Acceleration of healing and improvement of scarring: from laboratory discovery to clinical practice

Acceleration of wound healing and improvement of scarring at skin graft donor sites and trauma or surgical lesions are important clinical goals in human and veterinary medicine. It has been discovered that wounds made on early mouse embryos heal quickly and perfectly, with no scars. The cellular and molecular differences between scar‐free embryonic healing and scar‐forming adult healing have been investigated. As a result, molecules have been identified which can be experimentally manipulated during adult healing, both to accelerate the process and to improve scarring. Some of these molecules represent pharmaceutical targets to which novel therapeutic agents have been developed. For example, embryonic wounds have high levels of TGFβ3 from endogenous keratinocytes and fibroblasts, but relatively low levels of TGFβ1 and TGFβ2 derived from degranulating platelets and inflammatory cells, by comparison to adult wounds. Therapeutically elevating the level of TGFβ3 allows adult rodent and porcine wounds to heal significantly faster and with improved scarring. These experimental findings have progressed into further studies in humans. A number of clinical trials with novel pharmaceuticals designed to accelerate healing and prevent scarring have been successfully completed, and further large patient‐based trials are ongoing. These studies indicate that pharmaceutical treatment of healing wounds to accelerate the process (e.g. accelerated re‐epithelialization of graft sites) or improve scarring may soon supplement the current surgical and device approaches to wound management.     

Effect of growth factors on the migration of equine oral and limb fibroblasts using an in vitro scratch assay

The objective of this study was to determine the effect of platelet derived growth factor BB (PDGF), epidermal growth factor (EGF), transforming growth factor β1 (TGFβ1), insulin like growth factor-1 (IGF-1) and fibroblast growth factor-2 (FGF-2) on the proliferation and migration of equine oral mucosa and leg skin fibroblast cell lines, using an in vitro scratch assay. Fibroblasts from the two sites were firstly grown to confluence and then an area of cells removed (cell void area). Cell migration alone (with the addition of the mitosis inhibitor mitomycin-C to the culture media) and proliferation and migration combined (without mitomycin-C) into the cell void area were observed at 0, 5, 10, 24 and 36h. The presence of mitomycin-C in the culture media significantly slowed the closure of the cell void area, as mitosis was inhibited. For the oral cells only, TGFβ1 significantly slowed both migration (with mitomycin-C) and proliferation and migration combined (without mitomycin-C). For the limb cells only, both PDGF and FGF-2 significantly increased fibroblast proliferation and migration combined (without mitomycin-C). For both cell types, EGF significantly reduced migration (with mitomycin-C). IGF-1 had no effect on any of the parameters measured. It was concluded that TGFβ1, PDGF and FGF-2 have differential effects on the proliferation and migration of equine oral and limb fibroblasts. These differences in fibroblast responses to growth factors may in part form the basis of the different clinical outcomes for oral and limb wounds.     

Platelet-derived growth factor acts via both the Rho-kinase and p38 signaling enzymes to stimulate contraction in an in vitro model of equine wound healing

Horses are more prone to complications in the wound healing process than other species, and problems such as chronic inflammation, delayed epithelialization, poor wound contraction, and exuberant granulation tissue are commonly seen, particularly in wounds on the distal limbs. In comparison, wounds of the oral mucosa heal rapidly in a scarless fashion with a high degree of wound contraction. The effect of platelet-derived growth factor BB (PDGF), insulin-like growth factor (IGF)-1, and transforming growth factor β1 (TGFβ1) on the contraction of a fibroblast-populated collagen matrix (FPCM) as a model of equine wound contraction was investigated using equine oral fibroblasts. The fibroblasts were embedded into floating FPCM and treated with PDGF, IGF-1, and TGFβ1. The surface areas of the FPCM were determined daily for 5 d. Platelet-derived growth factor significantly stimulated the contraction of the FPCM at an optimal concentration of 10 ng/mL (P = 0.025). Insulin-like growth factor-1 and TGFβ1 did not significantly affect the contraction of the FPCM relative to the control. To elucidate the mechanisms by which PDGF stimulated contraction of FPCM, the Rho-kinase and p38 cell signaling pathways were blocked, resulting in a significant inhibition (P < 0.001) of PDGF-stimulated contraction. Platelet-derived growth factor BB is a potent stimulator of fibroblast migration, and hence the FPCM contraction generated here is probably a result of its effects on cell migration. The results of the present experiment suggest that this effect is stimulated via both the Rho-kinase and p38 signaling pathways in equine oral fibroblasts.     

Endothelial cell hypertrophy is associated with microvascular occlusion in horse wounds

Wound repair in horse limbs is often complicated by excessive fibroplasia and scarring. Occlusion of the microvessels populating the granulation tissue appears to be involved in the excessive accumulation of extracellular matrix during the repair of limb wounds. This study aimed to determine whether endothelial cell hypertrophy or hyperplasia, or both, contribute to microvascular occlusion and whether the pericyte is involved in this anomaly. We created 5 wounds, each 2.5 x 2.5 cm, on both forelimbs and on the body of 6 horses. One limb was bandaged to stimulate excessive wound fibroplasia. Weekly biopsy specimens were evaluated by transmission electron microscopy to measure microvessel luminal diameters and the surface area of endothelial cells and to count endothelial cells and pericytes. Microvessels were occluded significantly more often in limb wounds than in body wounds. The surface area of endothelial cells lining occluded microvessels (mean +/- standard error, 28.4013 +/- 1.5154 microm2) was significantly greater (P = 0.05) than that of cells lining patent microvessels (26.2220 +/- 1.5268 microm2). Conversely, neither the number of endothelial cells nor the number of pericytes differed between patent and occluded microvessels or between limb and body wounds. Furthermore, the wound location and the status of the microvessels (patent or occluded) did not alter the ratio of endothelial cells to pericytes. These data suggest that endothelial cell hypertrophy might play a role in the microvascular occlusion present in granulation tissue of limb wounds in horses, but the contribution of the pericyte remains obscure.     

The effect of aloe vera oral administration on cutaneous wound healing in type 2 diabetic rats

Delayed wound healing is one of the complications of diabetes mellitus. The present study was performed to investigate the effect of Aloe vera oral administration on open wounds in type 2 diabetic rats. Full thickness open wounds (1.5 × 1.5 cm) were created under general anesthesia on the backs of the rats. These rats were divided into two group, a control group (Group C) and an Aloe vera oral administration group (Group A). Each wound area was measured on days 1, 2, 4 and 8 postwounding. The stages of wound granulation tissues were evaluated histopathologically. The expression of transforming growth factor (TGF)-β1 and vascular endothelial growth factor (VEGF) were determined by immunohistochemically. The wounds were significantly contracted in Group A on days 2, 4 and 8 postwounding. Histological results revealed that the inflammatory cell infiltration, angiogenesis, extracellular matrix deposition and epithelialization were promoted in Group A, respectively. The immunohistochemical results revealed that both TGF-β1 and VEGF protein-positive cells increased in Group A on day 4 postwounding. We concluded that Aloe vera oral administration accelerated wound healing in type 2 diabetic rats.     

Spatial and temporal expression of types I and II receptors for transforming growth factor beta in normal equine skin and dermal wounds

OBJECTIVE : To describe immunolocalization of TGF-beta receptors (RI and RII) in normal equine skin and in thoracic or limb wounds, healing normally or with exuberant granulation tissue (EGT). STUDY DESIGN : Group A: six wounds on one metacarpus and one midthoracic area. Group B: six wounds on both metacarpi, one of which was bandaged to stimulate EGT. Immunohistochemistry was used to detect RI and RII expression in wound margins. ANIMALS : Eight horses, randomly assigned to one of two study groups. METHODS : Neutralizing polyclonal anti-rabbit RI and RII antibodies were used to detect spatial expression of RI and RII in biopsies obtained before wounding, at 12 and 24 hours, and 5, 10 and 14 days after wounding. RESULTS : RI and RII were co-localized in both unwounded and wounded skin. There were no differences in cell types staining positively between tissues obtained from the limb and the thorax, or from normally healing limb wounds and limb wounds with EGT, at any time. Because of increased cellularity within EGT, staining intensity of limb wounds with 'proud flesh' was greater than limb wounds healing normally, and thoracic wounds, during the proliferative phase of repair. CONCLUSIONS : Strong expression of RI and RII, particularly in limb wounds with EGT, suggested that signalling for stimulation of matrix proteins is in place to contribute to scarring. CLINICAL RELEVANCE : This information may help determine the appropriate time for using receptor antagonists to prevent scarring of limb wounds of horses.     

Efficacy and safety of mitomycin C as an agent to treat corneal scarring in horses using an in vitro model

Objective Mitomycin C (MMC) is used clinically to treat corneal scarring in human patients. We investigated the safety and efficacy of MMC to treat corneal scarring in horses by examining its effects at the early and late stages of disease using an in vitro model. Procedure An in vitro model of equine corneal fibroblast (ECF) developed was used. The ECF or myofibroblast cultures were produced by growing primary ECF in the presence or absence of transforming growth factor beta‐1 (TGFβ1) under serum‐free conditions. The MMC dose for the equine cornea was defined with dose‐dependent trypan blue exclusion and (3‐4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assays after applying MMC to the cultures once for 2 min. The efficacy of MMC to control corneal scarring in horses was determined by measuring mRNA and protein expression of corneal scarring markers (alpha‐smooth muscle actin and F‐actin) with western blotting, immunocytochemistry and/or quantitative real‐time polymerase chain reactions. Results A single 2‐min treatment of 0.02% or less MMC did not alter ECF phenotype, viability, or cellular proliferation whereas 0.05% or higher MMC doses showed mild‐to‐moderate cellular toxicity. The TGFβ1 at 1 ng/mL showed significant myofibroblast formation in ECF under serum‐free conditions. A single 2‐min, 0.02% MMC treatment 24 h (early) after TGFβ1 stimulation significantly reduced conversion of ECF to myofibroblasts, however, a single 0.02% MMC treatment 11 days after TGFβ1 stimulation showed moderate myofibroblast inhibition. Conclusions That MMC safely and effectively reduced scarring in ECF by reducing the degree of transdifferentiation of corneal fibroblasts to myofibroblasts in vitro. Further clinical in vivo investigations are warranted using MMC in horses.     

Effect of Gallus TGFβ1 on the Proliferation,Apoptosis, Migration and Invasion of MDCC-MSB1 Cells

This study aimed to detect the effect of Gallus TGFβ1 on the biological behavior of MDCC-MSB1 cells. MDCC-MSB1 cells were transiently transfected with Gallus TGFβ1 overexpression vector, interference expression vector, and the corresponding negative control. Then, the expression of Gallus TGFβ1, the cell proliferation, the cell cycle and apoptosis, the migration and invasion of each transfection groups were examined. Results showed that compared with the corresponding control, the MDCC-MSB1 cells transfected with overexpression vector of Gallus TGFβ1 could up-regulate the expression level of TGFβ1, the proliferation of MDCC-MSB1 cells was significantly inhibited, G1 phase cells were increased, S and G2 cells were decreased, the apoptosis rate of the cells was increased, the migration and invasion ability were decreased.However,the MDCC-MSB1 cells transfected with the interference expression vector of TGFβ1 significantly down-regulated the expression level of TGFβ1, cell proliferation was improved,G1 phase cells were decreased, S and G2 cells were increased, the cell apoptosis was decreased, the migration and invasion ability was increased. The results showed that Gallus TGFβ1 could inhibit the proliferation, migration and invasion of MDCC-MSB1 cells, and promote their apoptosis.     

鸡TGFβ1对MDCC-MSB1细胞增殖、凋亡、迁移与侵袭的影响

旨在探讨鸡TGFβ1对MDCC-MSB1细胞增殖、凋亡、迁移与侵袭的影响。作者将构建的鸡TGFβ1过表达载体、干扰表达载体以及相应阴性对照转染MDCC-MSB1细胞,然后检测转染后各组细胞鸡TGFβ1的表达水平、细胞增殖能力、细胞周期与凋亡,细胞的迁移与侵袭能力。结果显示,与相应阴性对照相比,转染TGFβ1过表达质粒可显著上调MDCC-MSB1细胞的TGFβ1表达水平,显著抑制MDCC-MSB1细胞的增殖,且使G1期细胞增加、S和G2期细胞减少,同时增加细胞凋亡率,降低细胞的迁移与侵袭能力;转染TGFβ1干扰表达质粒可显著下调MDCC-MSB1细胞的TGFβ1表达水平,显著促进MDCC-MSB1细胞的增殖,G1期细胞减少、S和G2期细胞增加,同时降低细胞凋亡率,增加细胞的迁移与侵袭能力。结果表明,鸡TGFβ1可抑制MDCC-MSB1细胞增殖、迁移与侵袭,促进其凋亡。     

OB‐cadherin cloning and expression in a model of wound repair in horses

Reason for performing study: Horses suffer from a debilitating impediment in repairing wounds located on the lower limb that leads to the development of a fibroproliferative disorder (exuberant granulation tissue). This condition is a source of wastage since it often forces retirement from competition. Treatments that resolve or prevent this condition are still lacking, maybe due to deficient knowledge of the underlying molecular mechanisms. Fibroblast‐to‐myofibroblast conversion is an essential step allowing contraction during wound repair and is accompanied by an increase in OB‐cadherin expression. Objectives: To clone equine cadherin‐11 (CDH11) cDNA and to study its spatiotemporal expression profile during the repair of body and limb wounds, thereby contributing to a better understanding of the repair process. Methods: Cloning was by a PCR technique. Expression was studied in intact skin and in 1, 2, 3, 4 and 6‐week‐old wounds of the body and limb. Temporal CDH11 gene expression was determined by RT‐PCR while OB‐cadherin protein expression was mapped immunohistochemically. Results: Equine CDH11 is a highly conserved gene and protein. mRNA was not expressed in equine skin whereas the wound repair process was characterised by a significantly higher expression in the thorax than in limb samples. mRNA expression pattern was paralleled by protein data as confirmed by immunohistochemistry. Conclusions: The data suggest that deficient OB‐cadherin expression in the first phases of wound repair contributes to the excessive proliferative response seen in horse limb wounds. Potential relevance: Future studies should verify the quantitative, temporal expression of this protein in order to provide the basis for targeted therapies that might prevent the development of EGT in horse wound repair.     

In vitro differentiation of bovine bone marrow‐derived mesenchymal stem cells into male germ cells by exposure to exogenous bioactive factors

Mesenchymal stem cells (MSC) are multipotent progenitor cells defined by their ability to self‐renew and give rise to differentiated progeny. Previous studies have reported that MSC may be induced in vitro to develop into different types of specialized cells including male gametes. In vitro gamete derivation technology has potential applications as an alternative method for dissemination of elite animal genetics, production of transgenic animals and conservation of endangered species. This study aimed at investigating the in vitro effect of BMP4, TGFβ1 and RA on the potential for germ cell (GC) differentiation of bovine foetal MSC (bfMSC) derived from bone marrow (BM). The effect of BMP4, TGFβ1 and RA was analysed on the expression of pluripotent, GC and male GC markers on bfMSC during a 21‐day culture period. bfMSC cultured under in vitro conditions expressed OCT4, NANOG and DAZL, but lacked expression of mRNA of VASA, STELLA, FRAGILIS, STRA8 and PIWIL2. Treatment with exogenous BMP4 and TGFβ1 induced a transient increase (p < .05) in DAZL and NANOG mRNA levels, respectively. However, exposure to RA was more effective in increasing (p < .05) expression of DAZL and regulating expression of OCT4 and mRNA levels of NANOG. These data suggest that bfMSC may possess potential for early GC differentiation, where OCT4, NANOG and specially DAZL may play significant roles in controlling progression along the GC lineage.     

An evaluation of methods for the assessment of healing of open wounds in the dog

Acceleration of wound healing and improvement of scarring at skin graft donor sites and trauma or surgical lesions are important clinical goals in human and veterinary medicine. It has been discovered that wounds made on early mouse embryos heal quickly and perfectly, with no scars. The cellular and molecular differences between scar-free embryonic healing and scar-forming adult healing have been investigated. As a result, molecules have been identified which can be experimentally manipulated during adult healing, both to accelerate the process and to improve scarring. Some of these molecules represent pharmaceutical targets to which novel therapeutic agents have been developed. For example, embryonic wounds have high levels of TGFβ3 from endogenous keratinocytes and fibroblasts, but relatively low levels of TGFβ1 and TGFβ2 derived from degranulating platelets and inflammatory cells, by comparison to adult wounds. Therapeutically elevating the level of TGFβ3 allows adult rodent and porcine wounds to heal significantly faster and with improved scarring. These experimental findings have progressed into further studies in humans. A number of clinical trials with novel pharmaceuticals designed to accelerate healing and prevent scarring have been successfully completed, and further large patient-based trials are ongoing. These studies indicate that pharmaceutical treatment of healing wounds to accelerate the process (e.g. accelerated re-epithelialization of graft sites) or improve scarring may soon supplement the current surgical and device approaches to wound management.     

Treatment of Exuberant Granulation Tissue in the Horse Evaluation of Four Methods

Four methods of treating granulating wounds on the dorsal aspect of the metacar-pophalangeal and metatarsophalangeal joints of ponies were evaluated. The following treatments were used: Group 1—excision of the granulation tissue with no further treatment; Group 2—cryosurgery; Group 3—excision of the granulation tissue and pressure bandage; and Group 4—excision of the granulation tissue and immobilization of the limb with a plaster cast. The wounds in Group 1 healed fastest, without producing exuberant granulation tissue and with only moderate scar fibrosis. The wounds in Group 2 healed without producing exuberant granulation tissue but with marked scarring. Wounds in Groups 3 and 4 took longer (p < 0.001) to heal compared to wounds in Groups 1 and 2. Wounds in Groups 3 and 4 produced exuberant granulation tissue, but the resultant scars were the least fibrotic.     

Tenogenic induction of equine mesenchymal stem cells by means of growth factors and low-level laser technology

Tendons regenerate poorly due to a dense extracellular matrix and low cellularity. Cellular therapies aim to improve tendon repair using mesenchymal stem cells and tenocytes; however, a current limitation is the low proliferative potential of tenocytes in cases of severe trauma. The purpose of this study was to develop a method useful in veterinary medicine to improve the differentiation of Peripheral Blood equine mesenchymal stem cells (PB-MSCs) into tenocytes. PB-MSCs were used to study the effects of the addition of some growth factors (GFs) as TGFβ3 (transforming growth factor), EGF2 (Epidermal growth factor), bFGF2 (Fibroblast growth factor) and IGF-1 (insulin-like growth factor) in presence or without Low Level Laser Technology (LLLT) on the mRNA expression levels of genes important in the tenogenic induction as Early Growth Response Protein-1 (EGR1), Tenascin (TNC) and Decorin (DCN). The singular addition of GFs did not show any influence on the mRNA expression of tenogenic genes whereas the specific combinations that arrested cell proliferation in favour of differentiation were the following: bFGF2 + TGFβ3 and bFGF2 + TGFβ3 + LLLT. Indeed, the supplement of bFGF2 and TGFβ3 significantly upregulated the expression of Early Growth Response Protein-1 and Decorin, while the use of LLLT induced a significant increase of Tenascin C levels. In conclusion, the present study might furnish significant suggestions for developing an efficient approach for tenocyte induction since the external administration of bFGF2 and TGFβ3, along with LLLT, influences the differentiation of PB-MSCs towards the tenogenic fate.     

Factors regulating collagen synthesis and degradation during second-intention healing of wounds in the thoracic region and the distal aspect of the forelimb of horses

OBJECTIVE: To determine significant molecular and cellular factors responsible for differences in second-intention healing in thoracic and metacarpal wounds of horses. ANIMALS: 6 adult mixed-breed horses. PROCEDURE: A full-thickness skin wound on the metacarpus and another such wound on the pectoral region were created, photographed, and measured, and tissue was harvested from these sites weekly for 4 weeks. Gene expression of type-I collagen, transforming growth factor (TGF)-beta1, matrix metalloproteinase (MMP)-1, and tissue inhibitor of metalloproteinase (TIMP)-1 were determined by quantitative in situ hybridization. Myofibroblasts were detected by immunohistochemical labeling with alpha-smooth muscle actin (alpha-SMA). Collagen accumulation was detected by use of picrosirius red staining. Tissue morphology was examined by use of H&E staining. RESULTS: Unlike thoracic wounds, forelimb wounds enlarged during the first 2 weeks. Myofibroblasts, detected by week 1, remained abundant with superior organization in thoracic wounds. Type-I collagen mRNA accumulated progressively in both wounds. More type-I collagen and TGF-beta1 mRNA were seen in forelimb wounds. Volume of MMP-1 mRNA decreased from day 0 in both wounds. By week 3, TIMP-1 mRNA concentration was greater in thoracic wounds. CONCLUSIONS AND CLINICAL RELEVANCE: Greater collagen synthesis in metacarpal than thoracic wounds was documented by increased concentrations of myofibroblasts, type-I collagen mRNA,TGF-beta1 mRNA, and decreased collagen degradation (ie, MMP-1). Imbalanced collagen synthesis and degradation likely correlate with development of exuberant granulation tissue, delaying healing in wounds of the distal portions of the limbs. Factors that inhibit collagen synthesis or stimulate collagenase may provide treatment options for horses with exuberant granulation tissue.     

Expression of transforming growth factor beta(1), beta(3), and basic fibroblast growth factor in full-thickness skin wounds of equine limbs and thorax

OBJECTIVE--To map the expression of transforming growth factor (TGF)-beta(1), TGF-beta(3), and basic fibroblast growth factor (bFGF) in full-thickness skin wounds of the horse. To determine whether their expression differs between limbs and thorax, to understand the pathogenesis of exuberant granulation tissue. STUDY DESIGN--Six wounds were created on one lateral metacarpal area and one midthoracic area of each horse. Sequential wound biopsies allowed comparison of the temporal expression of growth factors between limb and thoracic wounds. ANIMALS--Four 2- to 4-year-old horses. METHODS--Wounds were assessed grossly and histologically at 12 and 24 hours, and 2, 5, 10, and 14 days postoperatively. ELISAs were used to measure the growth factor concentrations of homogenates of wound biopsies taken at the same timepoints. RESULTS--TGF-beta(1) peaked at 24 hours in both locations and returned to baseline in thoracic wounds by 14 days but remained elevated in limb wounds for the duration of the study. Expression kinetics of TGF-beta(3) differed from those of TGF-beta(1). TGF-beta(3) concentrations gradually increased over time, showing a trend toward an earlier and higher peak in thoracic compared with limb wounds. bFGF expression kinetics resembled those of TGF-beta(1), but no statistically significant differences existed between limb and thoracic wounds. CONCLUSIONS--Growth factor expression is up-regulated during normal equine wound repair. TGF-beta(1) and TGF-beta(3) show a reciprocal temporal regulation. Statistically significant differences exist between limb and thoracic wounds with respect to TGF-beta(1) expression. CLINICAL RELEVANCE--The persistence of TGF-beta(1) expression in leg wounds may be related to the development of exuberant granulation tissue in this location, because TGF-beta(1) is profibrotic.     

Interactions between TGFβ1 and cyclic strain in modulation of myofibroblastic differentiation of canine mitral valve interstitial cells in 3D culture

ObjectivesThe mechanisms of myxomatous valve degeneration (MVD) are poorly understood. Transforming growth factor-beta1 (TGFβ1) induces myofibroblastic activation in mitral valve interstitial cells (MVIC) in static 2D culture, but the roles of more physiological 3D matrix and cyclic mechanical strain are unclear. In this paper, we test the hypothesis that cyclic strain and TGFβ1 interact to modify MVIC phenotype in 3D culture.Animals, materials and methodsMVIC were isolated from dogs with and without MVD and cultured for 7 days in type 1 collagen hydrogels with and without 5 ng/ml TGFβ1. MVIC with MVD were subjected to 15% cyclic equibiaxial strain with static cultures serving as controls. Myofibroblastic phenotype was assessed via 3D matrix compaction, cell morphology, and expression of myofibroblastic (TGFβ3, alpha-smooth muscle actin – αSMA) and fibroblastic (vimentin) markers.ResultsExogenous TGFβ1 increased matrix compaction by canine MVIC with and without MVD, which correlated with increased cell spreading and elongation. TGFβ1 increased αSMA and TGFβ3 gene expression, but not vimentin expression, in 15% cyclically stretched MVIC. Conversely, 15% cyclic strain significantly increased vimentin protein and gene expression, but not αSMA or TGFβ3. 15% cyclic strain however was unable to counteract the effects of TGFβ1 stimulation on MVIC.ConclusionsThese results suggest that TGFβ1 induces myofibroblastic differentiation (MVD phenotype) of canine MVIC in 3D culture, while 15% cyclic strain promotes a more fibroblastic phenotype. Mechanical and biochemical interactions likely regulate MVIC phenotype with dose dependence. 3D culture systems can systematically investigate these phenomena and identify their underlying molecular mechanisms.     

Temporal localization of immunoreactive transforming growth factor beta1 in normal equine skin and in full-thickness dermal wounds

OBJECTIVE: To describe the localization of immunoreactive transforming growth factor (TGF)-beta1 in both normal skin and full-thickness dermal wounds of the limb and the thorax of the horse. STUDY DESIGN: Six full-thickness excisional wounds were created on the lateral aspect of one metacarpal region and on the midthoracic area of each horse. Sequentially collected tissue specimens from wound margins were assessed for TGF-beta1 expression by immunohistochemistry. ANIMALS: Four horses (2 to 4 years of age). METHODS: A neutralizing monoclonal anti-human TGF-beta1 antibody was used to detect the spatial expression of TGF-beta1 protein by immunohistochemical localization in biopsies obtained before wounding and at 12 and 24 hours, and 5, 10, and 14 days. RESULTS: No differences in localization of immunoreactive TGF-beta1 were detected between limb and thorax, for either intact skin or wounds. Unwounded epidermis stained moderately for TGF-beta1 protein throughout all layers, whereas the dermis was relatively devoid of immunoreactivity. During the acute stage of repair, migrating epithelium lost its stain, whereas cells of epidermal appendages remained strongly immunoreactive. The epithelium recovered its TGF-beta1 immunoreactivity during wound remodeling, although cells of the stratum corneum remained negative. Macrophages of the inflammatory exudate had positive cytoplasmic staining that diminished with time. Immunoreactivity of granulation tissue fibroblasts was evident early on and increased throughout the repair process. CONCLUSIONS: TGF-beta1 is constitutively expressed in normal, unwounded equine epithelium. Its expression is upregulated within the skin on injury and is associated with the cells involved in wound repair. CLINICAL RELEVANCE: A more precise understanding of the temporal and spatial expression of TGF-beta1 during wound repair in horses should provide the groundwork for possible future manipulations of both normal and aberrant tissue repair.     

Application of Exogenous Esterified Hyaluronan to Equine Distal Limb Wounds

The objective of this study was to evaluate the efficacy of topical application of a hyaluronan (HA) derivative in wound healing with respect to the rate of epithelialization, fibroplasia, angiogenesis and contraction, magnitude of the local inflammatory response, local expression of transforming growth factor-β 1 and 3 (TGF-β 1 and 3), tumor necrosis factor-α (TNF-α), and collagen type III deposition. In six healthy adult horses, six full-thickness skin wounds were created on the dorsal aspect of both metacarpi using a sterile template. Sites were sampled at 0, 1, 2, 5, 14, 21, and 35 days following wounding. Wounds on one limb were dressed with commercially available esterified HA fleece under a nonadherent dressing. The opposite limb was covered with the nonadherent dressing alone (control). Images of the most proximal wounds were used to determine the area of total healing and the relative contributions of epithelialization and contraction to healing. At each sample time, a control and treatment biopsy were taken for histological evaluation and special stains. All samples were evaluated for degree of inflammation, fibroplasia and angiogenesis; in situ hybridization for type III collagen, TGFβ1 and 3, and immunohistochemistry for TNF-α. Mean percentages of total wound healing, epithelialization, and wound contraction were not significantly different between control and treatment groups. In treated horses, initial wound expansion was significantly decreased during the first 2 weeks. Mononuclear cell numbers, counted in the granulation tissue, increased in both control and treated limbs over the entire course of the study. However at day 35 the macrophage numbers counted in the treated horses were significantly increased compared with the control limbs (P < .05). Although not statistically significant, relative staining for type III collagen in the treated wounds was less than that of control wounds. Results of the present study do not support a benefit of an exogenous HA-derivative in the healing of distal limb wounds in horses. The shortcomings of the study design are discussed.     

Preliminary observations on expression of transforming growth factors beta1 and beta3 in equine full-thickness skin wounds healing normally or with exuberant granulation tissue

OBJECTIVE: To determine whether transforming growth factor (TGF)-beta1 and -beta3 expression differs between equine limb wounds healing normally and those healing with experimentally induced exuberant granulation tissue (EGT). STUDY DESIGN: Six wounds were created on the lateral aspect of both metacarpi of each horse; one forelimb was untreated, and the other was bandaged to stimulate the development of EGT. Sequential wound biopsies allowed comparison of growth factor expression between the two types of wound. ANIMALS: Four horses (2 to 4 years of age; 350 to 420 kg). METHODS: Wounds were assessed grossly, histologically, and by enzyme-linked immunosorbent assay (ELISA) for TGF-beta1 and -beta3 expression at 12 and 24 hours and 2, 5, 10, and 14 days postoperatively. RESULTS: Bandaged wounds developed EGT. In all wounds, TGF-beta1 peaked early and remained elevated at 14 days. Peak TGF-beta1 concentration was higher in wounds with EGT, but not significantly so. Expression of TGF-beta3 differed from TGF-beta1, with peak TGF-beta3 concentrations being delayed. Concentrations of TGF-beta3 were higher in wounds healing normally, but this difference was not significant. CONCLUSIONS: During both normal and exuberant wound repair, the expression of TGF-beta1 occurred earlier than TGF-beta3 expression. Wounds healing with EGT tended to have higher concentrations of fibrogenic TGF-beta1 and lower concentrations of antifibrotic TGF-beta3 than wounds healing normally, although these differences were not statistically significant. CLINICAL RELEVANCE: This study suggests that the production of EGT in bandaged wounds may be related to increased expression of fibrogenic TGF-beta1 and decreased expression of antifibrotic TGF-beta3. Further investigation of the roles of TGF-beta1 and -beta3 may be important in understanding the molecular control of EGT in horses.