasts were found higher in the messenger RNA (mRNA) levels of type 1 collagen, alpha smooth muscle actin, transforming growth factor beta, stromal cell-derived factor 1, and tissue inhibitor of metalloproteinase 1, an inhibitor of collagenase (matrix metalloproteinase 1). As well, deep dermal fibroblasts had low matrix metalloproteinase 1 mRNA, produced more collagen, and contracted collagen lattices significantly greater than superficial fibroblasts. By co-culturing layered fibroblasts with BM-MSCs in a transwell insert system, BM-MSCs enhanced the fibrotic behavior of deep dermal fibroblasts, which suggests a possible involvement of BM-MSCs in the pathogenesis of hypertrophic scarring.

Features of wound healing shown by fibroblasts obtained from the superficial and deep dermis

Dermal fibroblasts (DF) obtained from the superficial dermal layer and those from the deep dermal layer have different cellular functions. These differences are often associated with excessive scarring; they also influence early wound healing. We therefore investigated the differences between superficial and deep dermal fibroblasts with special emphasis on their contractile properties, and ability to produce connective tissue. We investigated their proliferation kinetics, ability to contract collagen lattices, and chronological mRNA expression of eight genes associated with wound healing. To estimate the changes in the differences between them during the early phase of wound healing, we investigated mRNA expression in bFGF supplemented medium because bFGF is a representative cytokine that is familiar to clinicians. Superficial DF proliferate faster than deep DF in culture, whereas deep DF are better at contracting collagen lattices than superficial ones. In realtime analysis of polymerase chain reaction, the expression of type I and III collagen, fibronectin, TGF β1 and β3, and connective tissue growth factor were higher in deep DF than in superficial DF, while the expression of TGF β2 was higher in superficial DF. After bFGF supplementation, the relative dominance of mRNA expression between superficial and deep DF remained constant except for the expression of collagenase. According to our analysis, deep DF are superior to superficial DF at promoting wound healing (particularly contraction and production of connective tissue). The intradermal distribution of DF is appropriate for efficient wound healing.

In vivo photoinduction of metallothionein in human skin by ultraviolet irradiation.

The aims of this study were to confirm and substantiate the in vivo cutaneous induction of metallothionein (MT) in human skin by UVR, which we have reported in brief previously, and to make a preliminary attempt to characterize the time course of this phenomenon. Buttock skin in 32 volunteers was irradiated with 2 MED of UVB and biopsies were taken at 24 h from matched non-irradiated and irradiated sites. In the kinetic study, skin biopsies from six volunteers were taken at 0, 2, 8, 24, and 48 h after 2 MED UVB irradiation. MT was immunolocalized in formalin-fixed, paraffin-embedded tissue with the monoclonal antibody E9 by an indirect immunoperoxidase method. Statistically significant differences between immunocytochemical scores were identified between non-irradiated (NI) and irradiated (I) skin within suprabasal keratinocytes (mean: NI = 1.2, I = 5.1; P = 0.01), superficial dermal fibroblasts (mean: NI = 2, I = 43; P < 0.001), mid-dermal fibroblasts (mean: NI = 0, I = 27; P < 0.001), and deep dermal fibroblasts (mean: NI = 0, I = 11; P < 0.001). In the kinetic study, no consistent rise in MT score with time was observed for the epidermal component. In dermal fibroblasts, however, the first statistically significant rise in immunocytochemically detectable MT was detected at 2 h and this was found to plateau beyond 8 h. These results confirm that ambient levels of UV irradiation are capable of inducing MT in human skin in vivo. Taken together with the relative rapidity of the response, this suggests a physiological photoprotective role for MT in human skin cells. The lack of a kinetic increase in epidermal MT may be due to high basal levels. Induction of MT in dermal fibroblasts may reflect the effects of a diffusible factor released from keratinocytes after UVR.