Interference with transforming growth factor-beta/ Smad3 signaling results in accelerated healing of wounds in previously irradiated skin

Transforming growth factor (TGF)-beta regulates many aspects of wound repair including inflammation, chemotaxis, and deposition of extracellular matrix. We previously showed that epithelialization of incisional wounds is accelerated in mice null for Smad3, a key cytoplasmic mediator of TGF-beta signaling. Here, we investigated the effects of loss of Smad3 on healing of wounds in skin previously exposed to ionizing radiation, in which scarring fibrosis complicates healing. Cutaneous wounds made in Smad3-null mice 6 weeks after irradiation showed decreased wound widths, enhanced epithelialization, and reduced numbers of neutrophils and myofibroblasts compared to wounds in irradiated wild-type littermates. Differences in breaking strength of wild-type and Smad3-null wounds were not significant. As shown previously for neutrophils, chemotaxis of primary dermal fibroblasts to TGF-beta required Smad3, but differentiation of fibroblasts to myofibroblasts by TGF-beta was independent of Smad3. Previous irradiation-enhanced induction of connective tissue growth factor mRNA in wild-type, but not Smad3-null fibroblasts, suggested that this may contribute to the heightened scarring in irradiated wild-type skin as demonstrated by Picrosirius red staining. Overall, the data suggest that attenuation of Smad3 signaling might improve the healing of wounds in previously irradiated skin commensurate with an inhibition of fibrosis.     

A comparative study of fibroblasts in healing freeze and burn injuries in rats.

In rats, the healing process of a full-thickness dermal freeze injury differs from that of a burn wound. Whereas burn wounds heal by wound contraction, the movement of surrounding normal skin over the defect, freeze wounds heal without wound contraction. That absence of contraction may be due to the freeze wound's lack of myofibroblasts, the cells reportedly associated with wound contraction. Myofibroblasts can be demonstrated histologically by staining the F-actin filaments of the stress fibers with NBD-phallacidin, a fluorescent reagent specific to F-actin filaments. Fibroblasts in normal dermis have no staining stress fibers. However, staining myofibroblasts are uniformly distributed in the granulation tissue of the healing burn and in the islands of granulation tissue between residual connective tissue fibers in the healing freeze wound. These residual dermal fibers were identified by their patterns of birefringence. Residual connective tissue matrix persists following cold trauma and acts like an internal splint. Burn trauma destroys cells and the connective tissue matrix, which is completely replaced with granulation tissue which undergoes wound contraction. Freeze trauma kills the cellular components of dermis, while some residual connective tissue fibers endure. This study shows that the connective tissue matrix can play an important role in the control of wound contraction.     

Platelet-derived growth factor-BB and transforming growth factor beta 1 selectively modulate glycosaminoglycans, collagen, and myofibroblasts in excisional wounds.

Recombinant platelet-derived growth factor (PDGF) and transforming growth factor beta 1 (TGF-beta 1) influence the rate of extracellular matrix formed in treated incisional wounds. Because incisional healing processes are difficult to quantify, a full-thickness excisional wound model in the rabbit ear was developed to permit detailed analyses of growth-factor-mediated tissue repair. In the present studies, quantitative and qualitative differences in acute inflammatory cell influx, glycosaminoglycan (GAG) deposition, collagen formation, and myofibroblast generation in PDGF-BB (BB homodimer)- and TGF-beta 1-treated wounds were detected when analyzed histochemically and ultrastructurally. Although both growth factors significantly augmented extracellular matrix formation and healing in 10-day wounds compared with controls (P less than 0.002). PDGF-BB markedly increased macrophage influx and GAG deposition, whereas TGF-beta 1 selectively induced significantly more mature collagen bundles at the leading edge of new granulation tissue (P = 0.007). Transforming growth factor-beta 1-treated wound fibroblasts demonstrated active collagen fibrillogenesis and accretion of subfibrils at the ultrastructural level. Myofibroblasts, phenotypically modified fibroblasts considered responsible for wound contraction, were observed in control, but were absent in early growth-factor-treated granulating wounds. These results provide important insights into the mechanisms of soft tissue repair and indicate that 1) PDGF-BB induces an inflammatory response and provisional matrix synthesis within wounds that is qualitatively similar but quantitatively increased compared with normal wounds; 2) TGF-beta 1 preferentially triggers synthesis and more rapid maturation of collagen within early wounds; and 3) both growth factors inhibit the differentiation of fibroblasts into myofibroblasts, perhaps because wound contraction is not required, due to increased extracellular matrix synthesis.     

Smad signaling determines chondrogenic differentiation of bone-marrow-derived mesenchymal stem cells: inhibition of Smad1/5/8P prevents terminal differentiation and calcification

The aim of this study was to investigate the roles of Smad2/3 and Smad1/5/8 phosphorylation in transforming growth factor-beta-induced chondrogenic differentiation of bone-marrow-derived mesenchymal stem cells (BMSCs) to assess whether specific targeting of different Smad signaling pathways offers possibilities to prevent terminal differentiation and mineralization of chondrogenically differentiated BMSCs. Terminally differentiated chondrocytes produced in vitro by chondrogenic differentiation of BMSCs or studied ex vivo during murine embryonic limb formation stained positive for both Smad2/3P and Smad1/5/8P. Hyaline-like cartilage produced in vitro by articular chondrocytes or studied in ex vivo articular cartilage samples that lacked expression for matrix metalloproteinase 13 and collagen X only expressed Smad2/3P. When either Smad2/3 or Smad1/5/8 phosphorylation was blocked in BMSC culture by addition of SB-505124 or dorsomorphin throughout culture, no collagen II expression was observed, indicating that both pathways are involved in early chondrogenesis. Distinct functions for these pathways were demonstrated when Smad signaling was blocked after the onset of chondrogenesis. Blocking Smad2/3P after the onset of chondrogenesis resulted in a halt in collagen II production. On the other hand, blocking Smad1/5/8P during this time period resulted in decreased expression of matrix metalloproteinase 13, collagen X, and alkaline phosphatase while allowing collagen II production. Moreover, blocking Smad1/5/8P prevented mineralization. This indicates that while Smad2/3P is important for continuation of collagen II deposition, Smad1/5/8 phosphorylation is associated with terminal differentiation and mineralization.     

Morphologic examination of mesenchymal cells in healing wounds of normal and tight skin mice.

The healing process of an open wound as effected by wound contraction is complete by 3 weeks in the normal mouse. In contrast, its onset is delayed by 3 weeks and complete healing requires 6 weeks in the tight skin mouse (TSM), a mutant mouse strain with the autosomal dominant gene for tight skin. Possible mechanisms for this delay were evaluated. The frequency and distribution of myofibroblasts were studied during the 3-week delay in wound contraction by actin staining and electron microscopy. It was determined, by electron microscopy and phalloidin staining, that myofibroblasts were found in high density in noncontracting TSM wounds. Electron microscopy showed, however, that these myofibroblasts were surrounded by a pericellular matrix that separated their surface from adjacent collagen fibers. No pericellular matrix was found around cells in granulation tissue of normal mice. At 3 weeks, as TSM wounds began to contract, the number and intensity of cells stained by phalloidin in this tissue was less than that seen earlier. The pericellular matrix was fragmented at this time, and cell surface and collagen fiber associations were apparent. Finally, at 5 weeks, when wound contraction was well developed in the TSM, only a small area in the center of the healing wound beneath the epidermis contained phalloidin-positive myofibroblasts. Electron-microscopic examination of the residual granulation tissue at this time revealed the complete absence of the pericellular matrix. It is postulated that during the 3-week delay in wound closure, the presence of a localized pericellular matrix prevents the interaction between cells and collagen fibers necessary for the reorganization of collagen. It is also thought that the tightly adherent uninjured skin surrounding the healing wound may cause delayed wound closure. There was no evidence that the absence of myofibroblasts is responsible for delayed wound contraction.     

Erythropoietin improves skin wound healing and activates the TGF-β signaling pathway

We could recently report that erythropoietin (EPO) accelerates skin wound healing in mice. Now, we provide insight into the molecular mechanisms of this non-hematopoietic property of EPO analyzing the transforming growth factor (TGF)-β signaling pathway. EPO receptor was found expressed in both non-wounded and wounded skin tissue as well as in fibroblasts and keratinocytes. In saline-treated control animals, wounds exhibited a significant upregulation of TGF-β1 and of α-smooth muscle actin (α-SMA) compared with non-wounded skin. EPO treatment accelerated wound epithelialization and induced mRNA expression of TGF-β1 and α-SMA. In addition, EPO significantly enhanced phosphorylation of Smad2 and Smad3 in fibroblasts and also elevated phosphorylation of Smad3 in wound tissue. Blockade of TGF-β using a neutralizing anti-TGF-β antibody attenuated EPO-induced acceleration of wound epithelialization in vivo and markedly reversed EPO effects on mRNA expression of TGF-β1 and α-SMA. In conclusion, EPO caused activation of the Smad-dependent TGF-β signaling pathway, enhanced differentiation of myofibroblasts, and accelerated skin wound closure.     

Wound healing in embryos: a review

 Skin wounds in young embryos heal rapidly, efficiently and perfectly without scar formation, an ability that is lost as development proceeds. The tissue movements of repair (re-epithelialisation and connective tissue contraction) are the same in embryos as adults, but the means by which these movements are achieved are very different. Whilst adult wound front epidermal cells crawl forwards over the exposed substratum to close a defect, a gap in the embryonic epidermis is closed by contraction of a rapidly assembled actin purse-string. In the adult wound situation connective tissue contraction is brought about by specialist contractile myofibroblasts, but in the embryo standard embryonic fibroblasts exert similar tractional forces to bring the wound margins together. We review what is known of the cellular sources and the levels of various growth factor signals that might activate wound closure movements during embryonic and adult repair and how this knowledge might help in designing therapeutic strategies to enhance adult healing. Finally we discuss how studies of the tissue movements of embryonic wound healing may guide our understanding of more natural tissue movements that occur during embryogenesis, such as gastrulation and neurulation. Accepted: 11 October 1996     

Higher numbers of autologous fibroblasts in an artificial dermal substitute improve tissue regeneration and modulate scar tissue formation

Cultured skin substitutes are increasingly important for the treatment of burns and chronic wounds. The role of fibroblast numbers present in a living-skin equivalent is at present unknown. The quality of dermal tissue regeneration was therefore investigated in relation to the number of autologous fibroblasts seeded in dermal substitutes, transplanted instantaneously or precultured for 10 days in the substitute. A full-thickness porcine wound model was used to compare acellular dermal substitutes (ADS) with dermal substitutes seeded with fibroblasts at two densities, 1x10(5) (0-DS10) and 5x10(5) cells/cm(2) (0-DS50), and with dermal substitutes seeded 10 days before operation at the same densities (10-DS10 and 10-DS50) (n=7 for each group, five pigs). After transplantation of the dermal substitutes, split-skin mesh grafts were applied on top. Wound healing was evaluated blind for 6 weeks. Cosmetic appearance was evaluated and wound contraction was measured by planimetry. The wound biopsies taken after 3 weeks were stained for myofibroblasts (alpha-smooth muscle actin), and after 6 weeks for scar tissue formation (collagen bundles organized in parallel and the absence of elastin staining). Collagen maturation was investigated with polarized light. For wound cosmetic parameters, the 10-DS50 and 0-DS50 treatments scored significantly better than the ADS treatment, as did the 10-DS50 treatment for wound contraction (p<0.05, paired t-test). Three weeks after wounding, the area with myofibroblasts in the granulation tissue, determined by image analysis, was significantly smaller for 0-DS50, 10-DS10, and 10-DS50 than for the ADS treatment (p<0.04, paired t-test). After 6 weeks, the wounds treated with 0-DS50, 0-DS10, and 10-DS50 had significantly less scar tissue and significantly more mature collagen bundles in the regenerated dermis. This improvement of wound healing was correlated with the higher numbers of fibroblasts present in the dermal substitute at the moment of transplantation. In conclusion, dermal regeneration of experimental full-skin defects was significantly improved by treatment with dermal substitutes containing high numbers of (precultured) autologous fibroblasts.     

丹参酮ⅡA抑制心肌细胞的纤维化

背景：转化生长因子β-Smads信号通路是心肌纤维化的关键通路;丹参酮ⅡA具有抑制心肌纤维化作用。 目的：验证丹参酮ⅡA对转化生长因子β1致心肌纤维化的作用并探讨其机制。 方法：取出生24 h内的SD大鼠心脏,利用酶消化法结合差速贴壁体外培养心肌成纤维细胞。取上述第3代心肌成纤维细胞,用5 μg/L转化生长因子β1培养15,30,60,120 min或6,12,24 h后收集细胞,用不同浓度(10-5 mol/L、10-4 mol/L)丹参酮ⅡA预处理2 h后再加入5 μg/L转化生长因子β1培养120 min或24 h后收集细胞,并设空白对照组。免疫细胞化学染色法进行细胞鉴定,反转录聚合酶链反应检测结缔组织生长因子及Ⅰ型胶原蛋白mRNA表达,Western Blot检测Smad7及磷酸化Smad3蛋白表达。免疫组织化学染色及免疫荧光法检测磷酸化Smad3、结缔组织生长因子及Ⅰ型胶原蛋白表达。 结果与结论：转化生长因子β1在一定范围内以时间依赖方式诱导结缔组织生长因子、Ⅰ型胶原、磷酸化Smad3及Smad7的表达,刺激终末结缔组织生长因子、Ⅰ型胶原mRNA的表达量明显上升(均P < 0.01);磷酸化Smad3及Smad7蛋白表达量在刺激后1 h达到峰值,表达量显著增加(均P < 0.01)。高浓度丹参酮ⅡA预处理可显著下调磷酸化Smad3、结缔组织生长因子及Ⅰ型胶原表达(均P < 0.01)。两种浓度的丹参酮ⅡA预处理均可上调Smad7蛋白表达(P < 0.05,P < 0.01)。提示丹参酮ⅡA对心肌纤维化有抑制作用,可能与其上调Smad7蛋白表达,抑制转化生长因子β1诱导的Smad3磷酸化,部分阻断转化生长因子β1-Smads信号通路有关。     

Platelet-derived growth factor-beta receptor activation is essential for fibroblast and pericyte recruitment during cutaneous wound healing

Connective tissue remodeling provides mammals with a rapid mechanism to repair wounds after injury. Inappropriate activation of this reparative process leads to scarring and fibrosis. Here, we studied the effects of platelet-derived growth factor receptor-beta blockade in vivo using the platelet-derived growth factor receptor (PDGFR)-beta inhibitor imatinib mesylate on tissue repair. After 7 days, healing of wounds was delayed with significantly reduced wound closure and concomitant reduction in myofibroblast frequency, expression of fibronectin ED-A, and collagen type I. Using a collagen type I transgenic reporter mouse, we showed that inhibiting PDGFR-beta activation restricted the distribution of collagen-synthesizing cells to wound margins and dramatically reduced cell proliferation in vivo. By 14 days, treated wounds were fully closed. Blocking PDGFR-beta signaling did not prevent the differentiation of myofibroblasts in vitro but potently inhibited fibroblast proliferation and migration. In addition, PDGFR-beta inhibition in vivo was accompanied by abnormal microvascular morphogenesis reminiscent of that observed in PDGFR-beta-/- mice with significantly reduced immunostaining of the pericyte marker NG2. Imatinib treatment also inhibited pericyte proliferation and migration in vitro. This study highlights the significance of PDGFR-beta signaling for the recruitment, proliferation, and functional activities of fibro-blasts and pericytes during the early phases of wound healing.     

Effect of collagen cross-linking inhibition by local application of beta-aminopropionitrile fumarate on wound contraction and healing (macroscopic study)

Wound contraction is a major component of second-intention wound healing. The mechanism of this process is not completely understood. Two theories have been described for the mechanism of the wound contraction. To evaluate the collagen cross-linking inhibition on wound contraction, the present study was carried out. Macroscopical aspects of second-intention healing of full-thickness, excisional wounds were studied in five normal male mixed-breed dogs. Under general anesthesia, two full-thickness skin wounds (20 × 20 mm) were created on the back of each dog symmetrically. Left-side wounds (test group) and right-side wounds (control group) were treated topically with beta-aminopropionitrile fumarate 5 mg/ml in methyl cellulose gel and methyl cellulose gel, respectively. Wounds were treated starting at 24 h after wounding and continued for ten successive days. The wounds were evaluated over a 4-week period. At the days 0, 3, 7, 10, 14, 17, 21, 24, and 28, digital photographs were taken of all wounds. Rulers were held vertically and horizontally close to the wound as a reference. The area of the epithelialization and granulation tissue were measured for each wound using Scion Image software. Percent of the wound contraction, epithelialization, and healing were calculated for each wounds. Wound contraction, epithelialization, and healing were significantly decreased in the wounds treated by beta-aminopropionitrile fumarate (P < 0.05). Our data demonstrated that the collagen and collagen cross-linking play a key role in the wound contraction and healing during the first 10 days of the wound healing.     

Effects of subcutaneous injection of ozone during wound healing in rats

Fibroblast growth factor 2 (FGF2) regulates the wound repair process and it is secreted by inflammatory and endothelial cells, and by myofibroblasts. This study aimed to establish the expression patterns of FGF2 and myofibroblastic differentiation during wound healing in rats treated with subcutaneous ozone injection. We created full-thickness excisional wounds in rats, and the healing process was analyzed through morphometric analyses and digital quantification of immunoreactivity of smooth muscle actin and FGF2. Ozone therapy-treated wounds presented granulation tissue with a reduced number of inflammatory cells and greater dermal cellularity, and intense collagen deposition. FGF2 immunoreactivity, microvessel density, and amount of myofibroblasts were significantly higher in treated wounds compared to controls. In conclusion, it was demonstrated that subcutaneous injections of ozone accelerate and ameliorate wound repairing process. Moreover, injectable ozone therapy’s action mechanism may be associated with FGF2 overexpression.     

Accelerated wound healing by smad3 antisense oligonucleotides-impregnated chitosan/alginate polyelectrolyte complex

Smad3 mediates the intracellular signaling of TGF-beta1 superfamily and plays a critical role in the cellular proliferation, differentiation and elaboration of matrix pivotal to cutaneous wound healing. Smad3 antisense oligonucleotides (ASOs) impregnated polyelectrolyte complex (PEC) containing chitosan and sodium alginate was prepared for accelerated wound healing. Physicochemical properties of PEC were characterized by zeta potential, scanning electron microscopy and bioadhesive test. Full-thickness, excisional wounds were made on the dorsum of C57BL6 mice. Then, smad3 ASOs-PEC, PEC alone, smad3 ASOs and gauze dressing were applied to determine concentration of TGF-beta1 and collagen in tissues and observe the wound contraction and histology of tissues. Zeta potentials and bioadhesive strengths of ASOs-PEC were increased as the chitosan ratio in PEC. In smad3 ASOs-PEC, the healing process suggested by wound closure and histological observation was faster than other groups because collagen contents increased and level of TGF-beta1 decreased. These results demonstrate that the smad3 ASOs-PEC composed of chitosan and sodium alginate could be applied for accelerated wound healing.     

Systemic vanadate ingestion modulates rat tendon repair

The chronic ingestion of vanadate prevents the appearance of myofibroblasts within granulation tissue of full excision wounds in rats, yet these wounds close at an optimal rate. Myofibroblasts are reported in the repair of transected tendons. Here we investigate tendon repair in the absence of myofibroblasts. Vanadate in saline drinking water was given to rats in the experimental group, while rats in the control group received saline alone. The Achilles tendon of the left leg of each rat was transected and suture repaired. On day 10, both repaired tendons and uninjured tendons from the right leg were harvested and processed for histology. By immunohistology the repaired tendons of control rats had myofibroblasts (fibroblasts with alpha smooth muscle actin positive stress fibers), while myofibroblasts were absent in healing tendons from vanadate-treated rats. By transmission electron microscopy and polarized light optics, repaired tendons of control rats demonstrated thin, loosely packed, immature collagen fiber bundles. Collagen fiber bundles from healing tendons of the vanadate-treated group were thicker, uniformly packed, and more mature. The chronic ingestion of vanadate promotes the more rapid organization of collagen fiber bundles of healing transected tendons in the absence of myofibroblasts.     

Platelet-rich plasma gel promotes differentiation and regeneration during equine wound healing

Nonhealing wounds of the lower equine limb represent a challenging model. The platelet is a natural source of a myriad of growth factors and cytokines that promote wound healing. This study evaluates the potential of platelet derived factors to enhance wound healing in the lower equine limb. Platelets were isolated from horse blood and activated with thrombin, a process known to induce growth factor release. This produced a platelet gel composed of platelet-rich plasma (PRP). To test this all-natural wound healant, 2.5-cm(2) full thickness cutaneous wounds were created below the knee and hock of a thoroughbred horse. Wounds were treated with PRP gel or left untreated. Sequential wound biopsies collected at Days 7, 36, and 79 postwounding permitted comparison of the temporal expression of differentiation markers and wound repair. To test the hypothesis that wounds treated with PRP gel exhibit more rapid epithelial differentiation and enhanced organization of dermal collagen compared to controls, tissues were stained for cytokeratin 10, a suprabasal differentiation marker, and the reestablishment of collagen was evaluated by trichrome staining. PRP gel-treated wounds at Day 7 expressed intense cytokeratin 10 staining near the wound junction in suprabasal epidermal layers, while staining in control tissues was less intense and restricted to apical epidermal layers distal to the wound junction. By Day 79, the staining was equal in both groups. However, PRP gel-treated wounds at Day 79 contained abundant, dense collagen bundles oriented parallel to each other and to the overlying epithelium, whereas control tissues contained fewer collagen fibers that were oriented randomly. Thus, treatment of wounds with PRP gel induced accelerated epithelial differentiation and produced tissue with organized, interlocking collagen bundles. This study reveals that this novel all-natural wound healant induced wound repair in injuries previously deemed untreatable.     

Comparative growth dynamics and actin concentration between cultured human myofibroblasts from granulating wounds and dermal fibroblasts from normal skin

The normal contraction of open wounds and many forms of pathologic contracture are related by the presence of a contractile fibroblast known as a myofibroblast. The function of this cell has been postulated as a result of previous pharmacological, immunological, and biochemical testing on strips of contracted connective tissue. The purpose of this study was to develop a specific assay that could measure the concentration of one contractile element (actin) within cultured myofibroblasts isolated from a contracting wound and in normal fibroblasts from uninjured dermis. Rates of growth and actin concentration through 15 days of culture were compared among populations of paired control fibroblasts from normal dermis and granulating wound myofibroblasts from three patients. Growth curves showed that myofibroblasts always grew slower than fibroblasts. An enzyme-linked immunosorbent assay showed that actin concentration was generally greater in mass cultures of granulating wound myofibroblasts than in fibroblasts from uninjured dermis. During exponential growth (1-6 days) the average actin concentration among myofibroblast lines ranged from 24 to 62 pg/cell. Average actin levels among control fibroblasts ranged from 3 to 47 pg/cell during the same interval. After 15 days of culture, actin concentration peaked twice. The first actin peak occurred within the period of exponential growth. At confluency, cellular actin levels dropped. Superconfluent cultures exhibited a second actin peak that displayed an irregular pattern of actin concentration. The latter observation suggested an artifact that might be the result of three-dimensional matrix of cells that altered points of cell adhesion and produced an irregular pattern of actin concentration. These data show that the phenotype of increased actin in cultured myofibroblasts was carried over by myofibroblasts from contracted skin wounds to culture. Because of a higher concentration of actin in myofibroblasts than in undifferentiated fibroblasts, these data suggest that the differentiation process of myofibroblasts may be associated with an increased availability of monomeric actin for filament synthesis. This study demonstrates that the use of tissue culture and our enzyme-linked immunosorbent assay will be a useful method to study factors affecting myofibroblast phenotypic modulation. Future studies should be directed toward developing procedures for isolation of pure populations of myofibroblasts as well as extracellular matrices that would maintain the morphology of both differentiated myofibroblasts and normal undifferentiated fibroblasts.     

Expression of Smad7 in mouse eyes accelerates healing of corneal tissue after exposure to alkali

Damage to the cornea from chemical burns is a serious clinical problem that often leads to permanent visual impairment. Because transforming growth factor (TGF)-beta has been implicated in the response to corneal injury, we evaluated the effects of altered TGF-beta signaling in a corneal alkali burn model using mice treated topically with an adenovirus (Ad) expressing inhibitory Smad7 and mice with a targeted deletion of the TGF-beta/activin signaling mediator Smad3. Expression of exogenous Smad7 in burned corneal tissue resulted in reduced activation of Smad signaling and nuclear factor-kappaB signaling via RelA/p65. Resurfacing of the burned cornea by conjunctival epithelium and its differentiation to cornea-like epithelium were both accelerated in Smad7-Ad-treated corneas with suppressed stromal ulceration, opacification, and neovascularization 20 days after injury. Introduction of the Smad7 gene suppressed invasion of monocytes/macrophages and expression of monocyte/macrophage chemotactic protein-1, TGF-beta1, TGF-beta2, vascular endothelial growth factor, matrix metalloproteinase-9, and tissue inhibitors of metalloproteinase-2 and abolished the generation of myofibroblasts. Although acceleration of healing of the burned cornea was also observed in mice lacking Smad3, the effects on epithelial and stromal healing were less pronounced than those in corneas treated with Smad7. Together these data suggest that overexpression of Smad7 may have effects beyond those of simply blocking Smad3/TGF-beta signaling and may represent an effective new strategy for treatment of ocular burns.     

Role of collagen cross-linking on equine wound contraction and healing

The present study was carried out to evaluate the effect of collagen cross-linking inhibition on equine wound contraction and healing. In five male adult donkeys, two full-thickness skin wounds (20 × 20 mm in diameter) were created on the lateral aspect of forelimbs, at the mid-point between the carpal and fetlock joints under general anesthesia. Two other wounds were created on the neck of each donkey symmetrically. Left-side wounds (test group) and right-side wounds (control group) were treated topically with beta-aminopropionitrile fumarate, 5 mg/ml, added to methyl cellulose gel and only methyl cellulose gel, respectively. Treatment of wounds were started at 24 h after wounding and continued every other day for ten successive days. The wounds were evaluated over a 3-week period. On days 0, 1, 3, 5, 7, 9, 12, 15, 18, and 21, digital photographs were taken of all wounds after careful shaving to visualize the wound margin. Rulers were held vertically and horizontally close to the wound as a reference. Epithelialization and granulation tissue formation were measured for each wound using Scion Image software. Percentage of the wound contraction, epithelialization, and healing were calculated for each wounds. At the end of the study, biopsy was taken from the center of each wound for hydroxyproline measurement and the same corner of each wound for histopathological examination. Macroscopic evaluation revealed significant differences in wound contraction and healing process between test and control groups in wounds located in neck (P < 0.05), but there was no significant difference in percent of epithelialization at the same area (P > 0.05). Significant differences were observed in the percent of wound contraction and healing between the test and control groups in wounds located in the forelimb (P < 0.05), but no significant difference was observed in percent of epithelialization at this area (P > 0.05). There were no significant differences between median of hydroxyproline levels of left and right wounds in forelimb and neck (P > 0.05). Histopathological examination revealed no significant differences between median of epithelialization, inflammatory infiltration, presence of dermal granulation tissue, fibroblast proliferation, arrangement of fibroblasts, collagen deposition, and collagen bundle formation scores in the specimens prepared from left and right wounds in forelimb and neck (P > 0.05). Our data demonstrated that collagen cross-linking could play a key role in equine wound contraction and healing at the limb and neck area.