Fibroblasts in Dermal Tissue Repair

ABSTRACT: The cellular dynamics of dermal regeneration were studied in nonsutured cutaneous wounds of female pigs and monkeys with electron microscopy and immunohistochemistry to demonstrate the origin and the development of fibroblasts forming granulation tissue. The results indicate that fibroblasts do not originate from histiocytes but from resting fibroblasts in the wound margins. These resting fibroblasts first become undifferentiated mesenchymal cells termed “X” cells. The “X” cells then multiply, migrate, and invade the wound defect in approximately 3 days, transforming into highly active fibroblasts. The active fibroblasts are endowed with the capacity of further transformation to fibroclasts and myofibroblasts. The latter two cell populations then effectively cause remodeling of newly formed tissue and contraction of wound margins.     

Effects of fat cells on keratinocytes and fibroblasts in a reconstructed rat skin model using collagen gel matrix culture

BACKGROUND: Fat cells (stromal tissue cells), not only have the function of lipid metabolism, but produce various cytokines that exert an influence on other cell types through paracrine or endocrine mechanisms. OBJECTIVES: To elucidate possible roles of fat cells in the skin, we examined their effects on the biological behaviour of keratinocytes and dermal fibroblasts in culture. METHODS: In the present study, focusing upon fat cell--keratinocyte or fat cell--dermal fibroblast interactions, we used a reconstructed skin system with rat skin cells in a three-dimensional collagen gel matrix culture. RESULTS: In this coculture system, fat cells promoted the proliferation and differentiation of keratinocytes. When keratinocytes were seeded directly on the fat cell layer without dermal fibroblasts, they proliferated extensively and formed a thick epidermal layer with a well-differentiated structure. Conversely, fat cells inhibited the proliferation of dermal fibroblasts. These effects of fat cells were presumed to be mediated by cytokines derived from the fat cells. CONCLUSIONS: The effects of fat cells could not be mimicked by the addition of leptin, tumour necrosis factor-alpha or insulin-like growth factor-II, suggesting that fat cells are mediating these activities via some other cytokines.     

Subpopulations of dermal skin fibroblasts secrete distinct extracellular matrix: implications for using skin substitutes in the clinic

Chronic wounds affect 1–2% of the world's population at any given time. These can be as a result of burns, or ulceration, and are essentially wounds which do not close. To facilitate closure, there are a number of biological products available which can be used as temporary skin replacements, or to promote tissue repair. These products usually replicate the two main layers found in human skin: the epidermis and dermis. Within the skin dermis the most abundant cell type are fibroblasts, whose primary role is to secrete extracellular matrix and support growth of cells in the adjacent epidermal layer. As fibroblasts within the skin are highly varied, the extracellular matrix in distinct locations of the dermis is also different; however skin substitutes do not usually reflect this diversity. In this study, from the UK, the researchers isolated three fibroblast sub‐types from human scalp skin dermis, and set about to characterise the extracellular matrix which the different sub‐types of fibroblasts synthesised in culture (i.e. developed in the lab, rather than on living skin). They found that the different fibroblast sub‐types produced extracellular matrix in culture reflective of the extracellular matrix found in distinct dermal locations in vivo (in living skin). They also found that certain fibroblast sub‐types were more proficient at supporting adjacent epithelial cells than others, which reflected the sub‐anatomical location from which the fibroblast sub‐types were originally isolated. The authors concluded that inspiration should be taken from the extracellular matrix which fibroblasts secrete to improve the design of biomimetic skin substitutes with improved therapeutic potential for skin tissue engineering.     

The differentiation and function of myofibroblasts is regulated by mast cell mediators.

Myofibroblasts are fibroblasts that express certain features of smooth muscle differentiation. Increased numbers of myofibroblasts and mast cells are frequently found together in a wide variety of settings, such as normal wound repair and scleroderma skin, which suggests that mediators produced by the mast cells could play a role in the regulation of myofibroblast differentiation and function. We used a human mast cell line, HMC-1, to determine if mast cells can induce normal human dermal fibroblasts to differentiate into functional myofibroblasts in vitro. We monitored the differentiation process by assaying two properties of the myofibroblast phenotype: expression of alpha-smooth muscle actin and functional capacity to contract a collagen matrix. In both a simple coculture system and in a skin-equivalent culture system, HMC-1 cells induced alpha-smooth muscle actin expression by fibroblasts. HMC-1 cells also stimulated fibroblast contraction of collagen gels, and the relative amount of contraction was dependent upon the number of HMC-1 cells present. To characterize the individual contributions made by specific mast cell products, we examined the effects of histamine, tumor necrosis factor alpha, and tryptase. Histamine induced a clear increase in alpha-smooth muscle actin expression, but it did not appear to stimulate fibroblast contraction. Tumor necrosis factor alpha had no effect in either assay. Purified human tryptase induced alpha-smooth muscle actin expression, and blocking the proteolytic activity of tryptase with specific inhibitors reduced that response. Tryptase inhibitors also eliminated the ability of HMC-1 cells to stimulate fibroblast contraction, suggesting that tryptase secreted by the HMC-1 cells may be one of the active mast cell mediators.     

Tocoretinate inhibited the contraction of collagen gel matrices by human dermal fibroblasts with tenascin-C expression

Retinoids are strong tissue modifiers and have been used to treat severe acne, keloids and photo-aged skin. Tocoretinate (TR), ester bound retinoic acid and tocopherol, has been topically applied for skin ulcers and, more recently, for sclerotic skin diseases. To clarify the mechanism of tissue softening by retinoids and TR, we investigated their effects on the contraction of hydrated type-1 collagen gel matrices by human dermal fibroblasts and on tenascin-C expression. TR, 13-cis-retinoic acid/isotretinoin and all trans-retinoic acid significantly inhibited collagen gel matrices contraction at concentrations from 10(-4) to 10(-8) M without significant changes of the fibroblast growth. TR and the other two retinoids dose-dependently induced tenascin-C expression in the fibroblasts. Since tenascin-C is involved in cellular detachment and tissue remodeling, these results suggest that TR and other retinoids down-regulated the tensile tension of fibroblasts in collagen gel matrices by the induction of tenascin-C.     

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.     

Indication of fibroblast apoptosis during the maturation of disc-shaped mechanically stressed collagen lattices

Human dermal fibroblasts seeded in tense disc-shaped collagen gels organized gradually into a tissue composed of three distinct superimposed layers of cells: a basal layer of aligned myofibroblasts, a middle layer of unoriented fibroblasts, and an upper layer of myofibroblasts oriented orthogonally to the basal myofibroblasts. Confocal observation of -smooth muscle actin (-SMA) immunolabelling as a marker of myofibroblasts showed that the upper myofibroblasts disappeared during maturation of the lattices. Observation of morphological modifications of cells, typical chromatin condensation identified by Hoechst 33258 staining, and detection of ssDNA after formamide-induced denaturation suggested the involvement of apoptosis in myofibroblast disappearance. It is suggested that the model of disc-shaped stressed collagen lattice is thus able to mimic conditions in wound repair: on the one hand, wound contraction during which fibroblasts undergo mechanical stress and, on the other, apoptotic disappearance of cells at the end of tissue retraction.     

A TGF-beta1-dependent autocrine loop regulates the structure of focal adhesions in hypertrophic scar fibroblasts

Following injury, fibroblasts migrate into wounds and differentiate into alpha smooth muscle cell actin (SMCA)-positive cells, termed myofibroblasts, that assemble and remodel the scar. Cultured myofibroblasts assemble larger focal adhesions than do normal dermal fibroblasts and these focal adhesions attach to alpha SMCA-rich stress fibers. Following severe traumatic or thermal injury to the dermis, hypertrophic scars (HTSs) often develop and these scar fibroblasts (HTSFs) express alpha SMCA persistently. We now report that HTSFs stably display large focal adhesions as a consequence of both the autocrine production and activation of transforming growth factor beta1 (TGF-beta1). We also observe that myofibroblasts elaborating larger focal adhesions adhere more tightly to fibronectin. Conditioned medium from HTSFs induces focal adhesion growth in normal fibroblasts and this is blocked by pre-incubation with a soluble TGF-beta1 receptor mimetic. Human foreskin fibroblasts transduced with a retrovirus encoding active TGF-beta1 elaborate large focal adhesions, whereas fibroblasts overexpressing normal, latent TGF-beta1 do not. We conclude that the large focal adhesions found in pathogenic myofibroblasts arise through an autocrine loop involving the production and activation of TGF-beta1; these adhesions likely mediate both tighter adhesion to wound matrix and the exuberant wound contraction observed in pathogenic scars.     

Fibroblasts and myofibroblasts in wound healing: Force generation and measurement

Fibroblasts are one of the most abundant cell types in connective tissues. These cells are responsible for tissue homeostasis under normal physiological conditions. When tissues are injured, fibroblasts become activated and differentiate into myofibroblasts, which generate large contractions and actively produce extracellular matrix (ECM) proteins to facilitate wound closure. Both fibroblasts and myofibroblasts play a critical role in wound healing by generating traction and contractile forces, respectively, to enhance wound contraction. This review focuses on the mechanisms of force generation in fibroblasts and myofibroblasts and techniques for measuring such cellular forces. Such a topic was chosen specifically because of the dual effects that fibroblasts/myofibroblasts have in wound healing process– a suitable amount of force generation and matrix deposition is beneficial for wound healing; excessive force and matrix production, however, result in tissue scarring and even malfunction of repaired tissues. Therefore, understanding how forces are generated in these cells and knowing exactly how much force they produce may guide the development of optimal protocols for more effective treatment of tissue wounds in clinical settings.     

Exogenous Smad3 accelerates wound healing in a rabbit dermal ulcer model

Exogenous administration of transforming growth factor-beta (TGF-beta) improves wound healing by affecting cellular and molecular events involved in tissue repair. But mice with a deficiency of a key TGF-beta signaling intermediate, Smad3, paradoxically showed accelerated cutanenous wound healing, suggesting that endogenous Smad3 had inhibitory effect on cutaneous wound healing. Here we investigated the effect of exogenous expression of Smad3 in dermal fibroblasts on cutaneous wound healing. Subcutaneous injection of adenovirus-containing Smad3 complementary DNA (AdCMV-Smad3) targeting mainly dermal fibroblasts accelerated tissue repair following full-thickness dermal round wounds in rabbit ear as judged by the size of granulation tissue area, number of capillaries, and re-epithelialization rate of the wounds. Expressions of alpha-smooth muscle actin (alpha-SMA), vascular endothelial growth factor (VEGF), and fibroblast growth factor receptor were upregulated in the wounded area injected with AdCMV-Smad3. Consistent with the in vivo findings, overexpression of Smad3 induced alpha-SMA, VEGF, and TGF-beta1 expression and augmented chemotactic response in cultured dermal fibroblasts. Therefore, exogenous administration of Smad3 targeting dermal fibroblasts accelerated tissue repair in a rabbit dermal ulcer model by affecting fibroblast responses associated with wound healing. The results suggest that Smad3, when overexpressed in dermal fibroblasts, can promote wound healing.     

Inducible lineage-specific deletion of TbetaRII in fibroblasts defines a pivotal regulatory role during adult skin wound healing

Previous attempts to delete type II TGFbeta receptor (TbetaRII) in fibroblasts have precluded examination of adult mice due to early mortality. We have selectively deleted TbetaRII postnatally in differentiated connective tissue fibroblasts using an inducible Cre-Lox strategy. Tamoxifen-dependent Cre recombinase linked to a fibroblast-specific regulatory sequence from the proalpha2(I)collagen gene permitted deletion of floxed TbetaRII alleles. After postnatal deletion of TbetaRII in fibroblasts, healing of excisional skin wounds in adults showed markedly attenuated dermal scar formation, defective wound contraction and enhanced epidermal proliferation. These findings support a pivotal role for transforming growth factor beta (TGFbeta) signalling in fibroblasts in regulating normal skin wound healing. Explanted dermal fibroblasts from TbetaRII-null-fib mice showed impaired migration and did not generate normal contractile biomechanical forces in fixed collagen gels nor develop alpha-smooth muscle antigen-rich stress fibers in response to TGFbeta1. Surprisingly, some TGFbeta-regulated proteins, including connective tissue growth factor (CTGF), were basally upregulated in TbetaRII-null fibroblasts and this was dependent on extracellular signal-regulated kinase 1/2 activity in these cells. This suggests that other intracellular pathways regulating CTGF expression may partially compensate for disruption of TGFbeta signalling in fibroblasts. Together, our data confirm that expression of TbetaRII in differentiated dermal fibroblasts is essential for normal wound healing and demonstrate a critical role in the development and function of myofibroblasts.     

Characterization of biologic properties of wound fluid collected during early stages of wound healing.

The clinical effects of occlusive dressings on wound healing are well documented. However, the underlying biologic mechanisms associated with moist healing are not well understood. Experimental studies and clinical experience have shown enhanced eschar and clot removal, re-epithelialization, and collagen synthesis under occlusion, suggesting the possibility of elevated activities of proteinases and other effectors, e.g., growth factors, in the moist wound environment. To gain an insight into the biology of early wounds under occlusion, we have carried out biologic and biochemical analyses on fluids from occluded full- and partial-thickness wounds. Metalloproteinase activities were detected in the wound fluid samples. When applied to cultured dermal fibroblasts, mitogenic activity was observed with fluids from full-thickness wounds. Wound fluid-stimulated accumulation of urokinase-type plasminogen activator by fibroblasts was also observed in a time-dependent manner. Stimulation of metalloproteinase accumulation by fibroblasts was also observed. We have further demonstrated the presence of platelet-derived growth factor-like and basic fibroblast growth factor-like factors in wound fluid by antibody neutralization of their biologic activities. Proteinase presence and proteinase stimulatory activity of wound fluid retained in the occluded wound may contribute to an enhanced proteolytic environment in these wounds in comparison to non-occluded "dry" wounds. The presence of growth factors and the potential abilities of proteinases to activate latent growth factors and generate chemotactic peptides through connective tissue breakdown may also contribute to the enhanced healing of occluded wounds.     

Decellularized dermis in combination with cultivated keratinocytes in a short- and long-term animal experimental investigation

Decellularized human dermis as a potentially ideal scaffold for dermal substitution in severe burns was examined in a two‐staged animal experiment. In an initial step, an in vitro generated composite graft consisting of human keratinocytes and decellularized dermis (AlloDerm®) was transplanted onto nude mice in a short‐term trial (n = 20, 14 days). Subsequently, a combined one‐step grafting of full thickness wounds with both decellularized dermis (in part preincubated with fibroblasts) and cultivated autologous keratinocytes as a cell suspension in fibrin glue was done in a long‐term porcine animal model (n = 10, 6 months). In both series, macroscopic wound healing was evaluated by planimetry. Histological investigations included morphological as well as immunohistochemical parameters. The short‐term study showed both successful integration of the composite grafts and reduction of wound contraction compared with the control group (epithelial grafts). The long‐term porcine study displayed reduced myofibroblast formation and contraction in the wounds that had been treated with fibroblast‐preincubated dermis. After 4 weeks, a decline of the structural integrity of the dermal matrix could be noticed. The utility of decellularized dermis as template for both dermal reconstitution and keratinocyte delivery vehicle was shown. The closure of full thickness wounds by a single‐step combination of an autologous keratinocyte fibrin sealant suspension and acellular dermis in a pig animal model could be shown. Incorporation of fibroblasts led to reduced wound contraction but could not prevent the loss of dermal integrity. The engineered ‘skin’ remained viable and stable over a period of 6 months.     

Keratinocytes suppress transforming growth factor-beta1 expression by fibroblasts in cultured skin substitutes

Transforming growth factor (TGF)-beta1 is a multipotent growth factor with an important role in tissue homeostasis. This growth factor regulates cell proliferation, adhesion, migration and differentiation, as well as extracellular matrix deposition. The temporal secretion and activation of latent TGF-beta1 is thus of major importance to physiological and pathological processes and in wound healing and tumour formation. Cultured skin substitutes, as used to treat extensive acute or chronic skin wounds, offer an attractive model to investigate cellular interactions in cytokine and growth factor expression and response in vitro. In the present investigation, expression of TGF-beta1 was analysed in keratinocyte, fibroblast and melanocyte monolayer cultures, as well as in the dermal vs. epidermal components of reconstituted human skin. Immunohistology, enzyme-linked immunosorbent assay (ELISA) and Northern blotting were used to demonstrate expression at the RNA and protein level. In the monolayer cultures, levels of TGF-beta1 synthesized by melanocytes were observed to be considerably elevated when compared with keratinocytes. Most TGF-beta1, however, was secreted by fibroblasts. The relative contribution of the epidermal and dermal components of the skin substitutes to overall TGF-beta1 levels was determined by comparing results obtained for either component in the presence and absence of fibroblasts and keratinocytes. From results obtained by ELISA it was apparent that TGF-beta1 levels generated predominantly by fibroblasts within the skin substitutes were greatly reduced over time in the presence of keratinocytes. Suppression of fibroblast TGF-beta1 expression in the presence of keratinocytes was also demonstrable at the RNA level by Northern blotting. Results obtained by immunohistochemistry suggest that most, if not all, of the growth factor was present in the latent form. It is therefore most likely that the observed effect results from a factor secreted by keratinocytes, which is capable of suppressing TGF-beta1 synthesis by fibroblasts. These results suggest that expression of TGF-beta1 by fibroblasts is downregulated by paracrine actions of keratinocytes in healing skin.     

Reduced wound contraction and scar formation in punch biopsy wounds. Native collagen dermal substitutes. A clinical study

In full-thickness skin wounds dermal regeneration usually fails, resulting in scar formation and wound contraction. We studied dermal regeneration by implantation of collagenous matrices in a human punch biopsy wound model. Matrices were made of native bovine collagen 1 fibres, and either hyaluronic acid, fibronectin, or elastin was added. Matrices were placed in 6-mm punch biopsy holes in seven patients (biopsies were used for the grafting of leg ulcers), and covered with a protective semi-permeable polyether urethane membrane. Histology, wound contraction and dermal architecture were studied. Dermal architecture was evaluated using a recently developed laser scatter technique. All collagen matrices showed a tendency to reduce wound contraction, compared with control wounds; elastin- and fibronectin-treated matrices showed significantly less contraction than control wounds. Only the addition of elastin had a clear beneficial effect on dermal architecture; collagen bundles were more randomly organized, compared with control wounds, and wounds treated with collagen matrices coated with fibronectin or hyaluronic acid, or without coating. We conclude that the punch biopsy wound model provides important information on dermal regeneration in humans. Native collagen matrices with elastin contributed to dermal regeneration and reduced wound contraction, in contrast with matrices coated with fibronectin or hyaluronic acid, or without coating. Future clinical studies of large-area, full-thickness wounds will be required to establish their clinical relevance for leg ulcer and burn treatment.     

Autologous full-thickness skin substitute for healing chronic wounds

BACKGROUND: Chronic wounds represent a major problem to our society. Therefore, advanced wound-healing strategies for the treatment of these wounds are expanding into the field of tissue engineering. OBJECTIVES: To develop a novel tissue-engineered, autologous, full-thickness skin substitute of entirely human origin and to determine its ability to heal chronic wounds. METHODS: Skin substitutes (fully differentiated epidermis on fibroblast-populated human dermis) were constructed from 3-mm punch biopsies isolated from patients to be treated. Acellular allodermis was used as a dermal matrix. After a prior 5-day vacuum-assisted closure therapy to prepare the wound bed, skin substitutes were applied in a simple one-step surgical procedure to 19 long-standing recalcitrant leg ulcers (14 patients; ulcer duration 0.5-50 years). RESULTS: The success rate in culturing biopsies was 97%. The skin substitute visibly resembled an autograft. Eleven of the 19 ulcers (size 1-10 cm2) healed within 8 weeks after a single application of the skin substitute. The other eight larger (60-150 cm2) and/or complicated ulcers healed completely (n = 5) or continued to decrease substantially in size (n = 3) after the 8-week follow-up period. Wound healing occurred by direct take of the skin substitute (n = 12) and/or stimulation of granulation tissue/epithelialization (n = 7). Skin substitutes were very well tolerated and pain relief was immediate after application. CONCLUSIONS: Application of this novel skin substitute provides a promising new therapy for healing chronic wounds resistant to conventional therapies.     

Periostin induces fibroblast proliferation and myofibroblast persistence in hypertrophic scarring

Hypertrophic scarring is characterized by the excessive development and persistence of myofibroblasts. These cells contract the surrounding extracellular matrix resulting in the increased tissue density characteristic of scar tissue. Periostin is a matricellular protein that is abnormally abundant in fibrotic dermis, however, its roles in hypertrophic scarring are largely unknown. In this report, we assessed the ability of matrix‐associated periostin to promote the proliferation and myofibroblast differentiation of dermal fibroblasts isolated from the dermis of hypertrophic scars or healthy skin. Supplementation of a thin type‐I collagen cell culture substrate with recombinant periostin induced a significant increase in the proliferation of hypertrophic scar fibroblasts but not normal dermal fibroblasts. Periostin induced significant increases in supermature focal adhesion formation, α smooth muscle actin levels and collagen contraction in fibroblasts cultured from hypertrophic scars under conditions of increased matrix tension in three‐dimensional type‐I collagen lattices. Inhibition of Rho‐associated protein kinase activity significantly attenuated the effects of matrix‐associated periostin on hypertrophic scar fibroblasts and myofibroblasts. Depletion of endogenous periostin expression in hypertrophic scar myofibroblasts resulted in a sustained decrease in α smooth muscle actin levels under conditions of reducing matrix tension, while matrix‐associated periostin levels caused the cells to retain high levels of a smooth muscle actin under these conditions. These findings indicate that periostin promotes Rho‐associated protein kinase‐dependent proliferation and myofibroblast persistence of hypertrophic scar fibroblasts and implicate periostin as a potential therapeutic target to enhance the resolution of scars.     

A novel model of wound healing in the SCID mouse using a cultured human skin substitute

Studies of skin graft behaviour in rodent excisional wound models are limited by the dominance of wound contracture and graft sloughing as primary healing responses. To slow skin contraction, polytetrafluoroethylene (Teflon) rings were inserted into dorso-lateral full-thickness wounds in SCID mice. Cultured skin substitutes (OrCel), composed of cultured human keratinocytes and fibroblasts in a bovine collagen sponge, were implanted within the rings. Examination and histology of grafts 14 days later showed graft take in four of six recipients, with 90% epithelialization and wound contraction of 31–47%. Immunohistochemical studies, using human-specific antisera to distinguish graft from host tissues, showed that regenerated tissue was predominantly human. Staining with anticytokeratin, revealed a multilayered, stratified neoepidermis. HBG were identified in keratinocytes in all epidermal layers. Langerhans cells were absent. Antihuman vimentin, used as a fibroblast marker, confirmed that cells of the neodermis were primarily of human origin. Neoepidermal keratinocytes, primarily in the basal and suprabasal layers, were also stained. Results suggest that the poly(tetrafluoroethylene) ring inhibited graft sloughing and provided a more favourable environment for the skin substitute to regenerate a substantially normal human skin.