Search

We hypothesised that a feedback mechanism of the transforming growth factor (TGF)-β1 signalling pathway, triggered by high-level TGF-β1, activates platelet-rich plasma (PRP) release to reduce connective tissue growth factor (CTGF) production and expression of CTGF mRNA in hypertrophic scar dermal fibroblasts. Primary dermal fibroblasts were isolated from cultures of hypertrophic scars. Cells were cultured after addition of serum-free Dulbecco's modified Eagle's medium supplemented with 5% (wt/vol) PRP or platelet-poor plasma (PPP). At 1, 4, 6, 8, 11, and 13 days after addition of PRP or PPP, the TGF-β1 and CTGF levels in supernatants were determined using solid-phase enzyme-linked immunosorbent assays. Quantitative reverse transcription polymerase chain reactions were performed to quantify TGF-β1 and CTGF mRNA expression levels. TGF-β1 mRNA expression in the PRP groups was lower than in the PPP groups from 4 to 13 days of culture, and there was statistically significant difference (P < .01). CTGF level and mRNA expression in the PRP groups was lower than in the PPP groups, and there were statistically significant differences (P < .01). Although further experiments will focus on clarifying the second messenger of the TGF-β1 negative feedback mechanism, the in vitro data presented show that PRP can potentially reduce CTGF and CTGF gene transcription by triggering the TGF-β1 signalling negative feedback mechanism., (© 2018 Medicalhelplines.com Inc and John Wiley & Sons Ltd.)

Mechanical strain is a key contributor in the pathogenesis of hypertrophic scarring, whose optimal stretch magnitudes to initiate the differentiation of normal skin fibroblasts into aberrant fibroblasts phenotype remains largely unresolved. Influence of varying cyclic strain magnitudes on cultured human normal skin fibroblasts and its transformation into hypertrophic scar fibroblast-like phenotype is investigated in this study. Cultured fibroblasts isolated from hypertrophic scar and normal skin tissue were subjected to cyclic mechanical stretching under individual 10%, 15%, and 20% strain magnitudes at a frequency of 0.1 Hz for 24 hours. Stretched normal skin fibroblasts demonstrated significantly increased rates of cell proliferation, and also apparently oriented away nearly perpendicular to the applied stretching direction. Interestingly, the applied 10% strains magnitude resulted in a markedly enhanced cell proliferative ability compared with that of 20% strain magnitude. Parameters involving the mechanotransduction signaling, such as integrin β1 and P130Cas, were significantly improved at both mRNA and protein levels in the stretched normal skin fibroblasts, which was demonstrated in a negative magnitude-dependent manner. In addition, 10% strains magnitude triggered the highest expression levels of growth factor TGF-β1 and collagen matrix in stretched normal skin fibroblasts. Collectively, these results indicate that the 10% stretching magnitude, of the 3 strain magnitudes studied, is most effective for triggering the optimal mechanotransduction effects and biological responses inside cultured skin fibroblasts. The demonstrable conversion of normal skin fibroblasts into hypertrophic scar fibroblasts was also observed when 10% stretching magnitude was applied to cultured fibroblasts in vitro.

Burn injury-mediated destruction of the skin barrier normally induces microbial invasion, in turn leading to the development of systemic infection and occasional septic shock by the release of endotoxins. The objective of this work was to study the influence of lipopolysaccharide (LPS) on the biological characteristics of normal skin fibroblasts and to elucidate the influence of LPS in the initial stage of skin wound healing. Twenty patients with hypertrophic scar in proliferative stage were selected randomly and primary cultures were established from fibroblasts derived from their hypertrophic scar tissue and normal skin. Normal skin fibroblasts of passage 3 were stimulated with different concentrations of LPS. LPS stimulated the proliferation and collagen synthesis of fibroblasts within a certain extent of concentrations (0.005-0.5 μg/mL) (P < 0.05), whereas at a concentration of 1 μg/mL inhibited the proliferation and collagen synthesis of fibroblasts (P < 0.05). Collagen synthesis by normal skin fibroblasts after LPS stimulation mimicked those derived from hypertrophic scar tissue. LPS of 0.1 μg/mL had significant effect on normal skin fibroblasts-continuous passage of these fibroblasts resulted in ultrastructural pattern similar to fibroblasts derived from hypertrophic scar tissue, and the findings was substantiated by hematoxylin and eosin staining and immunohistochemistry detection of proliferation cell nuclear antigen, type I procollagen and α-smooth muscle actin. Our results suggest that LPS might convert normal skin fibroblasts to hypertrophic scar tissue fibroblasts and participate in the formation of hypertrophic scar; hence, appropriate concentration of LPS may have no effect or be beneficial to skin wound healing, whereas excessive concentration of LPS may delay the time of wound healing., (Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.)

Background: The prominent inflammatory cell identified in excessive scarring is the mast cell. Hypertrophic scar exhibits myofibroblasts derived from the transformation of fibroblasts, increased collagen synthesis, and stationary nonmigratory resident cells. The co-culture of fibroblasts with an established rat mast cell line (RMC-1) was used to explore the hypothesis of whether mast cells through gap junctional intercellular communications guide fibroblasts in promoting excessive scarring., Methods: Human dermal fibroblasts were cultured alone or co-cultured with RMC-1 cells as is or with either blocked gap junctional intercellular communications or devoid of cytoplasmic granules. Collagen synthesis was analyzed by dot blot analysis; immunohistology identified myofibroblasts, and a cell migration assay measured fibroblast locomotion., Results: Fibroblasts co-cultured with RMC-1 cells transformed into myofibroblasts, had increased collagen synthesis, and showed retarded cell migration. In contrast, RMC-1 cells unable to form gap junctional intercellular communications were similar to fibroblasts alone, failing to promote these activities. Degranulated RMC-1 cells were as effective as intact RMC-1 cells., Conclusions: Mast cells induce fibroblast activities associated with hypertrophic scarring through gap junctional intercellular communications. Eliminating the mast cell or its gap junctional intercellular communications with fibroblasts may be a possible approach in preventing hypertrophic scarring or reducing fibrotic conditions.

Dermal papilla cells (DPCs) show phenotypic plasticity during wound healing. The multipotency of DPCs is well recognized, but the signaling pathways that regulate the differentiation of these cells into fibroblasts are poorly understood. A preliminary experiment showed that transforming growth factor beta1 (TGF-β1) can induce DPCs to differentiate into fibroblast-like cells, which suggests that DPCs may be a source of wound-healing fibroblasts. Bone morphogenetic protein-7 (BMP-7), a member of the TGF-β superfamily, can prevent and reverse fibrosis by counteracting the TGF-β1-mediated profibrotic effect. To determine whether BMP-7 attenuates the TGF-β1-induced differentiation of DPCs into fibroblasts, we established an in vitro system for DPC differentiation and recorded the gene expression patterns that distinguished DPCs from fibroblasts. The proportion of fibroblast-like cells was significantly enhanced in DPCs treated with TGF-β1, as evidenced by immunocytochemistry, flow cytometry, quantitative real-time reverse transcriptase polymerase chain reaction, and Western blot analysis. BMP-7 and TGF-β1 administration substantially decreased fibroblast-like differentiation, indicating inhibition of TGF-β1-induced differentiation. The antagonistic BMP-7- and TGF-β1-activated signaling pathways can be used to promote wound healing or suppress hypertrophic scarring., (© 2013 by the Wound Healing Society.)

To explore further the role of substance P (SP) in wound healing and scar formation, SP concentrations in wounds of scalded rats were assayed. Expressions of apoptosis-associated genes in fibroblasts cultured with SP were detected. SP concentrations in superficial wounds increased earlier than those in deep wounds. SP was associated with an increased proliferation and a decreased apoptosis of fibroblasts. It had a greater influence on keloid fibroblasts than on hypertrophic scar fibroblasts by elevating the expression of proliferating cell nuclear antigen and BCL-2 in fibroblasts. Spantide completely suppressed the effects of SP on hypertrophic scar fibroblasts, and partly inhibited its effects on keloid scar fibroblasts. SP may play an important role in wound healing by promoting wound fibroblast proliferation and inhibiting apoptosis. It may also participate in pathological scar formation by modulating the expression of apoptosis-associated genes. SP is postulated to play a dual role in wound repair.

Hypertrophic scar and keloids are fibroproliferative disorders of the skin which occur often unpredictably, following trauma and inflammation that compromise cosmesis and function and commonly recur following surgical attempts for improvement. Despite decades of research in these fibrotic conditions, current non-surgical methods of treatment are slow, inconvenient and often only partially effective. Fibroblasts from these conditions are activated to produce extracellular matrix proteins such as collagen I and III, proteoglycans such as versican and biglycan and growth factors, including transforming growth factor-beta and insulin like growth factor I. However, more consistently these cells produce less remodeling enzymes including collagenase and other matrix metalloproteinases, as well as the small proteoglycan decorin which is important for normal collagen fibrillogenesis. Recently, the systemic response to injury appears to influence the local healing process whereby increases in Th2 and possibly Th3 cytokines such as IL-2, IL-4 and IL-10 and TGF-beta are present in the circulating lymphocytes in these fibrotic conditions. Finally, unique bone marrow derived cells including mesenchymal and endothelial stem cells as well as fibrocytes appear to traffic into healing wounds and influence the healing tissue. On this background, clinicians are faced with patients who require treatment and the pathophysiologic basis as currently understood is reviewed for a number of emerging modalities.

Rho kinase is an enzyme that has been implicated in pathological wound healing. In our study, we aimed to characterize the dosages of Y-27632 (a competitive inhibitor of rho kinase) at which fibroblast stress fiber formation is inhibited, to determine the effect of Y-27632 on fibroblast confluence, and to determine the effect of varying concentrations of Y-27632 on fibroblast-mediated gel contraction. First, 3T3 fibroblasts were exposed to various concentrations of Y-27632, and stress fiber dissolution was assessed with immunofluorescence. Next, 10 microM Y-27632 was applied to 3T3 fibroblast monolayer cultures immediately after plating and at 8 hours after plating to determine the effect on fibroblast confluence. Finally, a three-dimensional model was used to assess the effect of Y-27632 on gel contraction. Increasing doses of Y-27632 resulted in a dose-dependent increase in stress fiber dissolution as well as a dose-dependent inhibition of gel contraction. Median number of days to confluence were 3, 5, and 6 after no, immediate, and delayed incubation with Y-27632, respectively (P < .05). Our results further characterize the dosages at which Y-27632 may be most effective in the modulation of wound contraction in burn patients. In addition, our confluence study suggests that patients need not necessarily be pretreated with Y-27632.

Background: Expression of connexins and other constituent proteins of gap junctions along with gap junctional intercellular communication are involved in cellular development and differentiation processes. In addition, an increasing number of hereditary skin disorders appear to be linked to connexins. Therefore, in this report, the authors studied in vitro gap junctional intercellular communication function and connexin expression in fibroblasts derived from keloid and hypertrophic scar patients., Methods: Fibroblasts harvested from each of six keloid and hypertrophic scar patients were used for this study. Gap junctional intercellular communication function was investigated using the gap fluorescence recovery after photobleaching method, and expression of connexin proteins was studied using quantitative confocal microscopic analyses., Results: Compared with normal skin, a decreased level of gap junctional intercellular communication was seen in fibroblasts derived from hypertrophic scar tissue, whereas an extremely low gap junctional intercellular communication level was detected in fibroblasts derived from keloid tissue. We also detected little connexin 43 (Cx43) protein localized in fibroblasts derived from keloids. Moreover, Cx43 protein levels were much lower in fibroblasts derived from hypertrophic scars than in those derived from normal skin., Conclusions: The authors' data suggest that the loss of gap junctional intercellular communication and connexin expression may affect intercellular recognition and thus break the proliferation and apoptosis balance in fibroblasts derived from keloid and hypertrophic scar tissue.

Fibrocytes are a unique leukocyte subpopulation implicated in wound healing. They are derived from peripheral blood mononuclear cells, display fibroblast-like properties, and synthesize extracellular matrix macromolecules. This study investigated whether fibrocytes are present in healing burn wounds and whether the number of fibrocytes in tissue correlates with the degree of burn injury and the development of hypertrophic scar. Proteins extracted from cultured fibrocytes and nonadherent lymphocytes were found to be similar using two-dimensional gel electrophoresis and quite distinct from those obtained from fibroblasts. However, one protein, identified as leukocyte-specific protein 1 using mass spectrometric peptide mapping, was found in significantly larger amounts in fibrocytes than in lymphocytes but was undetectable in fibroblasts. Double immunostaining with antibodies to leukocyte-specific protein-1 and to the N-terminal propeptide of type I collagen was performed on cryosections of hypertrophic scar, mature scar, and normal skin. Fibrocytes were seen in scar tissue as dual-labeled spindle-shaped cells but were absent from normal skin. Moreover, the number of fibrocytes was higher in hypertrophic than in mature scar tissue. We conclude that fibrocytes, which have been reported to be antigen-presenting cells, are recruited to wounds following extensive burn injury and could potentially upregulate the inflammatory response and synthesize collagen and other matrix macromolecules, thus contributing to the development of hypertrophic scarring.

Wound contraction in humans has both positive and negative effects. It is beneficial to wound healing by narrowing the wound margins, but the formation of undesirable scar contracture brings cosmetic and even functional problems. The entire mechanism of wound healing and scar contracture is not clear yet, but it is at least considered that both the fibroblasts and the myofibroblasts are responsible for contraction in healing wounds. The myofibroblast is a cell that possesses all the morphologic and biochemical characteristics of both a fibroblast and a smooth muscle cell. Normally, the myofibroblasts appear in the initial wound healing processes and generate contractile forces to pull both edges of an open wound until it disappears by apoptosis. But as an altered regulation of myofibroblast disappearance, they remain in the dermis and continuously contract the scar, eventually causing scar contracture. In this research, to compare and directly evaluate the influence on scar contracture of the myofibroblast versus the fibroblast, dermal tissues were taken from 10 patients who had highly contracted hypertrophic scars. The myofibroblasts were isolated and concentrated from the fibroblasts using the magnetic activating cell-sorting column to obtain the myofibroblast group, which contained about 28 to 41 percent of the myofibroblasts, and the fibroblast group, which contained less than 0.9 percent of the myofibroblasts. Each group was cultured in the fibroblast-populated collagen lattice for 13 days, and the contraction of the collagen gel was measured every other day. In addition, they were selectively treated with tranilast [N-(3',4'-dimethoxycinnamoyl) anthranilic acid] to evaluate the influence on the contraction of the collagen gel lattice. During the culture, the myofibroblast group, compared with the fibroblast group, showed statistically significant contraction of the collagen gel lattice day by day, except on the first day, and only the myofibroblast group was affected by tranilast treatment, showing significant inhibition of gel contraction. By utilizing an in vitro model, the authors have demonstrated that myofibroblasts play a more important role in the contracture of the hypertrophic scar.

The alteration of normal dermal fibroblast function that leads to the development of hypertrophic scar after thermal injury is unknown. To determine functional differences that might explain this process, fibroblasts were cultured from biopsies of post-thermal injury mature hypertrophic scars and patient-matched normal skin. The mitogenic responses of scar cells to fetal bovine serum, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and tumor necrosis factor alpha (TNF alpha) were determined and compared to normal skin cells. Collagen synthetic rate was also compared in the presence and absence of transforming growth factor beta 1 (TGF beta 1). Whereas both scar and normal cells responded with increased thymidine uptake to serum and cytokines, the stimulation to EGF and serum was significantly lower in scar cells. In contrast, synthesis of collagen, but not of non-collagenous proteins, was increased in scar relative to normal cells, both basally and when stimulated with low doses of TGF beta 1. Additionally, the fraction of protein synthesized as collagen was significantly higher in scar fibroblasts. These results suggest that fibroblasts from hypertrophic scars demonstrate stable phenotypic differences in cytokine responsiveness in comparison to cells from unaffected skin. The increased rate of collagen synthesis and decreased responsiveness to mitogens are consistent with the increased extracellular matrix content and decreased cellularity of hypertrophic scars.