Later Stages Of Wound Healing Research Articles

Fibroblast fate regulation by time dependent TGF-β1 and IL-10 stimulation in biomimetic 3D matrices.

The presentation of TGF-β1 during the early stage of wound healing is a prerequisite for extracellular matrix (ECM) synthesis and remodeling by activated fibroblasts, called myofibroblasts. At later stages, clearance of myofibroblasts is needed to avoid overshooting ECM production. Apoptosis of myofibroblasts and the macrophage-released anti-inflammatory cytokine IL-10 are controversially discussed as regulating cues in this context. To reveal the regulating cues, defined biomaterial scaffolds are needed to conduct in-depth in vitro studies in a physiologically relevant context. In this work, we used an in vitro biomimetic wound healing model. It consists of a 3D fibrillar matrix from collagen I and fibronectin and different temporal stimuli by TGF-β1 and IL-10. Human dermal fibroblast behavior was investigated in terms of myofibroblast differentiation (αSMA expression), matrix remodeling, proliferation and migration in the permanent or sequential presence of TGF-β1 and IL-10 over 4 days. We could show that removal of TGF-β1 after initial stimulation resulted in an increase of apoptosis of myofibroblasts. In contrast, TGF-β1 stimulation followed by IL-10 treatment did not result in increased cell apoptosis but instead led to a significant increase of cell motility and reduction of myofibroblasts. The findings suggest that myofibroblasts are a transiently "activated" fibroblastic phenotype and can be de-differentiated to fibroblasts in the presence of IL-10. Overall, our 3D ECM model allows mimicking the early and late stages of wound healing and highlights the temporal sequence of TGF-β1 and IL-10 as an important cue for completion of tissue formation and maintenance of tissue homeostasis.

Adipose Extracellular Matrix/Stromal Vascular Fraction Gel Secretes Angiogenic Factors and Enhances Skin Wound Healing in a Murine Model.

Mesenchymal stem cells are an attractive cell type for cytotherapy in wound healing. The authors recently developed a novel, adipose-tissue-derived, injectable extracellular matrix/stromal vascular fraction gel (ECM/SVF-gel) for stem cell therapy. This study was designed to assess the therapeutic effects of ECM/SVF-gel on wound healing and potential mechanisms. ECM/SVF-gel was prepared for use in nude mouse excisional wound healing model. An SVF cell suspension and phosphate-buffered saline injection served as the control. The expression levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and monocyte chemotactic protein-1 (MCP-1) in ECM/SVF-gel were analyzed at different time points. Angiogenesis (tube formation) assays of ECM/SVF-gel extracts were evaluated, and vessels density in skin was determined. The ECM/SVF-gel extract promoted tube formation in vitro and increased the expression of the angiogenic factors VEGF and bFGF compared with those in the control. The expression of the inflammatory chemoattractant MCP-1 was high in ECM/SVF-gel at the early stage and decreased sharply during the late stage of wound healing. The potent angiogenic effects exerted by ECM/SVF-gel may contribute to the improvement of wound healing, and these effects could be related to the enhanced inflammatory response in ECM/SVF-gel during the early stage of wound healing.

Negative pressure environment promotes angiogenesis and microvessel maturation

Objective To study the effect of negative pressure environment on angiogenesis and microvessel maturation and relevant signaling pathway, and explore the potential mechanisms of angiogenin/tyrosine kinase receptor-2 signal pathway promoting angiogenesis and microvessel maturation under negative pressure environment. Methods Successive 58 patients enrolled in this study were treated with negative pressure or petroleum gauze, and at 1st, 3rd, 7th, and 15th day blood flow perfusion was detected, as well as the Western boltting was applied to detect angiogenin-1 (Ang-1), the phosphorylation of tyrosine kinase receptor-2 (pTie-2), angiogenin-2 (Ang-2), α-smooth muscle actin (α-SMA) and collagen type Ⅳ respectively. Results Negative pressure environment could change the expression of related factorsin the early stages of wound healing (1-3 d) and late stage (7-15 d). In experimental group on day 3, the relative value of Ang-2 was 1.87±0.35 and significantly higher than control group (1.32±0.23, P<0.05), relative value of pTie-2 was 0.36±0.02 and significantly lower than control group (0.87±0.04, P<0.05), relative value of Ang-1 was 0.34±0.02 and significantly lower than control group (0.78±0.07, P<0.05), the ratio of Ang-1/Ang-2 was 0.34±0.02 and significantly lower than the control group (0.78±0.07, P<0.05). In the experimental group on day 15, negative pressure environment leadin to the relative value of Ang-1 was 1.89±0.24 and significantly higher than conrol group (1.35±0.22, P<0.05), relative value of α- smooth muscle actin was 2.56±0.42 and significantly higher than control group (1.68±0.31, P<0.05), relative value of collagen type Ⅳ was 2.32±0.35 and significantly higher than control group (1.36±0.24, P<0.05), the relative value of pTie-2 was 2.12±0.44 and significantly higher than control group (1.23±0.31, P<0.05), the ratio of Ang-1/Ang-2 was 4.86±0.67 and significantly higher than control group (1.87±0.54, P<0.05), the relative value of Ang-2 was 0.36±0.05 and significantly lower than control group (0.71±0.06, P<0.05). Conclusion Negative pressure could preferentially promote microvessel destabilization in the early stage of wound healing, consequently increase the number of angiogenesis. Subsequently, in the later stage of wound healing the negative pressure could preferentially promote microvessel stabilization, and thus promote microvessel maturation through the angiogenin/tyrosine kinase receptor-2 signal pathway. Negative pressure could increase blood flow perfusion because of abundant and mature microvessel pericytes in the wound. Key words: Negative pressure environment; Blood vessel maturation; Blood vessel stabilized; Blood flow perfusion

Corneal Wound Healing Requires IKB kinase β Signaling in Keratocytes.

IkB kinase β (IKKβ) is a key signaling kinase for inflammatory responses, but it also plays diverse cell type-specific roles that are not yet fully understood. Here we investigated the role of IKKβ in the cornea using IkkβΔCS mice in which the Ikkβ gene was specifically deleted in the corneal stromal keratocytes. The IkkβΔCS corneas had normal morphology, transparency and thickness; however, they did not heal well from mild alkali burn injury. In contrast to the IkkβF/F corneas that restored transparency in 2 weeks after injury, over 50% of the IkkβΔCS corneas failed to fully recover. They instead developed recurrent haze with increased stromal thickness, severe inflammation and apoptosis. This pathogenesis correlated with sustained myofibroblast transformation with increased α smooth muscle actin (α-SMA) expression, higher levels of senescence β-Gal activity and scar tissue formation at the late stage of wound healing. In addition, the IkkβΔCS corneas displayed elevated expression of hemo-oxygenase-1 (HO-1), a marker of oxidative stress, and activation of stress signaling pathways with increased JNK, c-Jun and SMAD2/3 phosphorylation. These data suggest that IKKβ in keratocytes is required to repress oxidative stress and attenuate fibrogenesis and senescence in corneal wound healing.

Negative pressure wound therapy promotes vessel destabilization and maturation at various stages of wound healing and thus influences wound prognosis.

Negative pressure wound therapy (NPWT) has been observed to accelerate the wound healing process in humans through promoting angiogenesis. However, the potential biological effect and relevant molecular mechanisms, including microvessel destabilization, regression and endothelial cell proliferation in the early stage (1–3 days), and the neovascular stabilization and maturation in the later stage (7–15 days), have yet to be fully elucidated. The current study aimed to research the potential effect of NPWT on angiogenesis and vessel maturation, and investigate relevant association between mature microvessels and wound prognosis, as well as the regulatory mechanisms in human wound healing. Patients in the present study (n=48) were treated with NPWT or a petrolatum gauze, and relevant growth factors and vessel changes were detected using various experimental methods. NPWT increased the expression levels of angiogenin-2 (Ang-2), and decreased the expression levels of Ang-1 and ratios of Ang-1/Ang-2 in the initial stages of wound healing. However, in the latter stages of wound healing, NPWT increased the expression levels of Ang-1 and ratios of Ang-1/Ang-2, as well as the phosphorylation level of tyrosine kinase receptor-2. Consequently, microvessel pericyte coverage was gradually elevated, and the basement membrane was gradually supplied with new blood at the later stage of wound healing. In conclusion, NPWT may preferentially stimulate microvessel destabilization and regression in the early stage of wound healing, and as a consequence, increase angiogenesis. Subsequently, in the later stage of wound healing, NPWT may preferentially promote microvessel stabilization, thereby promoting microvessel maturation in human wounds through the angiogenin/tyrosine kinase receptor-2 signaling pathway. The results of the present study results demonstrated that NPWT was able to accelerate wound healing speed, and thus influence wound prognosis, as a result of an abundance of mature microvessels in human wounds.

Combined Mulberry Leaf and Fruit Extract Improved Early Stage of Cutaneous Wound Healing in High-Fat Diet-Induced Obese Mice.

Obesity is a pandemic that causes many health challenges, including difficulties in achieving proper wound healing without complications. The current study investigated the role of NLRP3 inflammasome in the early stages of cutaneous wound healing and the effect of combined mulberry leaf and fruit extract (MLFE) on cutaneous NLRP inflammasome involvement in delayed wound healing mice with high-fat diet (HFD)-induced obesity. After obesity was induced by HFD for 10 weeks, the mice were supplemented with MLFE (at a dose of 500 mg/kg containing 333.3 mg/kg of mulberry leaf extract and 166.7 mg/kg of mulberry fruit extract) by gavage, 5 days/week for 12 weeks. MLFE supplementation ameliorated delayed wound closure in obese mice. While wound size was positively correlated with fasting blood glucose level during the early stage of wound healing, it was strongly correlated with body weight gain and body fat mass during the later stage of wound healing. Under obese conditions, the levels of NLRP3 inflammasome and its related markers (pro-caspase-1 and precursor/mature interleukin 1 beta) were increased at a basal level, but the NLRP3 inflammasome was suppressed during the inflammatory stage of cutaneous wound healing. However, MLFE supplementation stimulated cutaneous NLRP3 inflammasome in HFD-induced obese mice (day 3). Taken together, stimulating the NLRP3 inflammasome might be beneficial in the early inflammatory stage of cutaneous wound healing and MLFE could be a potential therapeutic intervention in delayed wound healing through activation of the NLRP3 inflammasome in obesity.

Acute suppression of TGF-ß with local, sustained release of tranilast against the formation of fibrous capsules around silicone implants

We propose the acute, local suppression of transforming growth factor beta (TGF-ß), a major profibrotic cytokine, to reduce fibrosis around silicone implants. To this end, we prepared silicone implants that were able to release tranilast, a TGF-ß inhibitor, in a sustained manner for 5days or 15days. We performed histologic and immunohistochemical analyses for 12weeks after the implantation of the implants in living rats. The capsule thicknesses and collagen densities significantly decreased compared with those around the non-treated silicone implants. Notably, early suppression of TGF-ß affected the fibrogenesis that actually occurs at the late stage of wound healing. This change may be ascribed to the decrease in monocyte recruitment mediated by early TGF-ß during the acute inflammatory reaction. Thus, a significant decrease in differentiated macrophages was observed along with a decrease in the quantity of TGF-ß and fibroblasts during the subsequent inflammation stage; these changes led to a diminished fibrotic capsule formation.

Epithelial cells are active participants in vocal fold wound healing: an in vivo animal model of injury.

Vocal fold epithelial cells likely play an important, yet currently poorly defined, role in healing following injury, irritation and inflammation. In the present study, we sought to identify a possible role for growth factors, epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGFβ1), in epithelial regeneration during wound healing as a necessary first step for uncovering potential signaling mechanisms of vocal fold wound repair and remodeling. Using a rat model, we created unilateral vocal fold injuries and examined the timeline for epithelial healing and regeneration during early and late stages of wound healing using immunohistochemistry (IHC). We observed time-dependent secretion of the proliferation marker, ki67, growth factors EGF and TGFβ1, as well as activation of the EGF receptor (EGFR), in regenerating epithelium during the acute phase of injury. Ki67, growth factor, and EGFR expression peaked at day 3 post-injury. Presence of cytoplasmic and intercellular EGF and TGFβ1 staining occurred up to 5 days post-injury, consistent with a role for epithelial cells in synthesizing and secreting these growth factors. To confirm that epithelial cells contributed to the cytokine secretion, we examined epithelial cell growth factor secretion in vitro using polymerase chain reaction (PCR). Cultured pig vocal fold epithelial cells expressed both EGF and TGFβ1. Our in vivo and in vitro findings indicate that epithelial cells are active participants in the wound healing process. The exact mechanisms underlying their roles in autocrine and paracrine signaling guiding wound healing await study in a controlled, in vitro environment.

Expression of Ski induces apoptosis and represses autophagy in cardiac myofibroblasts (868.8)

Following myocardial infarction (MI), relatively quiestent cardiac fibroblasts undergo phenoconversion to active, hyper‐synthetic myofibroblasts. During the late stages of wound healing, myofibroblasts are removed from the infracted region through apoptosis thus decreasing scar cellularity. Apoptosis and autophagy regulate cell fate. The role of apoptosis within the remodeling heart has been well described however the role that autophagy plays following an MI is relatively unknown. Ski is a negative regulator of TGF‐β signaling and has been implicated to play a role in the post‐MI heart. Herein we investigated the regulatory role that Ski has on apoptosis and autophagy using first passage primary rat cardiac myofibroblasts. Using an adenoviral approach for gene delivery, both MTT and Live/Dead viability/cytoxicity assays demonstrated a reduction in the number of viable cells and an increase in the number of dying cells due to the over‐expression of Ski. Additionally, over‐expression of Ski led to distinct morphological changes characteristic of apoptosis. These findings were confirmed via Western blot analysis, and caspase GLO analysis showing induction of executioner caspases‐3 and ‐7, as well as the mitochondrial regulator Bax. Furthermore, evidence of caspase‐9 cleavage, but not caspase‐8, indicated that this was primarily an intrinsic apoptotic response. Markers for autophagosome formation including LC3‐B and ATG‐7 were found to be significantly reduced following Ski over‐expression. In summary, we found that over‐expression of Ski in primary cardiac myofibroblasts leads to an induction of the intrinsic apoptotic pathway and reduction in autophagy leading to cell death.Grant Funding Source: Canadian Institutes of Health Research

Granulocyte/Macrophage Colony-Stimulating Factor Influences Angiogenesis by Regulating the Coordinated Expression of VEGF and the Ang/Tie System

Granulocyte/macrophage colony-stimulating factor (GM-CSF) can accelerate wound healing by promoting angiogenesis. The biological effects of GM-CSF in angiogenesis and the corresponding underlying molecular mechanisms, including in the early stages of primitive endothelial tubule formation and the later stages of new vessel maturation, have only been partially clarified. This study aimed to investigate the effects of GM-CSF on angiogenesis and its regulatory mechanisms. Employing a self-controlled model (Sprague-Dawley rats with deep partial-thickness burn wounds), we determined that GM-CSF can increase VEGF expression and decrease the expression ratio of Ang-1/Ang-2 and the phosphorylation of Tie-2 in the early stages of the wound healing process, which promotes the degradation of the basement membrane and the proliferation of endothelial cells. At later stages of wound healing, GM-CSF can increase the expression ratio of Ang-1/Ang-2 and the phosphorylation of Tie-2 and maintain a high VEGF expression level. Consequently, pericyte coverages were higher, and the basement membrane became more integrated in new blood vessels, which enhanced the barrier function of blood vessels. In summary, we report here that increased angiogenesis is associated with GM-CSF treatment, and we indicate that VEGF and the Ang/Tie system may act as angiogenic mediators of the healing effect of GM-CSF on burn wounds.

Use of ginsenoside Rg3-loaded electrospun PLGA fibrous membranes as wound cover induces healing and inhibits hypertrophic scar formation of the skin

Prevention of hypertrophic scar formation of the skin requires a complex treatment process, which mainly includes promoting skin regeneration in an early stage while inhibiting hypertrophic formation in a later stage. Electrospinning PLGA with the three-dimensional micro/nano-fibrous structure and as drugs carrier, could be used as an excellent skin repair scaffold. However, it is difficult to combine the advantage of nanofibrous membranes and drug carriers to achieve early and late treatment. In this study, Ginsenoside-Rg3 (Rg3) loaded hydrophilic poly(d,l-lactide-co-glycolide) (PLGA) electrospun fibrous membranes coated with chitosan (CS) were fabricated by combining electrospinning and pressure-driven permeation (PDP) technology. The PDP method was able to significantly improve the hydrophilicity of electrospun fibrous membranes through surface coating of the hydrophilic fibers with CS, while maintaining the Rg3 releasing rate of PLGA electrospun fibrous membranes. Experimental wounds of animal covered with PDP treated fibrous membranes completely re-epithelialized and healed 3–4 days earlier than the wounds in control groups. Scar elevation index (SEI) measurements and histologic characteristics revealed that Rg3 significantly inhibited scar formation 28 days post-surgery. Moreover, RT-PCR assays and western blot analysis revealed that at day 28 after wound induction the expression of VEGF, mRNA and Collagen Type I in the scars treated with Rg3 was decreased compared to control groups. Taken together PLGA-Rg3/CS electrospun fibrous membranes induced repair of tissue damage in the early stage and inhibited scar formation in the late stage of wound healing. These dual-functional membranes present a combined therapeutic approach for inhibiting hypertrophic scars of the skin.

Critical Role of Transforming Growth Factor Beta in Different Phases of Wound Healing

This review highlights the critical role of transforming growth factor beta (TGF-β)1-3 within different phases of wound healing, in particular, late-stage wound healing. It is also very important to identify the TGF-β1-controlling factors involved in slowing down the healing process upon wound epithelialization. TGF-β1, as a growth factor, is a known proponent of dermal fibrosis. Several strategies to modulate or regulate TGF's actions have been thoroughly investigated in an effort to create successful therapies. This study reviews current discourse regarding the many roles of TGF-β1 in wound healing by modulating infiltrated immune cells and the extracellular matrix. It is well established that TGF-β1 functions as a wound-healing promoting factor, and thereby if in excess it may lead to overhealing outcomes, such as hypertrophic scarring and keloid. Thus, the regulation of TGF-β1 in the later stages of the healing process remains as critical issue of which to better understand. One hypothesis is that cell communication is the key to regulate later stages of wound healing. To elucidate the role of keratinocyte/fibroblast cross talk in controlling the later stages of wound healing we need to: (1) identify those keratinocyte-released factors which would function as wound-healing stop signals, (2) evaluate the functionality of these factors in controlling the outcome of the healing process, and (3) formulate topical vehicles for these antifibrogenic factors to improve or even prevent the development of hypertrophic scarring and keloids as a result of deep trauma, burn injuries, and any type of surgical incision.

Influence of granulocyte macrophagecolony stimulating factor on the expression of angiopoietin-1 in deep partial thickness burn wound in rats

Objective To investigate the influence of granulocyte macrophage colony stimulatingfactor （ GM-CSF） on the expression of angiopoietin-1 （Ang-1） in deep partial thickness bum wound in 1rats. Methods The models of deep partial thickness bum wounds on the back in Spregue-Dawley （ SD）rats were established. The paired wounds on the same rats were randomly divided into GM-CSF treatment（test） and control groups by self-control. Tissue samples were harvested from the wounds on the 1st,3rd,7th, 14th and 21st day respectively after bums. The mRNA expression of Ang-1 was detected by using re-al-time reverse transcriptase-polymerase chain reaction （ FQ-PCR） . And the protein expression of Ang-1was detected by immunohistochemical and western blot, the protein expression of CD31 by using immuno-histochemical staining,and the microvascular density was calculated. Results The mRNA expression levelof Ang-1 in the bum wounds was decreased slightly on the 1st and 3rd postbum days and that in test group（1. 44 ±0. 20,0. 30 ± 0. 17 ） was lower than that in control group （2. 36 ± 0. 18 , 1. 34 ± 0. 24） （P 〈0.05）. Then the Ang-1 mRNA expression was gradually increased on the 7 th and 14 th postbum days anddecreased slightly on the 21st postbum day, and the expression level in the test group at the above timepoints （4. 52 ±0. 21,8. 47 ±0. 14, 7. 52 ±0. 20） was higher than that in control group （2. 71 ±0. 12,3.69 ±0. 24,3. 07 ±0. 17）（p 〈0. 05）. The protein expression of Ang-1 was declined remarkably on the 1stand 3rd postburn days, which in test group （0. 491 ±0. 055, 0. 181 ±0. 025） was lower than that in control group （0. 735 ± O. 080, O. 436 ± 0. 071 ） （ P 〈 O. 05 ）. Then the Ang-1 protein expression was gradually in- creased and reached the peak on the 14th postburn day and the expression in the test group （0. 821 ±0. 060, 1. 257 ± 0. 225, 1. 126 ±0. 131 ） was higher than that in the control group （0. 701 ± 0. 078, 0. 825 ± 0. 072, 0. 733 ±0. 073 ） （P 〈 0. 05 ）. The microvascular number with positive CD31 expression was increased evi- dently from the 3rd postburn day in test group, and the counting of MVD was remarkably higher in the test group （9.5±1.8, 23.4±2.9, 38.4±3. 1,24.6±2.5） than in the control group （4.0±1.9, 10.5±2.0, 22. 9 ±2. 7, 15.1 ±2. 6） on the 3rd, 7th, 14th and 21st days respectively after burns （P 〈0. 05）. Conclu- sion GM-CSF inhibits the mRNA and protein expression of Ang-1 during the early stage of wound healing, which can be beneficial to the angiogenesis. And GM-CSF boosts Ang-1 expression during the middle and late stages of wound healing. This would be helpful to wound vascular maturation and wound healinz. Key words: Granulocyte macrophage colony stimulating factor; Angiopoietin; Burn; Woundhealing

Involvement of mast cell chymase in burn wound healing in hamsters

Mast cells play a significant role in the late stage of wound healing following burn injuries. In the present study, the possible role of mast cell chymase in burn wound healing was examined using a mast cell membrane stabilizer, ketotifen, in hamsters. A total of 28 hamsters were randomly divided into two groups (n=14), termed as the control and ketotifen groups. A deep partial-thickness burn injury was made on the back skin of the hamsters. The control group was orally administered physiological saline (1 ml) and the ketotifen group was orally administered ketotifen (4 mg/kg) once daily, two days prior to and two days subsequent to the burn. The results showed that concentrations of angiotensin II (Ang II), TGF-β1, collagens I and III and interleukin (IL)-1β were significantly decreased in the ketotifen group compared with those in the control group. However, there was no significant difference in fibroblast apoptosis between the two groups. The release of mast cell chymase was inhibited by the mast cell membrane stabilizer ketotifen. Taken together, these results suggest that mast cell chymase may participate in the process of burn wound healing. Chymase may therefore be a promising therapeutic target for the treatment of burn wounds.

A comparison of two methods of endoscopic dilation of acute subglottic stenosis using a ferret model

Balloon dilation is accepted as a first line treatment of acute subglottic stenosis, but its effects on the subglottic tissue remain largely unknown. We aimed to develop an animal model of acute subglottic stenosis using endoscopic techniques. Once developed, this model was used to compare the immediate effects of balloon dilation and endotracheal tube dilation on subglottic tissue. Prospective randomized animal study. Acute subglottic injury was induced in 10 ferrets by endoscopic cauterization with silver nitrate. After 48-72 hours of observation, eight animals were randomized to undergo subglottic dilation with either a 5-mm balloon or endotracheal tubes of increasing diameter. These eight ferrets were euthanized within 10 minutes after dilation. The other two ferrets served as controls and were euthanized following observation only. The larynx from each ferret was harvested, and the subglottis was examined histologically by a pathologist blinded to the treatment arms. Acute subglottic stenosis was induced in all 10 ferrets using the endoscopic technique. Both balloon and endotracheal tube dilation resulted in comparable improvement in the subglottic airway diameter. A decreased thickness of submucosa/lamina propria was seen in the balloon dilation group. Acute subglottic stenosis can be reliably induced in ferrets using endoscopic techniques. Multiple dilation methods can be used to relieve acute obstruction. Balloon dilators seem to improve airway patency, in part, by decreasing the thickness of the submucosa and lamina propria. Further research is needed to determine how this impacts later stages of wound healing and final outcomes.

Modulation of cutaneous wound healing by silymarin in rats

To investigate the effect of topical application of silymarin on full-thickness cutaneous wounds in rats. A full-thickness cutaneous defect (2×2cm) was induced on the back of 85 male and female Wister rats. The animals were randomly divided into four groups (n=20 in each group), treated with 1ml basal cream (placebo group), low-dose (6mg/ml/rat) and high-dose (12mg/ml/rat) silymarin, and untreated (control). Five rats remained uninjured to serve as comparisons for biomechanical analysis. Wounds were evaluated 10, 20 and 30 days after injury, through histopathologic, biochemical and biomechanical analyses. There was a significant (p < 0.05) increase observed in the amount of glycosaminoglycans and collagen present on days 10, 20 and 30 for both low-dose and high-dose silymarin groups. Low-dose silymarin reduced the number of lymphocytes and enhanced the number of fibrocytes at the earlier stages of wound healing; however, high-dose silymarin reduced both lymphocytes and macrophages, and increased number of fibrocytes at the later stages of wound healing. Silymarin significantly improved alignment of the healing tissue, enhanced maturity of the collagen fibres and fibroblasts (p < 0,05), and increased the ultimate tensile strength and stress of the healing tissue. The results suggest that topical application of silymarin improved the morphological, biochemical and biomechanical properties of experimentally-induced wound defects in rats. There were no external sources of funding for this study. The authors have no conflicts of interest to declare.

Kallikrein-Related Peptidase 8–Dependent Skin Wound Healing Is Associated with Upregulation of Kallikrein-Related Peptidase 6 and PAR2

Kallikrein-related peptidase 8 (KLK8) is believed to be involved in the maintenance of skin homeostasis and pathogenesis of inflammatory skin diseases. Although previous studies have shown that KLK8 is expressed around incisional wounds, the exact role of KLK8 in wound healing remains obscure. In the present study, we compared wound healing in wild-type (WT) and Klk8 gene-disrupted (kallikrein-related peptidase 8 knockout, Klk8(-/-)) mouse skin. Wound healing in Klk8(-/-) mice was hampered with defective keratinocyte proliferation, differentiation, and migration in the early stages of wound healing. Compared with the prominent induction of Klk6 and protease-activated receptor 2 (PAR2) messenger RNA, and protein in WT mice after wounding, a much lower increase was observed in Klk8(-/-) skin. After skin wounding in WT mice, increased Klk6 was detected from the upper stratum spinosum to the stratum corneum. Moreover, in WT mice, Klk6 protein was processed. PAR2 was diffusely expressed in the cytoplasm of the stratum spinosum at day 7 post wounding in WT mice. These results suggest that Klk8 is involved in the proliferation and migration of keratinocytes through the upregulation and activation of Klk6 in the early stages of wound healing, and possibly in keratinocyte differentiation associated with the upregulation and activation of PAR2 in the late stages of wound healing.

The Polyvinyl Alcohol Sponge Model Implantation

Wound healing is a complicated, multistep process involving many cell types, growth factors and compounds1-3. Because of this complexity, wound healing studies are most comprehensive when carried out in vivo. There are many in vivo models available to study acute wound healing, including incisional, excisional, dead space, and burns. Dead space models are artificial, porous implants which are used to study tissue formation and the effects of substances on the wound. Some of the commonly used dead space models include polyvinyl alcohol (PVA) sponges, steel wire mesh cylinders, expanded polytetrafluoroethylene (ePTFE) material, and the Cellstick1,2.Each dead space model has its own limitations based on its material's composition and implantation methods. The steel wire mesh cylinder model has a lag phase of infiltration after implantation and requires a long amount of time before granulation tissue formation begins1. Later stages of wound healing are best analyzed using the ePTFE model1,4. The Cellstick is a cellulose sponge inside a silicon tube model which is typically used for studying human surgery wounds and wound fluid2. The PVA sponge is limited to acute studies because with time it begins to provoke a foreign body response which causes a giant cell reaction in the animal5. Unlike other materials, PVA sponges are easy to insert and remove, made of inert and non-biodegradable materials and yet are soft enough to be sectioned for histological analysis2,5.In wound healing the PVA sponge is very useful for analyzing granulation tissue formation, collagen deposition, wound fluid composition, and the effects of substances on the healing process1,2,5. In addition to its use in studying a wide array of attributes of wound healing, the PVA sponge has also been used in many other types of studies. It has been utilized to investigate tumor angiogenesis, drug delivery and stem cell survival and engraftment1,2,6,7. With its great alterability, prior extensive use, and reproducible results, the PVA sponge is an ideal model for many studies1,2.Here, we will describe the preparation, implantation and retrieval of PVA sponge disks (Figure 1) in a mouse model of wound healing.

The Polyvinyl Alcohol Sponge Model Implantation

Wound healing is a complicated, multistep process involving many cell types, growth factors and compounds1-3. Because of this complexity, wound healing studies are most comprehensive when carried out in vivo. There are many in vivo models available to study acute wound healing, including incisional, excisional, dead space, and burns. Dead space models are artificial, porous implants which are used to study tissue formation and the effects of substances on the wound. Some of the commonly used dead space models include polyvinyl alcohol (PVA) sponges, steel wire mesh cylinders, expanded polytetrafluoroethylene (ePTFE) material, and the Cellstick1,2. Each dead space model has its own limitations based on its material's composition and implantation methods. The steel wire mesh cylinder model has a lag phase of infiltration after implantation and requires a long amount of time before granulation tissue formation begins1. Later stages of wound healing are best analyzed using the ePTFE model1,4. The Cellstick is a cellulose sponge inside a silicon tube model which is typically used for studying human surgery wounds and wound fluid2. The PVA sponge is limited to acute studies because with time it begins to provoke a foreign body response which causes a giant cell reaction in the animal5. Unlike other materials, PVA sponges are easy to insert and remove, made of inert and non-biodegradable materials and yet are soft enough to be sectioned for histological analysis2,5. In wound healing the PVA sponge is very useful for analyzing granulation tissue formation, collagen deposition, wound fluid composition, and the effects of substances on the healing process1,2,5. In addition to its use in studying a wide array of attributes of wound healing, the PVA sponge has also been used in many other types of studies. It has been utilized to investigate tumor angiogenesis, drug delivery and stem cell survival and engraftment1,2,6,7. With its great alterability, prior extensive use, and reproducible results, the PVA sponge is an ideal model for many studies1,2. Here, we will describe the preparation, implantation and retrieval of PVA sponge disks (Figure 1) in a mouse model of wound healing.

Involvement of upper torso stress amplification, tissue compression and distortion in the pathogenesis of keloids

Keloids are benign tumours composed of fibrous tissue produced during excessive tissue repair triggered by minor injury, trauma or surgical incision. Although it is recognized that keloids have a propensity to form in the upper torso of the body, the predisposing factors responsible for this have not been investigated. It is crucial that the aetiopathoical factors implicated in keloid formation be established to provide guidelines for well-informed more successful treatment. We compared keloid-prone and keloid-protected skin, identified pertinent morphological differences and explored how inherent structural characteristics and intrinsic factors may promote keloid formation. It was determined that keloid prone areas were covered with high tension skin that had low stretch and a low elastic modulus when compared with skin in keloid protected areas where the skin was lax with a high elastic modulus and low pre-stress level. Factors contributing to elevated internal stress in keloid susceptible skin were the protrusion of hard connective tissue such as bony prominences or cartilage into the dermis of skin as well as inherent skin characteristics such as the bundled arrangement of collagen in the reticular dermis, the existent high tension, the low elastic modulus, low stretch ability, contractile forces exerted by wound healing fibroblastic cells and external forces.Stress promotes keloid formation by causing dermal distortion and compression which subsequently stimulate proliferation and enhanced protein synthesis in wound healing fibroblastic cells. The strain caused by stress also compresses and occludes microvessels causing ischaemic effects and reperfusion injury which stimulate growth when blood rich in growth factors returns to the tissue. The growth promoting effects of increased internal stress, primarily, and growth factors released by reperfusing blood, manifest in keloid formation. Other inherent skin characteristics promoting keloid growth during the late stages of wound healing in the upper torso are the thinner epidermis, the presence of vellus hairs, the absence of protective immunoglobulin A (IgA), and the thick fragile quality of upper torso skin. As it is not known why there is a predilection for keloids to form in the upper torso of the body, this hypothesis implicating and associating inherent morphological characteristics and elevated stress in the aetiopathogenesis of keloids is of potential value in terms of prevention, management and treatment of these enigmatic tumours.