Alpha-smooth muscle actin is crucial for focal adhesion maturation in myofibroblasts

Cultured myofibroblasts are characterized by stress fibers, containing alpha-smooth muscle actin (alpha-SMA) and by supermature focal adhesions (FAs), which are larger than FAs of alpha-SMA-negative fibroblasts. We have investigated the role of alpha-SMA for myofibroblast adhesion and FA maturation. Inverted centrifugation reveals two phases of initial myofibroblast attachment: during the first 2 h of plating microfilament bundles contain essentially cytoplasmic actin and myofibroblast adhesion is similar to that of alpha-SMA-negative fibroblasts. Then, myofibroblasts incorporate alpha-SMA in stress fibers, develop mature FAs and their adhesion capacity is significantly increased. When alpha-SMA expression is induced in 5 d culture by TGFbeta or low serum levels, fibroblast adhesion is further increased correlating with a "supermaturation" of FAs. Treatment of myofibroblasts with alpha-SMA fusion peptide (SMA-FP), which inhibits alpha-SMA-mediated contractile activity, reduces their adhesion to the level of alpha-SMA negative fibroblasts. With the use of flexible micropatterned substrates and EGFP-constructs we show that SMA-FP application leads to a decrease of myofibroblast contraction, shortly followed by disassembly of paxillin- and beta3 integrin-containing FAs; alpha5 integrin distribution is not affected. FRAP of beta3 integrin-EGFP demonstrates an increase of FA protein turnover following SMA-FP treatment. We conclude that the formation and stability of supermature FAs depends on a high alpha-SMA-mediated contractile activity of myofibroblast stress fibers.     

Inhibition of myofibroblasts by skin grafts.

The myofibroblast population was studied by electron microscopy in rat wounds healing by (1) contraction of granulation tissue, (2) by coverage with split-skin grafts, and (3) by coverage with full-thickness skin grafts. In all 3 types of wounds, myofibroblasts appeared early and reached a peak number at two weeks after wounding. At this time, 40 to 50 percent of the wound fibroblasts had myofibroblast characteristics. The granulating wounds contracted rapidly and completely, and had long persistence of myofibroblasts. Split-skin grafted wounds contracted less and had a more rapid decrease in myofibroblasts. The wounds covered with full thickness skin grafts had a minimum of contraction with a very rapid decrease in the number of myofibroblasts until by 4 weeks no myofibroblasts were present. Full-thickness skin grafts thus appeared to influence contracting wounds not by preventing the formation of myofibroblasts, but by speeding up completion of their life cycle.     

Myofibroblast contraction activates latent TGF-beta1 from the extracellular matrix

The conjunctive presence of mechanical stress and active transforming growth factor β1 (TGF-β1) is essential to convert fibroblasts into contractile myofibroblasts, which cause tissue contractures in fibrotic diseases. Using cultured myofibroblasts and conditions that permit tension modulation on the extracellular matrix (ECM), we establish that myofibroblast contraction functions as a mechanism to directly activate TGF-β1 from self-generated stores in the ECM. Contraction of myofibroblasts and myofibroblast cytoskeletons prepared with Triton X-100 releases active TGF-β1 from the ECM. This process is inhibited either by antagonizing integrins or reducing ECM compliance and is independent from protease activity. Stretching myofibroblast-derived ECM in the presence of mechanically apposing stress fibers immediately activates latent TGF-β1. In myofibroblast-populated wounds, activation of the downstream targets of TGF-β1 signaling Smad2/3 is higher in stressed compared to relaxed tissues despite similar levels of total TGF-β1 and its receptor. We propose activation of TGF-β1 via integrin-mediated myofibroblast contraction as a potential checkpoint in the progression of fibrosis, restricting autocrine generation of myofibroblasts to a stiffened ECM.     

Urokinase receptor cleavage: a crucial step in fibroblast-to-myofibroblast differentiation

Fibroblasts migrate into and repopulate connective tissue wounds. At the wound edge, fibroblasts differentiate into myofibroblasts, and they promote wound closure. Regulated fibroblast-to-myofibroblast differentiation is critical for regenerative healing. Previous studies have focused on the role in fibroblasts of urokinase plasmingen activator/urokinase plasmingen activator receptor (uPA/uPAR), an extracellular protease system that promotes matrix remodeling, growth factor activation, and cell migration. Whereas fibroblasts have substantial uPA activity and uPAR expression, we discovered that cultured myofibroblasts eventually lost cell surface uPA/uPAR. This led us to investigate the relevance of uPA/uPAR activity to myofibroblast differentiation. We found that fibroblasts expressed increased amounts of full-length cell surface uPAR (D1D2D3) compared with myofibroblasts, which had reduced expression of D1D2D3 but increased expression of the truncated form of uPAR (D2D3) on their cell surface. Retaining full-length uPAR was found to be essential for regulating myofibroblast differentiation, because 1) protease inhibitors that prevented uPAR cleavage also prevented myofibroblast differentiation, and 2) overexpression of cDNA for a noncleavable form of uPAR inhibited myofibroblast differentiation. These data support a novel hypothesis that maintaining full-length uPAR on the cell surface regulates the fibroblast to myofibroblast transition and that down-regulation of uPAR is necessary for myofibroblast differentiation.     

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.     

Transforming growth factor beta stimulates fibroblast-collagen matrix contraction by different mechanisms in mechanically loaded and unloaded matrices

Studies were carried out to test the idea that transforming growth factor beta (TGFbeta) stimulated fibroblast contraction of collagen matrices by different mechanisms depending on mechanical loading on the cells and matrix. Under mechanically unloaded conditions (floating matrices), TGFbeta stimulated contraction directly as an agonist and indirectly by preactivating cells to express the myofibroblast phenotype. Increased contraction of floating matrices by preactivated cells appeared to result in part from an autocrine mechanism. Under mechanically loaded conditions (stressed matrices), TGFbeta had no direct agonist effect on contraction. Fibroblasts preactivated to become myofibroblasts showed increased ability to transfer tension to stressed matrices, and tension persisted even after the cells' actin cytoskeleton was disrupted. Our findings are consistent with the idea that fibroblasts activated to become myofibroblasts in vitro have increased contractile activity and indicate that multiple mechanisms that differ depending on mechanical loading on the cells and matrix are involved.     

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.     

Role of MMP3 and fibroblast-MMP14 in skin homeostasis and repair

Early lethality of mice with complete deletion of the matrix metalloproteinase MMP14 emphasized the proteases’ pleiotropic functions. MMP14 deletion in adult dermal fibroblasts (MMP14^Sf-/-) caused collagen type I accumulation and upregulation of MMP3 expression. To identify the compensatory role of MMP3, mice were generated with MMP3 deletion in addition to MMP14 loss in fibroblasts. These double deficient mice displayed a fibrotic phenotype in skin and tendons as detected in MMP14^Sf-/- mice, but no additional obvious defects were detected. However, challenging the mice with full thickness excision wounds resulted in delayed closure of early wounds in the double deficient mice compared to wildtype and MMP14 single knockout controls. Over time wounds closed and epidermal integrity was restored. Interestingly, on day seven, post-wounding myofibroblast density was lower in the wounds of all knockout than in controls, they were higher on day 14. The delayed resolution of myofibroblasts from the granulation tissue is paralleled by reduced apoptosis of these cells, although proliferation of myofibroblasts is induced in the double deficient mice. Further analysis showed comparable TGFβ1 and TGFβR1 expression among all genotypes. In addition, in vitro, fibroblasts lacking MMP3 and MMP14 retained their ability to differentiate into myofibroblasts in response to TGFβ1 treatment and mechanical stress. However, in vivo, p-Smad2 was reduced in myofibroblasts at day 5 post-wounding, in double, but most significant in single knockout, indicating their involvement in TGFβ1 activation. Thus, although MMP3 does not compensate for the lack of fibroblast-MMP14 in tissue homeostasis, simultaneous deletion of both proteases in fibroblasts delays wound closure during skin repair. Notably, single and double deficiency of these proteases modulates myofibroblast formation and resolution in wounds.     

K+ currents regulate the resting membrane potential, proliferation, and contractile responses in ventricular fibroblasts and myofibroblasts

Despite the important roles played by ventricular fibroblasts and myofibroblasts in the formation and maintenance of the extracellular matrix, neither the ionic basis for membrane potential nor the effect of modulating membrane potential on function has been analyzed in detail. In this study, whole cell patch-clamp experiments were done using ventricular fibroblasts and myofibroblasts. Time- and voltage-dependent outward K(+) currents were recorded at depolarized potentials, and an inwardly rectifying K(+) (Kir) current was recorded near the resting membrane potential (RMP) and at more hyperpolarized potentials. The apparent reversal potential of Kir currents shifted to more positive potentials as the external K(+) concentration ([K(+)](o)) was raised, and this Kir current was blocked by 100-300 muM Ba(2+). RT-PCR measurements showed that mRNA for Kir2.1 was expressed. Accordingly, we conclude that Kir current is a primary determinant of RMP in both fibroblasts and myofibroblasts. Changes in [K(+)](o) influenced fibroblast membrane potential as well as proliferation and contractile functions. Recordings made with a voltage-sensitive dye, DiBAC(3)(4), showed that 1.5 mM [K(+)](o) resulted in a hyperpolarization, whereas 20 mM [K(+)](o) produced a depolarization. Low [K(+)](o) (1.5 mM) enhanced myofibroblast number relative to control (5.4 mM [K(+)](o)). In contrast, 20 mM [K(+)](o) resulted in a significant reduction in myofibroblast number. In separate assays, 20 mM [K(+)](o) significantly enhanced contraction of collagen I gels seeded with myofibroblasts compared with control mechanical activity in 5.4 mM [K(+)](o). In combination, these results show that ventricular fibroblasts and myofibroblasts express a variety of K(+) channel alpha-subunits and demonstrate that Kir current can modulate RMP and alter essential physiological functions.     

The Maguk protein, Pals1, functions as an adapter, linking mammalian homologues of Crumbs and Discs Lost

Chemokines are small cytokines primarily known for their roles in inflammation. More recently, however, they have been implicated in processes involved in development of the granulation tissue of wounds, but little is known about their functions during this process. Fibroblasts play key roles in this phase of healing: some fibroblasts differentiate into myofibroblasts, alpha-smooth muscle actin (SMA)-producing cells that are important in wound closure and contraction. Here we show that the CXC chemokine chicken chemotactic and angiogenic factor (cCAF) stimulates fibroblasts to produce high levels of alpha-SMA and to contract collagen gels more effectively than do normal fibroblasts, both characteristic properties of myofibroblasts. Specific inhibition of alpha-SMA expression resulted in abrogation of cCAF-induced contraction. Furthermore, application of cCAF to wounds in vivo increases the number of myofibroblasts present in the granulation tissue and accelerates wound closure and contraction. We also show that these effects in culture and in vivo can be achieved by a peptide containing the NH2-terminal 15 amino acids of the cCAF protein and that inhibition of alpha-SMA expression also results in inhibition of N-peptide-induced collagen gel contraction. We propose that chemokines are major contributors for the differentiation of fibroblasts into myofibroblasts during formation of the repair tissue. Because myofibroblasts are important in many pathological conditions, and because chemokines and their receptors are amenable to pharmacological manipulations, chemokine stimulation of myofibroblast differentiation may have implications for modulation of functions of these cells in vivo.     

Cardiac myofibroblasts differentiated in 3D culture exhibit distinct changes in collagen I production, processing, and matrix deposition

Myofibroblasts are a differentiated fibroblast cell type characterized by increased contractile capacity and elevated production of extracellular matrix (ECM) proteins. In the heart, myofibroblast expression is implicated in fibrosis associated with pressure-overload hypertrophy, among other pathologies. Although enhanced expression of ECM proteins by myofibroblasts is established, few studies have addressed the nature of the ECM deposited by myofibroblasts. To characterize ECM production and assembly by cardiac myofibroblasts, we developed a three-dimensional (3D) culture system using primary cardiac fibroblasts seeded into a nylon mesh that allows us to reversibly interconvert between myofibroblast and fibroblast phenotypes. We report that an increase in collagen I production by myofibroblasts was accompanied by a significant increase in collagen deposition into insoluble ECM. Furthermore, myofibroblasts exhibited increased levels of procollagen alpha1(I) with C-propeptide attached (and N-propeptide removed) relative to procollagen alpha1(I) compared with fibroblast cultures. An increase in production of the myofibroblast-associated splice variant of fibronectin (EDA-Fn) was seen in myofibroblast 3D cultures. Because the regulation of procollagen I processing is known to have profound effects on ECM assembly, differences in procollagen I secretion and maturation coupled with expression of EDA-Fn are shown to contribute to the production of a distinct ECM by the cardiac myofibroblast.     

Effects of the isothiocyanate sulforaphane on TGF-β1-induced rat cardiac fibroblast activation and extracellular matrix interactions

An important step in many pathological conditions, particularly tissue and organ fibrosis, is the conversion of relatively quiescent cells into active myofibroblasts. These are highly specialized cells that participate in normal wound healing but also contribute to pathogenesis. These cells possess characteristics of smooth muscle cells and fibroblasts, have enhanced synthetic activity secreting abundant extracellular matrix components, cytokines, and growth factors, and are capable of generating contractile force. As such, these cells have become potential therapeutic targets in a number of disease settings. Transforming growth factor β (TGF-β) is a potent stimulus of fibrosis and myofibroblast formation and likewise is an important therapeutic target in several disease conditions. The plant-derived isothiocyanate sulforaphane has been shown to have protective effects in several pathological models including diabetic cardiomyopathy, carcinogenesis, and fibrosis. These studies suggest that sulforaphane may be an attractive preventive agent against disease progression, particularly in conditions involving alterations of the extracellular matrix and activation of myofibroblasts. However, few studies have evaluated the effects of sulforaphane on cardiac fibroblast activation and their interactions with the extracellular matrix. The present studies were carried out to determine the potential effects of sulforaphane on the conversion of quiescent cardiac fibroblasts to an activated myofibroblast phenotype and associated alterations in signaling, expression of extracellular matrix receptors, and cellular physiology following stimulation with TGF-β1. These studies demonstrate that sulforaphane attenuates TGF-β1-induced myofibroblast formation and contractile activity. Sulforaphane also reduces expression of collagen-binding integrins and inhibits canonical and noncanonical TGF-β signaling pathways.     

Survivin expression induced by endothelin-1 promotes myofibroblast resistance to apoptosis

Fibrosis of the lungs and other organs is characterized by the accumulation of myofibroblasts, effectors of wound-repair that are responsible for the deposition and organization of new extracellular matrix (ECM) in response to tissue injury. During the resolution phase of normal wound repair, myofibroblast apoptosis limits the continued deposition of ECM. Mounting evidence suggests that myofibroblasts from fibrotic wounds acquire resistance to apoptosis, but the mechanisms regulating this resistance have not been fully elucidated. Endothelin-1 (ET-1), a soluble peptide strongly associated with fibrogenesis, decreases myofibroblast susceptibility to apoptosis through activation of phosphatidylinositol 3′-OH kinase (PI3K)/AKT. Focal adhesion kinase (FAK) also promotes myofibroblast resistance to apoptosis through PI3K/AKT-dependent and -independent mechanisms, although the role of FAK in ET-1 mediated resistance to apoptosis has not been explored. The goal of this study was to investigate whether FAK contributes to ET-1 mediated myofibroblast resistance to apoptosis and to examine potential mechanisms downstream of FAK and PI3K/AKT by which ET-1 regulates myofibroblast survival. Here, we show that ET-1 regulates myofibroblast survival by Rho/ROCK-dependent activation of FAK. The anti-apoptotic actions of FAK are, in turn, dependent on activation of PI3K/AKT and the subsequent increased expression of Survivin, a member of the inhibitor of apoptosis protein (IAP) family. Collectively, these studies define a novel mechanism by which ET-1 promotes myofibroblast resistance to apoptosis through upregulation of Survivin.     

Growth dynamics of cultured myofibroblasts from human breast cancer and nonmalignant contracting tissues

Myofibroblasts were successfully grown in tissue culture from the connective tissue stroma of three human breast adenocarcinomas. These cells had slower growth kinetics than fibroblasts from normal human dermis, as did myofibroblasts from two granulating wounds. Electron microscopy of breast cancer slices and tissue cultures of these specimens confirmed the presence of myofibroblasts in both. In early passages, the specificity of carcinoma-derived myofibroblast growth kinetics is preserved. The exact role of myofibroblasts in breast cancer, whether helping or hindering tumor growth, remains undetermined.     

Contractile activity and smooth muscle alpha-actin organization in thrombin-induced human lung myofibroblasts

Activated fibroblasts, or myofibroblasts, are crucial players in tissue remodeling, wound healing, and various fibrotic disorders, including interstitial lung fibrosis associated with scleroderma. Here we characterize the signaling pathways in normal lung fibroblasts exposed to thrombin as they acquire two of the main features of myofibroblasts: smooth muscle (SM) alpha-actin organization and collagen gel contraction. Our results show that the small G protein Rho is involved in lung myofibroblast differentiation. Thrombin induces Rho-35S-labeled guanosine 5'-O-(3-thiotriphosphate) binding in a dose-dependent manner. It potently stimulates Rho activity in vivo and initiates protein kinase C (PKC)-epsilon-Rho complex formation. Toxin B, which inactivates Rho by ADP ribosylation, inhibits thrombin-induced SM alpha-actin organization, collagen gel contraction, and PKC-epsilon-SM alpha-actin and PKC-epsilon-RhoA coimmunoprecipitation. However, it has no effect on PKC-epsilon activation or translocation of PKC-epsilon to the membrane. Overexpression of constitutively active PKC-epsilon and constitutively active RhoA induces collagen gel contraction or SM alpha-actin organization, whereas, individually, they do not perform these functions. We therefore conclude that the contractile activity of myofibroblasts induced by thrombin is mediated via PKC-epsilon- and RhoA-dependent pathways and that activation of both of these molecules is required. We postulate that PKC-epsilon-RhoA complex formation is an early event in thrombin activation of lung fibroblasts, followed by PKC-epsilon-SM alpha-actin coimmunoprecipitation, which leads to the PKC-epsilon-RhoA-SM alpha-actin ternary complex formation.     

K+ channels and the cAMP-PKA pathway modulate TGF-beta1-induced migration of rat vascular myofibroblasts

Our previous studies have indicated that TGF-beta1 exerts its effect on the expression of A-type potassium channels (I(A)) in rat vascular myofibroblasts by activation of protein kinase C during the phenotypic transformation of vascular fibroblasts to myofibroblasts. In the present study, patch-clamp whole-cell recording and transwell-migration assays were used to examine the effects of TGF-beta1- and phorbol 12-myristate 13-acetate (PMA)-induced expression of I(A) channels on myofibroblast migration and its modulation by the protein kinase A (PKA) pathway. Our results reveal that incubation of fibroblasts with TGF-beta1 or PMA up-regulates the expression of I(A) channels and increases myofibroblast migration. Blocking I(A) channel expression by 4-aminopyridine (4-AP) significantly inhibits TGF-beta1- and PMA-induced myofibroblast migration. Incubation of fibroblasts with forskolin does not result in increased expression of I(A) channels but does cause a slight increase in fibroblast migration at higher concentrations. In addition, forskolin increases the TGF-beta1- and PMA-induced myofibroblast migration but inhibits TGF-beta1- and PMA-induced the expression of I(A) channels. Whole-cell current recordings showed that forskolin augments the delayed rectifier outward K(+) (I(K)) current amplitude of fibroblasts, but not the I(A) of myofibroblasts. Our results also indicate that TGF-beta1- and PMA-induced expression of I(A) channels might be related to increase TGF-beta1- or PMA-induced myofibroblast migration. Promoting fibroblast and myofibroblast migration via the PKA pathway does not seem to involve the expression of I(A) channels, but the modulation of I(K) and I(A) channels might be implicated.     

Antifibrotic action of Cu/Zn SOD is mediated by TGF-beta1 repression and phenotypic reversion of myofibroblasts

Skin fibrosis is characterized by the proliferation and accumulation of activated fibroblasts called myofibroblasts. They exhibit specific cytoskeletal differentiation, overexpress the fibrogenic cytokine TGF-beta1, synthesize excess extracellular matrix compounds and exhibit a depleted antioxidant metabolism. Recently, SOD was successfully used as an antifibrotic agent in vivo, thus challenging the postulate of established fibrosis irreversibility. We postulated that myofibroblasts could be a direct target for this therapeutic effect. To test this hypothesis, we used three-dimensional co-culture models of skin, in which specific phenotypes of normal fibroblasts versus myofibroblasts are retained. These 3-D models were treated with liposomal and carrier-free Cu/Zn SOD, and examined for their effects on cell number, cell death, and phenotypic differentiation. The results show that SOD did not induce myofibroblast cell death, whereas it significantly reduced TGF-beta1 expression, thus demonstrating that SOD might be proposed as a potent antagonist of this major fibrogenic growth factor. We also found that SOD significantly lowered the levels of the myofibroblast marker alpha-sm actin, of beta-actin, and of the extracellular matrix components alpha1(I) collagen and tenascin-C. In conclusion, our results suggest that SOD antifibrotic action occurred in vitro through the reversion of myofibroblasts into normal fibroblasts.     

Electron microscopic immunochemical localization of actin in fibroblasts in healing skin and palate wounds of beagle dog

Wound contraction in soft tissue has been attributed to the activity of contractile fibroblasts containing actin microfilaments. Immunochemical staining at the electron microscopic level was used to demonstrate the presence of such cells in healing wounds from skin and oral mucosa. Biopsies of granulation tissue from 10 and 16 day old excision wounds in beagle palate mucoperiosteum and skin were fixed and 10 micrometer sections were treated with antiactin serum, peroxidase-anti peroxidase (PAP) and then incubated to reveal the localization of actin. Controls were prepared using non-immune serum or preabsorbed immune serum. Thin sections examined with the electron microscope revealed the presence of PAP particles associated with microfilament bundles beneath the plasma membrane and in processes of fibroblasts. Reaction was also associated with micropinocytotic vesicles at the cell surface. More reactive cells were seen in 16 day than in 10 day old wounds and there were greater numbers of these cells in skin than in oral mucoperiosteum. The results indicate that actin containing cells with the ultrastructural characteristics of contractile fibroblasts (myofibroblasts) are present in the granulation tissue of healing skin and oral mucosal wounds. Such cells may be responsible for the wound contraction observed clinically in the healing palatal mucoperiosteum.     

Contractility of single human dermal myofibroblasts and fibroblasts

Human dermal myofibroblasts, characterised by the expression of alpha-smooth muscle actin, are part of the granulation tissue and implicated in the generation of contractile forces during normal wound healing and pathological contractures. We have compared the contractile properties of single human dermal fibroblasts and human dermal myofibroblasts by culturing them on flexible silicone elastomers. The flexibility of the silicone substratum permits the contractile forces exerted by the cells to be measured [Fray et al., 1998: Tissue Eng. 4:273-283], without changing their expression of alpha-smooth muscle actin. The mean contractile force produced by myofibroblasts (2.2 microN per cell) was not significantly different from that generated by fibroblasts (2.0 microN per cell) when cultured on a substrata with a low elastomer stiffness. Forces produced by fibroblasts were unaffected by increases in elastomer stiffness, but forces measured for myofibroblasts increased to a mean value of 4.1 microN/cell. This was associated with a higher proportion of myofibroblasts being able to produce wrinkles on elastomers of high stiffness compared to fibroblasts. We discuss the force measurements at the single cell level, for both fibroblast and myofibroblasts, in relation to the proposed role of myofibroblasts in wound healing and pathological contractures.