In vitro analysis of adhesion molecule expression and gel contraction of human granulation fibroblasts

The objective of this study was to determine whether human fibroblasts obtained at different times from normally healing wounds were phenotypically distinct with respect to cell-matrix interactions. We cultured human granulation fibroblasts obtained from repeated biopsies from the same punch area. On days 3, 6, 9, and 14 the expression of key adhesion molecules and extracellular matrix components at the protein level by flow cytometry analysis were compared with quiescent fibroblasts. Intercellular adhesion molecule-1 levels were significantly elevated only around day 3, which implies a phenotypic change during the early phase of wound healing. Homing cell adhesion molecule, as well as the alpha4 integrin subunit (CD49d), were essentially unaltered. The alpha5 integrin subunit (CD49e), which imparts the specificity of the fibronectin receptor and alphav (CD51), a vitronectin receptor component, are upregulated around days 3 and 6; the alpha2 and 3 integrin subunits (CD49b/c) were only increased on day 6. In granulation fibroblasts, at days 3 and 14, the level of the beta1 integrin subunit was enhanced. In addition, collagen gel contraction with granulation fibroblasts increases constantly from day 3 to day 14. These results show that human granulation fibroblasts differentially express cell surface adhesion molecules depending on the age of the healing wound. Further, changes in the ability of these cells to contract collagen gels indicate a synchronicity between wound bed contraction and different stages of wound healing.     

Differential responses of collagen and glycosaminoglycan syntheses and cell proliferation to exogenous transforming growth factor beta 1 in the developing mouse skin fibroblasts in culture.

Skin fibroblast cultures were established from mouse foetuses at days 14, 15, 16 and 18 of gestation and from newborn mice. Modulations of mitotic and biosynthetic phenotypes of the fibroblasts by transforming growth factor beta1 (TGFbeta1) were studied. Treatment of the fibroblasts with TGFbeta1 at doses of 1 and 10 ng/ml for 48 h resulted in significant stimulation of cell proliferation in the 15-, 16- and 18-day foetal fibroblasts and a slight stimulation in the 14-day foetal fibroblasts. Treatment with TGFbeta1 resulted in stimulation of collagen synthesis approximately 2-fold in the 18-day foetal and newborn fibroblasts, but failed to stimulate it in the 14-, 15- and 16-day foetal fibroblasts. TGFbeta1 stimulated glycosaminoglycan (GAG) synthesis throughout all developmental stages approximately 1.8-2.6 fold. Histological study demonstrated that skin wounds made at day 16 of gestation were replaced with normal-appearing dermis, but at day 18 the wounds left dermal fibrosis and lack of hair follicles. These results indicate that the modulations of fibroblast phenotypes (proliferation and syntheses of collagen and GAG) in response to TGFbeta1 occur at different stages of gestation. Ontogenic transitions of skin wound healing and collagen synthetic phenotype with TGFbeta1 treatment in cultured fibroblasts occurred between days 16 and 18 of gestation, suggesting that the unresponsiveness of collagen synthesis to exogenous TGFbeta1 in cell culture may be related to the phenomenon of scarless wounds in the foetus.     

In vitro migration and adhesion of fibroblasts from different phases of palatal wound healing

Cleft palate patients often show mid-facial growth impairment after surgical closure of the defect. This is a consequence of palatal wound healing, and more specifically of wound contraction and scar tissue formation. Cells of the fibroblast lineage are responsible for these processes and they display different phenotypes in the course of the wound healing process. The aim of this study was to analyze the in vitro adhesion and migration of wound fibroblasts, isolated during the palatal wound healing process in the rat. Additionally, we analyzed the expression of beta1 integrins and vinculin, the key players in adhesion and migration. Palatal fibroblasts from age-matched controls were analyzed to measure the effects of normal aging. Palatal fibroblasts from unwounded tissue showed a low migratory behavior (<25 microm), a strong capability to adhere (>80%) and a low expression of beta1 integrins and vinculin. In contrast, fibroblasts obtained from healing palatal wounds were highly migratory (>200 microm) coupled to a weak capability to adhere (<65%) and a high expression of vinculin and beta1 integrins. These data show that the palatal wound healing process induces a change in fibroblast phenotype from "quiescent" to "activated," which persists in vitro.     

肉芽组织中NK1受体表达与创面愈合关系的实验研究

目的：研究创面愈合过程中NK1受体（NK1R）在肉芽组织成纤维细胞上的表达及其与创面愈合之间的关系。方法：新生Sprague-Dawley仔鼠20只，随机分为两组，每组10只。其中一组注射辣椒素将皮肤内感觉神经纤维破坏，制成“失感觉神经支配模型“，另一组作对照。两组动物均在腹部制作圆形皮肤缺损，并各选5只分别在一定时间点沿创面周缘及底部取材，进行NK1受体的免疫组织荧光染色及HE染色，观察其相应的时间、空间规律；另外5只不取材，而是在一定时间点测量未愈合的残留创面面积，并进行比较。结果：创面愈合过程中正常大鼠与去感觉神经支配大鼠在肉芽组织成纤维细胞均有NK1R表达；损伤后NK1R表达阳性的成纤维细胞形态变肥厚，胞体增大，车增殖旺盛表现；NK1R在感觉神经支配的大鼠成纤维细胞上的表达较正常对照升高；去感觉神经的大鼠与正常大鼠相比，创面愈合速度较慢。结论：大鼠皮肤NK1R表达与创面愈合之间关系密切，由感觉神经末梢所释放的P物质等神经递质可能是调节促进创面愈合的重要因子之一。     

血小板衍生生长因子对成纤维细胞合成胶原的影响

目的 探讨血小板衍生生长因子（ＰＤＧＦ－ＡＢ）促进伤口愈合的作用机理及其在瘢痕增生中的作用。方法 取体外培养的人正常皮肤及增生性瘢痕成纤维细胞,用原位杂交法观察ＰＤＧＦ对两种细胞表达Ｐｒｏα１（Ⅲ）ｍＲＮＡ的影响。结果;ＰＤＧＦ－ＡＢ对两种成纤维细胞表达Ｐｒｏ α１（Ⅲ）ｍＲＮＡ均有明显促进作用,且均呈剂量依赖性关系,但作用有差异。     

093Prostaglandin E2 Inhibits Fibroblast Migration: Implications for Wound Healing

Background : Wound healing is a complex process involving multiple cell types, extracellular matrix components and soluble mediators. Prostaglandin E2 is an important component of the inflammatory response to injury. PGE2 can regulate the fibroblast response to injury via the EP receptor family. Here, we examine PGE2 regulation of fibroblast migration. Our analysis extends to fibroblasts representing a spectrum of wound healing phenotypes. Hypothesis : Prostaglandin E2 mediated inhibition of fibroblast migration is conserved across multiple fibroblast phenotypes. Methods : Primary cultures of human fetal, adult and keloid fibroblasts were used. Analysis of the EP receptor profile for each fibroblast phenotype was conducted using real‐time PCR, Western blot and immunohistochemistry. Fibroblast migration was quantified using a well established in vitro scratch assay. Results : Prostaglandin E2, via EP2/EP4 receptors, inhibits fibroblast migration in all fibroblast phenotypes. Fetal fibroblasts retain a more robust migratory phenotype when compared to normal adult and keloid fibroblasts. Normal adult fibroblasts exhibit a dramatic destabilization of the actin cytoskeleton which accompanies PGE2 inhibition of cell migration. This effect was not observed in fetal or keloid fibroblasts. Conclusions : Fibroblast activity in the wound bed can be altered by inflammatory mediators. The effects of prostaglandin E2 appear to be partially conserved across various fibroblast phenotypes. Variability in the response of these cells, however, indicates that fibroblasts derived from fetal tissue may retain intrinsic altered response mechanisms to endogenous inflammatory mediators.     

ORIGINAL RESEARCH – BASIC SCIENCE: Primary dermal fibroblasts derived from sdc‐1 deficient mice migrate faster and have altered αv integrin function

The goal of this study is to determine whether dermal fibroblasts lacking syndecan‐1 (sdc1) show differences in integrin expression and function that could contribute to the delayed skin and corneal wound healing phenotypes seen in sdc‐1 null mice. Using primary dermal fibroblasts, we show that after 3 days in culture no differences in α‐smooth muscle actin were detected but sdc‐1 null cells expressed significantly more αv and β1 integrin than wildtype (wt) cells. Transforming growth factor β1 (TGFβ1) treatment at day 3 increased αv‐ and β1‐integrin expression in sdc‐1 null cells at day 5 whereas wt cells showed increased expression only of αv‐integrin. Using time‐lapse studies, we showed that the sdc‐1 null fibroblasts migrate faster than wt fibroblasts, treatment with TGFβ1 increased these migration differences, and treatment with a TGFβ1 antagonist caused sdc‐1 null fibroblasts to slow down and migrate at the same rate as untreated wt cells. Cell spreading studies on replated fibroblasts showed altered cell spreading and focal adhesion formation on vitronectin and fibronectin‐coated surfaces. Additional time lapse studies with β1‐ and αv‐integrin antibody antagonists, showed that wt fibroblasts expressing sdc‐1 had activated integrins on their surface that impeded their migration whereas the null cells expressed αv‐containing integrins which were less adhesive and enhanced cell migration. Surface expression studies showed increased surface expression of α2β1 and α3β1 on the sdc‐1 null fibroblasts compared with wt fibroblasts but no significant differences in surface expression of α5β1, αvβ3, or αvβ5. Taken together, our data indicates that sdc‐1 functions in the activation of αv‐containing integrins and support the hypothesis that impaired wound healing phenotypes seen in sdc‐1 null mice could be due to integrin‐mediated defects in fibroblast migration after injury.     

Double‐edged effects of neuropeptide substance P on repair of cutaneous trauma

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.     

Glucan stimulates human dermal fibroblast collagen biosynthesis through a nuclear factor-1 dependent mechanism

Glucan, an immunomodulator, has been reported to increase collagen deposition and tensile strength in experimental models of wound repair. Previous data suggest that glucan modulates wound healing via an indirect mechanism in which macrophages are stimulated to release growth factors and cytokines. However, recent data have shown the presence of glucan receptors on normal human dermal fibroblasts, suggesting that glucans may be able to directly stimulate fibroblast collagen biosynthesis. To test this hypothesis, we examined the effect of glucan on collagen biosynthesis in normal human dermal fibroblasts. We assessed nuclear factor-1 (NF-1) activation, procollagen mRNA expression, collagen biosynthesis, and whether there was a causal link between glucan treatment, NF-1 activation, and collagen expression. Glucan (1 microg/ml) increased NF-1 binding activity by 46% (8 hours), 64% (24 hours), 215% (36 hours), and 119% (48 hours) in cultured normal human dermal fibroblasts. Alpha 1(I) and alpha1 (III) procollagen mRNA were increased in glucan-treated normal human dermal fibroblasts when compared with the untreated fibroblasts. Collagen synthesis was increased at 24 hours and 48 hours following glucan treatment of normal human dermal fibroblasts. Down-regulation of NF-1 by pentifylline inhibited glucan-induced procollagen mRNA expression. These data indicate that glucan can directly stimulate human fibroblast collagen biosynthesis through an NF-1-dependent mechanism.     

整合素连接激酶在创面愈合中的作用

目的 观察整合素连接激酶(ILK)在大鼠创面中的表达,探讨其在创面愈合中的作用及机制.方法 健康SD大鼠20只,随机选取16只建立浅Ⅱ度烫伤模型,4只作为正常对照,烫伤后第1、5、10、15天分别随机选取4只大鼠,运用免疫组织化学检测创面中ILK的表达.7例人正常皮肤成纤维细胞,转化生长因子(TGF)-β1组给予TGF-β1刺激72 h,同一细胞培养相同时间作为对照,荧光定量聚合酶链反应(PCR)检测各组ILK和纤维连接蛋白(Fn)的表达.结果 烫伤后第5、10天大鼠烫伤创面中ILK的表达显著高于正常皮肤和愈合后(第15天,P<0.05).TGF-β1组细胞中ILK和Fn mRNA表达均显著强于对照组(P<0.05);对照组ILK与Fn mRNA表达强度呈正相关(r=0.57).结论 ILK可能同时通过参与整合素和TGF-β1信号通路,在创面愈合过程中发挥一定作用.     

Fascial fibroblast kinetic activity is increased during abdominal wall repair compared to dermal fibroblasts

Abdominal wall fascial wound healing failure is a common clinical problem for general surgeons, manifesting in early postoperative fascial dehiscence as well as delayed development of incisional hernias. We previously reported that abdominal wall fascial incisions normally recover breaking strength faster than simultaneous dermal incisions in a rodent model. The accelerated fascial repair was associated with greater fibroblast cellularity within fascial wounds and increased wound collagen deposition. The current study was designed to determine whether accelerated fascial healing is the result of increased fascial fibroblast kinetic activity as measured by a more efficient fibroblast phenotype for binding to and remodeling a collagen matrix. Using a new model of abdominal wall repair, fibroblast cell cultures were developed from uninjured and wounded fascia and compared to dermal fibroblasts in order to define the fibroproliferative kinetic properties of abdominal wall fibroblasts. Fascial wound fibroblasts produced a more efficient and greater overall collagen lattice compaction compared to dermal fibroblasts. Acute fascial wound fibroblasts also showed enhanced cell proliferation compared to dermal fibroblasts but no significant differences in collagen production when normalized to cell number. These results suggest that fascial fibroblasts express distinct acute repair phenotypes and therefore a specific mechanism for fascial repair following injury.     

Interleukin-10 suppresses proliferation and remodeling of extracellular matrix of cultured human skin fibroblasts

When we previously examined the participation of local expression of interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNFalpha) in wound healing of an intestinal anastomosis under septic conditions in mice, we found that IL-10 and TNFalpha expressions were markedly enhanced around the anastomosis and that wound healing was impaired in this animal model. The purpose of the present study was to investigate the combined effect of IL-10 on proliferation and remodeling of the extracellular matrix (ECM) of cultured human skin fibroblasts. Human skin fibroblasts were cultured for 48 h with IL-10 and/or TNFalpha at various concentrations, then the proliferation rates were determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The concentration of transforming growth factor-beta1 (TGFbeta1) in cell culture supernatants was measured by enzyme-linked immunosorbent assay, and type I collagen protein and matrix metalloproteinase-I (MMP-I) were detected by indirect immunofluorescence in cultured cells incubated for 48 h with 10 ng/ml of IL-10 and/or 10 ng/ml of TNFalpha. IL-10 itself had no effect on fibroblast proliferation, but reduced TNFalpha-induced fibroblast proliferation. The concentration of TGFbeta1 in cell culture supernatants was significantly lower in the presence of TNFalpha and IL-10 than in the presence of TNFalpha alone. Immunolabeling of fibroblasts for type I collagen protein was decreased in cells incubated with IL-10 and/or TNFalpha compared to controls. MMP-I immunolabeling was increased in cells incubated with IL-10, IL-10 and TNFalpha compared to control and cells incubated with TNFalpha. It is suggested that IL-10 is an inhibitory factor for the remodeling of the ECM during wound healing.     

A novel implantable collagen gel assay for fibroblast traction and proliferation during wound healing

BACKGROUND: A novel implantable assay for studying cellular behavior in the wound environment was developed. The assay is unique in that it combines the more quantitative nature of in vitro assays with the greater physiological relevance of in vivo wound healing models. MATERIALS AND METHODS: Cells were seeded in a physiologically relevant biological matrix, a collagen gel, contained within a semipermeable tube, and then exposed to soluble factors of the wound environment at different stages of the wound healing response. Gels were harvested at prescribed time points, and cell proliferation rates and gel compaction were measured. These data were combined with our theory for cell-matrix mechanical interactions to estimate the cell traction exerted by the cells leading to gel compaction. Cell morphology and alpha-smooth muscle actin expression were also characterized. RESULTS: The proliferation of and traction exerted by fibroblasts exposed to the soluble wound environment were different from those in similar collagen gels maintained in culture in complete medium. Proliferation and traction also varied over the course of the wound healing response. Traction was higher and proliferation lower in day 1-5 wounds compared to day 7-11 wounds. Recovered cells no longer stained for alpha-smooth muscle actin, in contrast to cells maintained in culture. CONCLUSIONS: Changes in the soluble wound environment that occur as the wound healing response proceeds alter fibroblast traction and migration. We have developed a new assay that employs a physiologically relevant biological matrix and allows the effects of the dynamic soluble wound environment on cellular traction, proliferation, and other phenomena such as protein expression to be quantified.     

血竭素高氯酸盐对成纤维细胞增殖与成纤维细胞生长因子mRNA表达的影响

目的 观察血竭素高氯酸盐(Dp)对人皮肤成纤维细胞(Fb)生物学特性的影响,探讨其促进创而愈合的机制.方法 分离培养人正常Fb,将Dp以不同浓度(0.063、0.125、0.250、0.625、1.250、2.500 mg/L)分别加入培养液,采用噻唑蓝(MTT)比色法检测Dp在不同浓度以及不同时间点对体外培养的Fb增殖作用的影响.通过流式细胞仪,实时荧光定量聚合酶链式反应( real-time PCR)分别检测最适浓度培养条件下Fb的细胞周期变化以及成纤维细胞生长因子(FGF) mRNA的合成表达.结果 Dp在0.625～1.250 mg/L浓度范围内,其吸光度值(0.237±0.012、0.243±0.017)均高于对照组(0.208±0.011)差异有统计学意义(t值分别为2.11、2.23,P＜0.05),此浓度范围可促进Fb增殖,且呈剂量依赖性,在浓度为1.250 mg/L时(A值0.243±0.017),促增殖作用最为显著(t =2.23,P＜0.01),流式细胞仪结果显示,在浓度为1.250 mg/L时,Dp可明显促进Fb通过G1/S及S/G2期限制点,S期及G2/M期细胞与对照组比较明显增多,G0/G1期细胞与对照组比较明显减少(t值分别为4.32、7.53、3.27,P＜0.05).细胞因子mRNA表达测定中,1.250 mg/L Dp组与对照组比较表达明显上调,差异亦有统计学意义(=1.48,p＜0.05).结论 Dp能显著促进Fb增殖,加快Fb周期进程,同时可促进FGF的mRNA表达,可能与血竭促进创面愈合的机制有关.     

Effect of a calcium-channel blocker (verapamil) on the morphology, cytoskeleton and collagenase activity of human skin fibroblasts

The effects of verapamil modulating collagen biosynthesis have prompted us to study the role of this drug in cultured fibroblasts. In this article, we describe the effects of verapamil on fibroblast behaviour, with special emphasis to phenotypic modifications, reorganisation of actin filaments and secretion of MMP1. Human dermal fibroblasts treated with 50-μM verapamil changed their normal spindle-shaped morphology to stellate. Treated cells showed discrete reorganisation of actin filaments, as revealed by fluorescein isothiocyanate (FITC)-phalloidin staining and confocal microscopy. We hypothesised that these effects would be associated to lower levels of cytosolic Ca²⁺. Indeed, short time loading with calcium green confirmed that verapamil-treated fibroblasts exhibited lower intracellular calcium levels compared to controls. We also observed that verapamil increases the secretion of MMP1 in cultured fibroblasts, as demonstrated by zymography, specific substrate assays and immunoblot. The morphological alterations induced by verapamil are neither cytotoxic nor associated with other dramatic cytoskeleton alterations. Thus we may conclude that this drug enhances collagenase secretion and does not disrupt the major tracks necessary to deliver these enzymes in the extracellular space. The present results suggested that verapamil could be used at physiological levels to enhance collagen I breakdown, and may be considered a potential candidate for intralesional therapy of wound healing and fibrocontractive diseases.     

Effect of heparin on production of transforming growth factor (TGF)-beta1 and TGF-beta1 mRNA expression by human normal skin and hyperplastic scar fibroblasts

Heparin affects both dermal fibroblast proliferation and collagen and may mediate these effects by altering the levels of transforming growth factor-beta1 (TGF-beta1) production and TGF-beta1 mRNA expression as a wound healing modulator. The purpose of this study is to probe the effect of heparin on TGF-beta1 and TGF-beta1 mRNA production by human normal skin and hyperplastic scar fibroblasts. This research investigates the effect of heparin on TGF-beta1 and TGF-beta1 mRNA production by human normal skin and hyperplastic scar fibroblasts with exposure to 0 microg/mL, 100 microg/mL, 300 microg/mL, or 600 microg/mL heparin for 24, 48, 72, or 96 hours in a serum-free in vitro model. Levels of TGF-beta1 in the supernatants and TGF-beta1 mRNA expression of fibroblasts were determined by enzyme-linked immunosorbent assay (ELISA) and real time RT-PCR, respectively. Heparin (300 microg/mL and 600 microg/mL) stimulated TGF-beta1 production by normal skin (26% to 83%) and hyperplastic scar fibroblasts (63% to 85%), with statistical significance (P < 0.05) at various time points. Heparin (300 microg/mL and 600 microg/mL) also stimulated TGF-beta1 mRNA expression by normal skin (12% to 53%) and hyperplastic scar fibroblasts (33% to 52%), with statistical significance (P < 0.05) at various time points. These effects of heparin on normal skin and hyperplastic scar fibroblasts may have implications for hyperplastic scar formation and wound healing in vivo.     

Interleukin-1alpha gene expression during wound healing.

Interleukin-1alpha is known to be constitutively produced by epidermal keratinocytes under normal conditions, and injection of this cytokine enhances wound reepithelialization. However, no studies have characterized the temporal sequence of interleukin-1alpha gene expression over the time course of wound healing, and the cellular sources of this cytokine have not been identified. In the present studies, levels of interleukin-1alpha messenger RNA in wound tissue isolated from SKH-1 hairless mice were characterized and the cells that produced interleukin-1alpha immunoreactive protein over a 10-day time course of wound healing were defined. A time-dependent upregulation in interleukin-1alpha gene expression occurred immediately (4 hours) after a full-thickness wound was made, which represented a four-fold increase over levels of cytokine gene expression detected in nonwounded skin. Upregulation of cytokine gene expression correlated with an immediate increase in plasma interleukin-1alpha levels and was followed by an increase in interleukin-1alpha immunoreactive protein localized to keratinocytes within the leading edge of the wound and epidermis, as well as to neutrophils within the dermis. The rapid increase in local and systemic interleukin-1alpha levels correlated with the infiltration of a significant number of neutrophils into the wound site and with the proliferation of both basal keratinocytes and dermal fibroblasts. Given the known ability of interleukin-1alpha to regulate proliferation and migration of epidermal keratinocytes and to indirectly induce leukocyte chemotaxis, the results of the present studies suggest that interleukin-1alpha may be an important cytokine with both local and systemic actions that are linked to the initiation of critical cellular events early in wound healing.     

Expression of growth factors in early wound healing in rat skin

Growth factors are a group of hormone-like polypeptides that have been shown to play a central role in different phases of wound healing. The expression of these growth factors in early wound healing has not been quantified, and the pattern and distribution of these growth factors in early wound healing has not been described completely. Furthermore the quantity and pattern of distribution of these growth factors have not been investigated in early wounds produced by various methods of surgical incision. Comparison of the rate of healing between the CO₂ laser wound and the scalpel wound has produced conflicting results. The present immunohistochemical study uses polyclonal antibodies specific for epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor p (TGF-β), and basic fibroblast growth factor (bFGF) to observe the pattern and distribution of these growth factors in rat skin wound and elucidate whether there are differences in the expression of these growth factors which might account for the delayed healing of the CO₂ laser wounds compared to the scalpel as has been observed by some authors. Our results indicate that EGF, TGF-β, PDGF, and bFGF are expressed and distributed in same areas of the early skin wound. The area of expression of these growth factors was associated with presence of wound inflammatory cells and wound fibroblasts. Our study found that there were no significant differences in the expression of growth factors in the majority of time points between the CO₂ laser wounds and the scalpel wounds. © 1994 wiley-Liss, Inc.     

Inhibition of wound contraction by papaverine: in vitro analysis with a submucosal tissue model.

We studied the effect of topical application of papaverine, a smooth-muscle relaxant, on the contraction of open, dorsal skin wounds in rats. Wound contraction was inhibited significantly in papaverine-applied wounds compared with saline-dressed control wounds. The in vitro effect of papaverine on wound contraction was studied by using human oral fibroblasts cultured three-dimensionally in the hydrated collagen gels containing papaverine. Papaverine inhibited collagen gel contraction in a dose-dependent manner. Any change in actin filament organization in fibroblasts cultured three-dimensionally in the collagen gels was observed by staining the filaments with fluorescent dye-conjugated phalloidin. In the control cultures, well-organized stress fibers were formed in the cell projections; in papaverine-treated fibroblasts, during the early stages of the treatment, the stress fibers were disrupted and actin filament aggregation was observed. In addition, papaverine induced a delay in the formation of the processes in fibroblasts cultured in collagen gels. These results indicate that wound contraction inhibition by papaverine is mediated by its effects on the organization of actin filaments of fibroblasts.