Sensitivity of a two-dimensional biomorphoelastic model for post-burn contraction

We consider a two-dimensional biomorphoelastic model describing post-burn scar contraction. This model describes skin displacement and the development of the effective Eulerian strain in the tissue. Besides these mechanical components, signaling molecules, fibroblasts, myofibroblasts, and collagen also play a significant role in the model. We perform a sensitivity analysis for the independent parameters of the model and focus on the effects on features of the relative surface area and the total strain energy density. We conclude that the most sensitive parameters are the Poisson’s ratio, the equilibrium collagen concentration, the contraction inhibitor constant, and the myofibroblast apoptosis rate. Next to these insights, we perform a sensitivity analysis where the proliferation rates of fibroblasts and myofibroblasts are not the same. The impact of this model adaptation is significant.

     

A mathematical model for the simulation of the formation and the subsequent regression of hypertrophic scar tissue after dermal wounding

A continuum hypothesis-based model is presented for the simulation of the formation and the subsequent regression of hypertrophic scar tissue after dermal wounding. Solely the dermal layer of the skin is modeled explicitly and it is modeled as a heterogeneous, isotropic and compressible neo-Hookean solid. With respect to the constituents of the dermal layer, the following components are selected as primary model components: fibroblasts, myofibroblasts, a generic signaling molecule and collagen molecules. A good match with respect to the evolution of the thickness of the dermal layer of scars between the outcomes of simulations and clinical measurements on hypertrophic scars at different time points after injury in human subjects is demonstrated. Interestingly, the comparison between the outcomes of the simulations and the clinical measurements demonstrates that a relatively high apoptosis rate of myofibroblasts results in scar tissue that behaves more like normal scar tissue with respect to the evolution of the thickness of the tissue over time, while a relatively low apoptosis rate results in scar tissue that behaves like hypertrophic scar tissue with respect to the evolution of the thickness of the tissue over time. Our ultimate goal is to construct models with which the properties of newly generated tissues that form during wound healing can be predicted with a high degree of certainty. The development of the presented model is considered by us as a step toward their construction.     

Pirfenidone attenuates the profibrotic contractile phenotype of differentiated human dermal myofibroblasts

Dysregulated wound healing after burn injury frequently results in debilitating hypertrophic scarring and contractures. Myofibroblasts, the main effector cells for dermal fibrosis, develop from normal fibroblasts via transforming growth factor beta 1 (TGF-β1). During wound healing, myofibroblasts produce extracellular matrix (ECM) proteins, modulate ECM stability, and contract the ECM using alpha smooth muscle actin (α-SMA) in contractile stress fibers. The antifibrotic pirfenidone has previously been shown to inhibit the initial differentiation of fibroblasts into myofibroblasts in vitro and act as a prophylactic measure against hypertrophic scar development in a mouse burn model. To test whether pirfenidone affects differentiated myofibroblasts, we investigated the in vitro effects of pirfenidone treatment after three to five days of stimulation with TGF-β1. In assays for morphology, protein and gene expression, and contractility, pirfenidone treatment produced significant effects. Profibrotic gene expression returned to near-normal levels, further α-SMA protein expression was prevented, and cell contraction within a stressed collagen matrix was reduced. These in vitro results promote pirfenidone as a promising antifibrotic agent to treat existing scars and healing wounds by mitigating the effects of differentiated myofibroblasts.     

A biomechanical mathematical model for the collagen bundle distribution-dependent contraction and subsequent retraction of healing dermal wounds

A continuum hypothesis-based, biomechanical model is presented for the simulation of the collagen bundle distribution-dependent contraction and subsequent retraction of healing dermal wounds that cover a large surface area. Since wound contraction mainly takes place in the dermal layer of the skin, solely a portion of this layer is included explicitly into the model. This portion of dermal layer is modeled as a heterogeneous, orthotropic continuous solid with bulk mechanical properties that are locally dependent on both the local concentration and the local geometrical arrangement of the collagen bundles. With respect to the dynamic regulation of the geometrical arrangement of the collagen bundles, it is assumed that a portion of the collagen molecules are deposited and reoriented in the direction of movement of (myo)fibroblasts. The remainder of the newly secreted collagen molecules are deposited by ratio in the direction of the present collagen bundles. Simulation results show that the distribution of the collagen bundles influences the evolution over time of both the shape of the wounded area and the degree of overall contraction of the wounded area. Interestingly, these effects are solely a consequence of alterations in the initial overall distribution of the collagen bundles, and not a consequence of alterations in the evolution over time of the different cell densities and concentrations of the modeled constituents. In accordance with experimental observations, simulation results show furthermore that ultimately the majority of the collagen molecules ends up permanently oriented toward the center of the wound and in the plane that runs parallel to the surface of the skin.

     

Biomedical implications from a morphoelastic continuum model for the simulation of contracture formation in skin grafts that cover excised burns

A continuum hypothesis-based model is developed for the simulation of the (long term) contraction of skin grafts that cover excised burns in order to obtain suggestions regarding the ideal length of splinting therapy and when to start with this therapy such that the therapy is effective optimally. Tissue is modeled as an isotropic, heterogeneous, morphoelastic solid. With respect to the constituents of the tissue, we selected the following constituents as primary model components: fibroblasts, myofibroblasts, collagen molecules, and a generic signaling molecule. Good agreement is demonstrated with respect to the evolution over time of the surface area of unmeshed skin grafts that cover excised burns between outcomes of computer simulations obtained in this study and scar assessment data gathered previously in a clinical study. Based on the simulation results, we suggest that the optimal point in time to start with splinting therapy is directly after placement of the skin graft on its recipient bed. Furthermore, we suggest that it is desirable to continue with splinting therapy until the concentration of the signaling molecules in the grafted area has become negligible such that the formation of contractures can be prevented. We conclude this study with a presentation of some alternative ideas on how to diminish the degree of contracture formation that are not based on a mechanical intervention, and a discussion about how the presented model can be adjusted.     

The effect of myofibroblast on contracture of hypertrophic scar

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.     

Agent-based modelling and parameter sensitivity analysis with a finite-element method for skin contraction

In this paper, we extend the model of wound healing by Boon et al. (J Biomech 49(8):1388–1401, 2016). In addition to explaining the model explicitly regarding every component, namely cells, signalling molecules and tissue bundles, we categorized fibroblasts as regular fibroblasts and myofibroblasts. We do so since it is widely documented that myofibroblasts play a significant role during wound healing and skin contraction and that they are the main phenotype of cells that is responsible for the permanent deformations. Furthermore, we carried out some sensitivity tests of the model by modifying certain parameter values, and we observe that the model shows some consistency with several biological phenomena. Using Monte Carlo simulations, we found that there is a significant strong positive correlation between the final wound area and the minimal wound area. The high correlation between the wound area after 4 days and the final/minimal wound area makes it possible for physicians to predict the most probable time evolution of the wound of the patient. However, the collagen density ratio at the time when the wound area reaches its equilibrium and minimum, cannot indicate the degree of wound contractions, whereas at the 4th day post-wounding, when the collagen is accumulating from null, there is a strong negative correlation between the area and the collagen density ratio. Further, under the circumstances that we modelled, the probability that patients will end up with 5% contraction is about 0.627.

     

Dermal substitution in acute burns and reconstructive surgery: a subjective and objective long-term follow-up.

Tissue engineering and dermal substitution are currently prominent topics of wound-healing research. However, no extensive clinical trials with objective evaluation criteria have been published so far that support the clinical effectiveness of dermal equivalents in the long term. The dermal substitute that is discussed here is derived from bovine collagen and elastin-hydrolysate and has been shown to improve skin elasticity during a short-term clinical follow-up of scar reconstructions. In this study we will present the long-term outcome by means of objective and subjective scar assessment tools for dermal substitution in acute burn wounds and scar reconstructions.In a clinical trial, an intraindividual comparison was performed between the conventional split-thickness autograft and a combination of the collagen/elastin substitute with an autograft. After 1 year, scars were evaluated by the Cutometer SEM 474 for objective elasticity measurements and by planimetry to establish scar contraction. An independent observer subjected scars to a generally accepted clinical scar assessment tool: the Vancouver Scar Scale. In addition, patients gave their impression of the outcome. Forty-two paired burn wounds and 44 paired scar reconstructions were included and evaluated 1 year after surgery.Although substituted scar reconstructions demonstrated an elasticity improvement of approximately 20 percent compared with control wounds, no statistically significant differences were found for skin elasticity, scar contraction, Vancouver Scar Scale, and patient's impression in both categories after 1 year. An extensive long-term follow-up shows that the dermal substitute, which was proven effective in a clinical trial on a short-term basis, did not yield statistical evidence for a long-term clinical effectiveness of dermal substitution.     

A two-compartment mechanochemical model of the roles of transforming growth factor β and tissue tension in dermal wound healing

The repair of dermal tissue is a complex process of interconnected phenomena, where cellular, chemical and mechanical aspects all play a role, both in an autocrine and in a paracrine fashion. Recent experimental results have shown that transforming growth factor -β (TGFβ) and tissue mechanics play roles in regulating cell proliferation, differentiation and the production of extracellular materials. We have developed a 1D mathematical model that considers the interaction between the cellular, chemical and mechanical phenomena, allowing the combination of TGFβ and tissue stress to inform the activation of fibroblasts to myofibroblasts. Additionally, our model incorporates the observed feature of residual stress by considering the changing zero-stress state in the formulation for effective strain. Using this model, we predict that the continued presence of TGFβ in dermal wounds will produce contractures due to the persistence of myofibroblasts; in contrast, early elimination of TGFβ significantly reduces the myofibroblast numbers resulting in an increase in wound size. Similar results were obtained by varying the rate at which fibroblasts differentiate to myofibroblasts and by changing the myofibroblast apoptotic rate. Taken together, the implication is that elevated levels of myofibroblasts is the key factor behind wounds healing with excessive contraction, suggesting that clinical strategies which aim to reduce the myofibroblast density may reduce the appearance of contractures.     

Fetal and postnatal sera differentially modulate human dermal fibroblast phenotypic and functional features in vitro

Fetal wounds heal without scar formation, fibrosis, or contracture. Compared with adult wounds, they are characterized by major differences in the extracellular matrix and the absence of myofibroblastic cells. The reasons for these differences are not well known and determination of factors affecting the absence of scarring in the fetus may lead to strategies for controlling adult pathological scarring. In the present study, we have assessed the effects of serum on the behavior of normal human dermal fibroblasts. Using an in vitro approach, we investigated the effects of fetal and adult serum on cell properties such as growth rate, collagen synthesis, gelatinase activities, and differentiation to myofibroblasts using biochemical, morphological, and ultrastructural parameters. We studied the induction of α-smooth muscle (α-SM) actin in fibroblasts, and its correlation with increased collagen gel contraction by the cells. Our results showed that, compared with FBS (fetal bovine serum), postnatal calf serum (PCS) decreased mitogenic activity and collagenase synthesis but not collagen synthesis. Furthermore, cells cultured with PCS differentiated to myofibroblasts with an increase in cell diameter, number of stress fibers, α-SM actin expression, and collagen gel contraction. To characterize the molecules involved in this differentiation process, the amount of transforming growth factor β (TGFβ) in FBS and PCS was determined and the effect of neutralizing anti-TGFβ antibody was evaluated. It was determined that FBS contained more TGFβ than PCS, but that essentially all the TGFβ was latent in both sera. However, results obtained with anti-TGFβ antibody show that active TGFβ is present when human dermal fibroblasts are cultured with medium containing PCS. These results suggest that, in the presence of PCS but not FBS, the cells either produce active TGFβ or an enzyme that is able to activate latent serum TGFβ. Alternatively, sera may contain two different forms of latent TGFβ, the PCS form being activated by the dermal fibroblast cells. A similar mechanism may be involved, at least in part, in skin wound healing and may underlie the appearance of myofibroblasts in postnatal wounds. J. Cell. Physiol. 171:1–10, 1997. © 1997 Wiley-Liss, Inc.     

Investigating the Role of P311 in the Hypertrophic Scar

The mechanisms of hypertrophic scar formation are not fully understood. We previously screened the differentially expressed genes of human hypertrophic scar tissue and identified P311 gene as upregulated. As the activities of P311 in human fibroblast function are unknown, we examined the distribution of it and the effects of forced expression or silencing of expression of P311. P311 expression was detected in fibroblast-like cells from the hypertrophic scar of burn injury patients but not in peripheral blood mononuclear cells, bone marrow mesenchymal stem cells, epidermal cells or normal skin dermal cells. Transfection of fibroblasts with P311 gene stimulated the expression of alpha-smooth muscle actin (α-SMA), TGF-β1 and α1(I) collagen (COL1A1), and enhanced the contraction of fibroblast populated collagen lattices (FPCL). In contrast, interference of fibroblast P311 gene expression decreased the TGF-β1 mRNA expression and reduced the contraction of fibroblasts in FPCL. These results suggest that P311 may be involved in the pathogenesis of hypertrophic scar via induction of a myofibroblastic phenotype and of functions such as TGF-β1 expression. P311 could be a novel target for the control of hypertrophic scar development.     

IL-10 alleviates lipopolysaccharide-induced skin scarring via IL-10R/STAT3 axis regulating TLR4/NF-κB pathway in dermal fibroblasts

Hypertrophic scar (HS) is a severe fibrotic skin disease. It has always been a major problem in clinical treatment, mainly because its pathogenesis has not been well understood. The roles of bacterial contamination and prolonged wound inflammation were considered significant. IL-10 is a potent anti-inflammatory cytokine and plays a pivotal role in wound healing and scar formation. Here, we investigate whether IL-10 alleviates lipopolysaccharide (LPS)-induced inflammatory response and skin scarring and explore the possible mechanism of scar formation. Our results showed that the expression of TLR4 and pp65 was higher in HS and HS-derived fibroblasts (HSFs) than their counterpart normal skin (NS) and NS-derived fibroblasts (NSFs). LPS could up-regulate the expression of TLR4, pp65, Col I, Col III and α-SMA in NSFs, but IL-10 could down-regulate their expression in both HSFs and LPS-induced NSFs. Blocking IL-10 receptor (IL-10R) or the phosphorylation of STAT3, their expression was up-regulated. In addition, in vitro and in vivo models results showed that IL-10 could alleviate LPS-induced fibroblast-populated collagen lattice (FPCL) contraction and scar formation. Therefore, IL-10 alleviates LPS-induced skin scarring via IL-10R/STAT3 axis regulating TLR4/NF-κB pathway in dermal fibroblasts by reducing ECM proteins deposition and the conversion of fibroblasts to myofibroblasts. Our results indicate that IL-10 can alleviate the LPS-induced harmful effect on wound healing, reduce scar contracture, scar formation and skin fibrosis. Therefore, the down-regulation of inflammation may lead to a suitable scar outcome and be a better option for improving scar quality.     

Topical application of ALK5 inhibitor A-83-01 reduces burn wound contraction in rats by suppressing myofibroblast population

Burn scar contracture that follows the healing of deep dermal burns causes severe deformation and functional impairment. However, its current therapeutic interventions are limited with unsatisfactory outcomes. When we treated deep second-degree burns in rat skin with activin-like kinase 5 (ALK5) inhibitor A-83-01, it reduced wound contraction and enhanced the area of re-epithelialization so that the overall time for wound closing was not altered. In addition, it reduced myofibroblast population in the dermis of burn scar with a diminished deposition of its biomarker proteins such as α-SMA and collagen. Treatment of rat dermal fibroblast with A-83-01 inhibited transforming growth factor-β1 (TGF-β1)-dependent induction of α-SMA and collagen type I. Taken together, these results suggest that topical application of ALK5 inhibitor A-83-01 could be effective in preventing the contraction of burn wound without delaying the wound closure by virtue of its inhibitory activity against the TGF-β-induced increase of myofibroblast population.     

Differences between scar and dermal cultured fibroblasts derived from a patient with recurrent abdominal incision wound herniation.

Fibroblasts were derived from dermis and scar of a 47-year-old white man with a recurrent incisional hernia as a result of fractured ribs. The scar was thin and stretched, suggesting a defect in the maturation of granulation tissue. After surgical repair, biopsy specimens of discarded scar and skin were used to generate fibroblast cell lines. Fibroblasts maintained in medium containing 10% fetal bovine serum and antibiotic were studied between their third and eighth passage. By phase contrast microscopy, no structural differences were obvious, but it was noted that to pass scar fibroblasts, a more aggressive trypsin regimen was required. Immunohistologic and Western blot analysis of patient scar fibroblasts showed (1) more a smooth muscle actin within stress fibers, (2) increased expression of the vitronectin integrin receptor alpha(v) (CD 51), and (3) reduced expression of the collagen integrin receptor alpha2 (CD 49b). The expression of vinculin from focal adhesions or a tubulin from microtubules was the same among cell lines. Contractions of scar and dermal fibroblast-populated collagen lattice were compared. At 24 hours, contractions were 69 percent with newborn fibroblasts (normal); 68 percent for patient dermal fibroblasts; and only 48 percent for patient scar fibroblasts. The retarded contraction of scar fibroblast-populated collagen lattice was significant (p > or = 0.002). Myosin ATPase activity, critical for lattice contraction, and cell migration were equivalent among all cell lines. A plausible mechanism for the retardation of scar lattice contraction is disruption of fibroblasts and collagen interactions, for which the attachment of cells to collagen is altered. It is proposed that either the decrease in the expression of collagen integrin receptor alpha2 (CD 49b), an increase in the expression of the vitronectin receptor alpha(v) (CD 51), or a combination of both is responsible for disruption of collagen fibroblast interactions.     

Quantitative analysis of collagen gel contractile forces generated by dermal fibroblasts and the relationship to cell morphology

The force generated in granulation tissue during wound contraction is thought to be cell mediated; however, it is unclear whether contractile forces are generated by fibroblast locomotion or contraction of myofibroblasts. To help clarify this question the force of this contraction can now be determined accurately in a human dermal fibroblast collagen lattice system using a novel instrument known as a Culture Force Monitor. Three distinct phases of contraction of such collagen gels could be identified over the first 24 hours. Most of the force generated by human dermal fibroblasts was produced during the first stage in parallel with cell attachment and associated changes in cell shape, and the appearance of cell processes. During this initial 24 hours no evidence could be found for the presence of myofibroblasts, but stereoscopic and electron microscopic analysis at a range of time points indicated that migratory fibroblasts were present in the system. Comparison of the contraction profiles of cells extracted from other tissues (tendon and articular cartilage), and extracted by different means from the same tissue specimen, indicated that different populations of fibroblasts can be distinguished on the basis of their pattern of contractions. It would seem that most of the force generated in this model is a result of fibroblast attachment and movement within the collagen lattice. Furthermore, different groups of fibroblasts, even within the same tissue, may vary in their contraction (hence locomotory) activity. © 1996 Wiley-Liss, Inc.     

Endothelial cells enhance adipose mesenchymal stromal cell‐mediated matrix contraction via ALK receptors and reduced follistatin: Potential role of endothelial cells in skin fibrosis

Abnormal cutaneous wound healing can lead to formation of fibrotic hypertrophic scars. Although several clinical risk factors have been described, the cross‐talk between different cell types resulting in hypertrophic scar formation is still poorly understood. The aim of this in vitro study was to investigate whether endothelial cells (EC) may play a role in skin fibrosis, for example, hypertrophic scar formation after full‐thickness skin trauma. Using a collagen/elastin matrix, we developed an in vitro fibrosis model to study the interaction between EC and dermal fibroblasts or adipose tissue‐derived mesenchymal stromal cells (ASC). Tissue equivalents containing dermal fibroblasts and EC displayed a normal phenotype. In contrast, tissue equivalents containing ASC and EC displayed a fibrotic phenotype indicated by contraction of the matrix, higher gene expression of ACTA2, COL1A, COL3A, and less secretion of follistatin. The contraction was in part mediated via the TGF‐β pathway, as both inhibition of the ALK4/5/7 receptors and the addition of recombinant follistatin resulted in decreased matrix contraction (75 ± 11% and 24 ± 8%, respectively). In conclusion, our study shows that EC may play a critical role in fibrotic events, as seen in hypertrophic scars, by stimulating ASC‐mediated matrix contraction via regulation of fibrosis‐related proteins.     

Knock‐out of MicroRNA 145 impairs cardiac fibroblast function and wound healing post‐myocardial infarction

Prevention of infarct scar thinning and dilatation and stimulation of scar contracture can prevent progressive heart failure. Since microRNA 145 (miR‐145) plays an important role in cardiac fibroblast response to wound healing and cardiac repair after an myocardial infarction (MI), using a miR‐145 knock‐out (KO) mouse model, we evaluated contribution of down‐regulation of miR‐145 to cardiac fibroblast and myofibroblast function during adverse cardiac remodelling. Cardiac function decreased more and the infarct size was larger in miR‐145 KO than that in WT mice after MI and this phenomenon was accompanied by a decrease in cardiac fibroblast‐to‐myofibroblast differentiation. Quantification of collagen I and α‐SMA protein levels as well as wound contraction revealed that transdifferentiation of cardiac fibroblasts into myofibroblasts was lower in KO than WT mice. In vitro restoration of miR‐145 induced more differentiation of fibroblasts to myofibroblasts and this effect involved the target genes Klf4 and myocardin. MiR‐145 contributes to infarct scar contraction in the heart and the absence of miR‐145 contributes to dysfunction of cardiac fibroblast, resulting in greater infarct thinning and dilatation. Augmentation of miR‐145 could be an attractive target to prevent adverse cardiac remodelling after MI by enhancing the phenotypic switch of cardiac fibroblasts to myofibroblasts.     

Post‐transcriptional regulation of α‐smooth muscle actin determines the contractile phenotype of Dupuytren's nodular cells

The objective was to study Dupuytren's myofibroblast cells in constrained collagen matrices in order to more closely emulate their in vivo environment and, to correlate their contractility with α‐smooth muscle actin (α‐SMA) expression and determine if dermal fibroblasts regulate Dupuytren's myofibroblast phenotype. Isotonic and isometric force contraction by cells isolated from Dupuytren's nodules, palmar and non‐palmar skin fibroblasts was measured in collagen matrices. The effect of co‐culturing nodule cells with dermal fibroblasts on isometric contraction was examined. Isometric contraction was correlated with levels of α‐SMA mRNA by pcr and protein by Western blotting, and α‐SMA distribution assessed by immunofluorescence. Dupuytren's nodule cells exhibited similar levels of isotonic contraction to both palmar and non‐palmar dermal fibroblasts. However, nodule cells generated high levels of isometric force (mean: 3.5 dynes/h), which continued to increase over 24 h to a maximum of 173 dynes. In contrast, dermal fibroblasts initially exhibited low levels of contraction (mean: 0.5 dynes/h) and reached tensional homeostasis on average after 15 h (range: 4–20 h), with a maximum force of 52 dynes. Although all three cell types had similar α‐SMA mRNA levels, increased levels of α‐SMA protein were observed in nodule cells compared to dermal fibroblasts. α‐SMA localised to stress fibres in 35% (range: 26–50%) of nodule cells compared to only 3% (range:0–6%) of dermal fibroblasts. Co‐cultures of Dupuytren's cells and dermal fibroblasts showed no contractile differences. The contractile phenotype of Dupuytren's myofibroblasts is determined by increased α‐SMA protein distributed in stress fibres, not by cellular mRNA levels. Dupuytren's cell contractility is not influenced by dermal fibroblasts. J. Cell. Physiol. 224: 681–690, 2010. © 2010 Wiley‐Liss, Inc.     

Effects of TRAP-1-Like Protein (TLP) Gene on Collagen Synthesis Induced by TGF-β/Smad Signaling in Human Dermal Fibroblasts

Background

Hypertrophic scars are pathologic proliferations of the dermal skin layer resulting from excessive collagen deposition during the healing process of cutaneous wounds. Current research suggests that the TGF-β/Smad signaling pathway is closely associated with normal scar and hypertrophic scar formation. TRAP-1-like protein (TLP), a cytoplasmic protein, has been reported to efficiently regulate Smad2- and Smad3-dependent signal expression in the TGF-β pathway. The relationship between TLP and Type I/III collagen (Col I/III) synthesis explored in the present study provides an effective target for wound healing and gene therapy of hypertrophic scarring.

Objective

To investigate the effects of TLP on collagen synthesis in human dermal fibroblasts.

Methods

Lentiviral vectors encoding TLP was constructed to transfect fibroblasts derived from normal human skin. The expression of Col I/III and phosphorylation of Smad2 and Smad3 in fibroblasts were examined after TLP treatment. In addition, the comparison of TLP expression in normal skin tissues and in hypertrophic scar tissues was performed, and the effect of TLP on cell viability was analyzed by MTT assay.

Results

TLP expression in hypertrophic scar tissue was markedly higher than in normal skin tissue. The Real Time PCR and Western blot test results both revealed that the synthesis of Col I/III was positively correlated with the expression of TLP. TLP also facilitate Smad2 phosphorylation while, conversely, inhibiting Smad3 phosphorylation. TLP may play a cooperative role, along with the cytokine TGF-β1, in improving the overall cell viability of skin fibroblasts.

Conclusions

TLP likely acts as a molecular modulator capable of altering the balance of Smad3- and Smad2-dependent signaling through regulation of phosphorylation, thus facilitating collagen synthesis in fibroblasts. Based on genetic variation in TLP levels in different tissues, these results suggest that TLP plays a key role in the process of TGF-β1/Smad3 signaling that contributes to wound healing and genesis of pathologic scars.     

Calpain activity is essential in skin wound healing and contributes to scar formation

Wound healing is a multistep phenomenon that relies on complex interactions between various cell types. Calpains are ubiquitously expressed proteases regulating several processes including cellular adhesion and motility as well as inflammation and angiogenesis. Calpains can be targeted by inhibitors, and their inhibition was shown to reduce organ damage in various disease models. We aimed to assess the role of calpains in skin healing and the potential benefit of calpain inhibition on scar formation. We used a pertinent model where calpain activity is inhibited only in lesional organs, namely transgenic mice overexpressing calpastatin (CPST), a specific natural calpain inhibitor. CPST mice showed a striking delay in wound healing particularly in the initial steps compared to wild types (WT). CPST wounds displayed reduced proliferation in the epidermis and delayed re-epithelization. Granulation tissue formation was impaired in CPST mice, with a reduction in CD45+ leukocyte infiltrate and in CD31+ blood vessel density. Interestingly, wounds on WT skin grafted on CPST mice (WT/CPST) showed a similar delayed healing with reduced angiogenesis and inflammation compared to wounds on WT/WT mice demonstrating the implication of calpain activity in distant extra-cutaneous cells during wound healing. CPST wounds showed a reduction in alpha-smooth muscle actin (αSMA) expressing myofibroblasts as well as αSMA RNA expression suggesting a defect in granulation tissue contraction. At later stages of skin healing, calpain inhibition proved beneficial by reducing collagen production and wound fibrosis. In vitro, human fibroblasts exposed to calpeptin, a pan-calpain inhibitor, showed reduced collagen synthesis, impaired TGFβ-induced differentiation into αSMA-expressing myofibroblasts, and were less efficient in a collagen gel contraction assay. In conclusion, calpains are major players in granulation tissue formation. In view of their specific effects on fibroblasts a late inhibition of calpains should be considered for scar reduction.