Early Stages Of Wound Healing Research Articles

Variations in expressions of periostin and related factors in early stage of wound healing and scar remodeling in rats

To investigate the expressions of periostin (PN), angiopoietin-1 (Ang-1), vascular epithelial growth factor (VEGF) and fetal liver kinase-1 (Flk-1) during the processes of scar formation and modulation in rat cutaneous wounds and probe into their roles in wound healing and scaring. Eighty-two male Sprague-Dawley (SD) rats were randomly divided into 10 groups with 8-9 rats in each group. Two 2 cm×2 cm full-thickness excisional wounds in the back were created in each rat. The wound surface was observed, and the healing area was measured. The pathological change was observed after hematoxylin and eosin (HE) staining. The expressions of PN, Ang-1, VEGF and Flk-1 in wound surface scar at 4-8 weeks were determined with immunohistochemistry. The expressions of PN, Ang-1 and VEGF were determined by Western blotting. The normal skin was served as control. HE staining showed that the wound surface tissue had healed with epithelization at 4-8 weeks. Immunohistochemistry results showed that there was no significant difference in Flk-1 expression between wound surface tissue and normal skin. The PN expression (A value/μm(2)) in wound surface tissue was significantly lower than that in normal skin at 5 weeks (2.43±0.44 vs. 4.24±0.50, P<0.05), and the expression of Ang-1 and VEGF (A value/μm(2)) at 4, 5, 6, 8 weeks was significantly lower than that in normal skin (Ang-1: 3.51±0.93, 3.10±0.57, 2.77±0.59, 2.77±1.26 vs. 4.89±0.48; VEGF: 1.76±0.68, 1.75±0.49, 1.99±0.42, 1.94±0.86 vs. 4.86±1.63, all P<0.05). In wound surface scar, PN and Flk-1 positive signal was found in cell, and the Ang-1 and VEGF positive signal in extracellular matrix. Western blotting data demonstrated that the expressions of PN, Ang-1 and VEGF peaked at the 10th day after excision with increases to 7.90-22.56 folds compared with normal skin (PN: 2.45±1.51 vs. 0.31±0.19, Ang-1: 18.43±15.20 vs. 1.53±1.42, VEGF: 6.09±4.66 vs. 0.27±0.13, P<0.05 or P<0.01), and then followed with a decrease. PN, Ang-1, VEGF and Flk-1 are transiently overexpressed in early stage of full-thickness cutaneous wound healing in rats. Their expressions vary in wounds and scars. They participate in the healing of full-thickness cutaneous wounds together and may be essential for the proliferation stage during wound healing.

The syndecan binding sequence KKLRIKSKEK in laminin α3 LG4 domain promotes epidermal repair.

Epithelialization of normal wounds occurs by an orderly series of events whereby keratinocytes migrate, proliferate, and differentiate to restore the barrier function. Keratinocyte migration is one of the most earliest and crucial event determining the efficiency of the overall wound repair process. Laminin 332, composed by the association of α3, β3 and γ2 chains, is a major adhesion substrate for keratinocytes and is known for its role in supporting cell adhesion and migration during wound repair. The α3 chain comprises a large globular region in its carboxyl-terminal end, which consists of five homologous globular domains (LG1-LG5), known to be involved in cellular interactions. Recent findings have suggested that the α3 chain C-terminal domains LG45 may have a role to play during the epithelialization phase in wound repair. In the present study, we have analyzed whether a peptide mimicking the major heparin binding sequence KKLRIKSKEK in α3LG45 may interact with keratinocytes to promote cell adhesion and migration. In vitro experiments supported this hypothesis and revealed that the KKLRIKSKEK peptide induces human primary keratinocyte adhesion and has the ability to promote keratinocyte migration when added in the culture medium. To examine the peptide efficacy in vivo, the KKLRIKSKEK peptide was applied over partial-thickness cutaneous wounds in pigs. Compared with vehicle-treated cutaneous wounds, the peptide application significantly promoted early-stage wound healing by accelerating re-epithelialization. Additional beneficial effects such as reduced inflammatory response and decreased granulation tissue formation were also noticed in the peptide-treated wounds.

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

Scar formation of laser skin lesions after cold atmospheric pressure plasma (CAP) treatment: A clinical long term observation

CAP treatment is likely to be of benefit in wound healing. In a clinical study, 20 laser lesions in five individuals have been treated with argon plasma 10, 30 or three times for 10s, with untreated as control. The scar formation was followed for 10 days, six and 12 months. In early stages of wound healing, plasma treatment seems to support the inflammation needed for tissue recovery. In later stages, plasma treatment shows better results in terms of avoiding post-traumatic skin disorders. Plasma treatment shows superior aesthetics during scar formation. No precancerous skin features occurred up to 12 months.

Wound‐associated macrophages control collagen 1α2 transcription during the early stages of skin wound healing

Wound-associated fibrosis is important to provide tensile strength upon wound healing but at the same time is detrimental to proper tissue regeneration. To date, there is no clear evidence of the role of macrophages and their subpopulations in the control of the kinetics of collagen production during wound healing. To evaluate in vivo the contribution of macrophages in collagen transcription, we depleted macrophages after wounding luciferase reporter mice of the collagen 1 alpha 2 (Col 1α2) promoter activity. Our data reveal that Col 1α2 starts to be transcribed at D2 after wounding, reaching a plateau after 7 days. Sustained macrophage depletion significantly reduced collagen 1α2 transcription from D4, indicating that the control of fibrosis by macrophages occurs during the early stages of the wound healing process. In conclusion, our results demonstrate an important role of wound macrophages in the control of collagen production during wound healing.

Diabetes induces stable intrinsic changes to myeloid cells that contribute to chronic inflammation during wound healing in mice

SUMMARYAcute inflammation in response to injury is a tightly regulated process by which subsets of leukocytes are recruited to the injured tissue and undergo behavioural changes that are essential for effective tissue repair and regeneration. The diabetic wound environment is characterised by excessive and prolonged inflammation that is linked to poor progression of healing and, in humans, the development of diabetic foot ulcers. However, the underlying mechanisms contributing to excessive inflammation remain poorly understood. Here we show in a murine model that the diabetic environment induces stable intrinsic changes in haematopoietic cells. These changes lead to a hyper-responsive phenotype to both pro-inflammatory and anti-inflammatory stimuli, producing extreme M1 and M2 polarised cells. During early wound healing, myeloid cells in diabetic mice show hyperpolarisation towards both M1 and M2 phenotypes, whereas, at late stages of healing, when non-diabetic macrophages have transitioned to an M2 phenotype, diabetic wound macrophages continue to display an M1 phenotype. Intriguingly, we show that this population predominantly consists of Gr-1+ CD11b+ CD14+ cells that have been previously reported as ‘inflammatory macrophages’ recruited to injured tissue in the early stages of wound healing. Finally, we show that this phenomenon is directly relevant to human diabetic ulcers, for which M2 polarisation predicts healing outcome. Thus, treatments focused at targeting this inflammatory cell subset could prove beneficial for pathological tissue repair.

Role of sensory and motor intensity of electrical stimulation on fibroblastic growth factor-2 expression, inflammation, vascularization, and mechanical strength of full-thickness wounds

Electrical stimulation (ES) profoundly affects angiogenesis by modulating the production of angiogenic factors. We evaluated the effect of sensory (direct current, 600 microamperes) and motor (monophasic pulse current, 2.5 to 3 milliamperes, 300-microsecond pulse duration, 100 Hz) intensities of cathodal current on the release of fibroblastic growth factor-2 (FGF-2) at the wound site and also the biomechanical and histological properties of healed skin. Ninety-six male, Sprague-Dawley rats were randomly assigned into one control and two experimental groups. A full-thickness skin incision was made on the dorsal region of each animal. The experimental groups received 10 sessions of ES (sensory or motor) for 1 hour per day every other day. The results showed that FGF-2 levels in the sensory group were significantly greater than in the other groups on the third day. In the motor group, FGF-2 levels were significantly decreased compared with the control group. There were no significant differences between the normalized ultimate strength and stiffness in the groups, but they tended to be higher in the motor ES group. We conclude that the application of sensory ES during the early stage of wound healing may have a beneficial effect on wound healing by inducing the release of angiogenic factors and decreasing the duration of the inflammation phase.

Dephosphorylation of myristoylated alanine-rich C kinase substrate accelerates wound-induced migration of SH-SY5Y cells

Inflammation, which is induced after infection of bacteria and tissue injury, is one of the important early stages of wound healing. Bradykinin is increased during acute and chronic inflammation. We previously reported that bradykinin stimulation induces dephosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) after phosphorylation by ROCK leading neurite outgrowth in neuroblastoma SH-SY5Y cells. In this report we showed that knock-down of MARCKS by RNAi reduced cell migration. Wild-type MARCKS-overexpressed SH-SY5Y cells migrated faster than the control cells. Unphosphorylatable MARCKS-overexpressed cells notably migrated fast. Moreover, chronic MARCKS dephosphorylation by a ROCK inhibitor HA-1077 promoted the cell migration, on the other hand a PKC inhibitor Ro-31-8220 did not. After wounding, MARCKS was transiently phosphorylated and dephospho-rylated by 20 min. Immunocytochemistry showed that the dephosphorylated MARCKS was localized at neurite tips. These findings suggest that MARCKS dephosphorylation is important in wound-induced migration of SH-SY5Y cells. It indicates the possibility that MARCKS is associated with wound repair after inflammation.

MiR-21 Regulates Skin Wound Healing by Targeting Multiple Aspects of the Healing Process

With the clarification of the important roles of microRNAs (miRNAs) in diverse physiologic and pathologic processes, the effects of miRNAs in wound healing have attracted more attention recently. However, the global pattern of miRNA expression in wound tissue is still unknown. In the present study, we depicted the miRNA profile and identified at least 54 miRNAs, including miR-21, changed for more than twofold at the stage of granulation formation during wound healing. These miRNAs were closely related to the major events of wound healing, including cell migration and proliferation, angiogenesis, and matrix remolding. Furthermore, we found that miR-21 was up-regulated after skin injury, mainly in activated and migrating epithelial cells of epidermis and mesenchymal cells of dermis. Locally antagonizing miR-21 by directly injecting antagomir to wound edge caused significant delay of wound closure with impaired collagen deposition. Unexpectedly, we found wounds treated with miR-21 antagomir had an obvious defect in wound contraction at an early stage of wound healing. The significant role of miR-21 in wound contraction was further confirmed by in vivo gain-of-function and in vitro loss-of-function experiments. In conclusion, the present study has for the first time depicted miRNA profiling of wound healing and demonstrated the involvement of miR-21 in regulating the wound contraction and collagen deposition. These results suggest that miR-21 may be a new medical target in skin wound manipulation.

Notch Inhibition during Corneal Epithelial Wound Healing Promotes Migration

To determine the role of Notch signaling in corneal epithelial migration and wound healing. Immunolocalization of Notch1 was performed during epithelial wound healing in vivo in mouse corneal epithelial debridement wounds and in vitro in primary human corneal epithelial cells following a linear scratch wound. The effects of Notch inhibition, using the γ-secretase inhibitor N-(N-[3,5-difluorophenacetyl]-l-alanyl)-S-phenylglycine t-butyl ester (DAPT) or following stable transfection with Notch1-short hairpin RNA (shRNA), was evaluated in a scratch assay and transwell migration assay. Likewise, in vitro adhesion, proliferation and the actin cytoskeleton was examined. The DAPT effect was also evaluated in vivo in a mouse model of corneal epithelial wound healing. The expression of Notch1 was reduced at the leading edge of a healing corneal epithelium both in vivo and in vitro. Notch inhibition using DAPT and using Notch1-shRNA both enhanced in vitro migration in scratch and transwell migration assays. Consistent with this increased migratory behavior, Notch inhibited cells demonstrated decreased cell-matrix adhesion and enhanced lamellipodia formation. Notch inhibition by DAPT was also found to accelerate corneal epithelial wound closure in an in vivo murine model without affecting proliferation. The results highlight the role of Notch in regulating corneal epithelial migration and wound healing. In particular, Notch signaling appears to decrease in the early stages of wound healing which contributes to cytoskeletal changes with subsequent augmentation of migratory behavior.

Negative pressure therapy: theory and practice

NPT(negative pressure therapy)which also can be called VAC(vacuum-assisted closure)is a treatment which can promote wound healing.It can not only be used to one stage treatment of chronic and complex wounds but also can assist to temporary close the wound and to the preparatory work of pre-operation wound area such as dermatoplasty. Although the physiological basis of VAC has been not fully expounded, the clinical effects have been proved.We analysed the relevant researches that based on randomized clinical trial, VAC and topical negative pressure which are chosen from Pubmed-medline and assessed the scientific data which derived from animal experiment, randomized clinical trial and observing in clinical application. According to the data, NPT has remarkable effects in inducing wound healing especially in early stage of wound healing. Besides, NPT also has positive effect in anti-infective therapy. Experimental data have presented that VAC has positive effect in promoting wound healing. But the functional mechanism of it has not been clarified because of evidence-based medicine data could not explain the positive effects of clinical treatment. Although there still need further random and blind method to explain its effects, we deem that reasonable using VAC especially using by prectised surgeon is a commendable treatment to promoting wound healing.

Facile fabrication of the glutaraldehyde cross-linked collagen/chitosan porous scaffold for skin tissue engineering

Porous scaffold is one of the key factors in skin tissue engineering. In this study, a facile method was developed to prepare the glutaraldehyde (GA) cross-linked collagen/chitosan porous scaffold (S2). The properties of S2 were compared with the scaffolds prepared by the traditional method (S1). Compared to the rough surface and collapsed inner structure of S1, S2 showed a smooth surface and controlled size. After treated by GA with same concentration, S1 and S2 showed the similar swelling ratios, which are big enough to ensure the nutrient supply in the early stage of wound healing. The effects of the fabrication methods as well as the GA concentration on the cross-linking degree and in vitro degradation degree of the scaffolds were studied. It was found that the cross-linking degree of S2-0.25% was much higher than that of S1. Investigation of the tensile and compression properties of the scaffolds found that the mechanical property of S2-0.04% is closest to that of S1. High performance liquid chromatography (HPLC) was applied to determine the residual GA. The results proved that, compared to water rinse, oven drying is a feasible and effective method to remove the residual GA. Finally, the cytocompatibility of S2 was evaluated by in vitro culture of fibroblasts. The results of cell morphology and cell viability proved that S2-0.04% could retain the original good cytocompatibility of S1 to accelerate cell infiltration and proliferation effectively. All these results indicate that it is a feasible method to prepare the GA cross-linked collagen/chitosan scaffold.

The effect of monochromatic infrared energy on diabetic wound healing

This study examined the effect of monochromatic infrared energy (MIRE) on diabetic wound healing. Fifteen diabetic rats were given MIRE intervention on their skin wounds located on the dorsum and compared with 15 control diabetic rats. Assessments were conducted for each group at weeks 1, 2 and 4 post wounding (five rats at each time point) by calculating the percentage of wound closures (WCs) and performing histological and immunohistochemical staining on sections of wound tissue. Evaluations of WCs and histological examinations of reepithelialisation, cellular content and granulation tissue formation showed no significant difference between the MIRE and the control group at each time point. Through semi-quantitative immunohistochemical staining, the deposition of type I collagen in the MIRE group was found to have improved when compared with the control group at the end of week 2 (P = 0.05). No significant differences in the myofibroblast population were detected between the two groups. In conclusion, MIRE appeared to promote collagen deposition in the early stage of wound healing in diabetic rats, but the overall wound healing in the MIRE group was not significantly different from that of the control group.

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.

Topical application of Acalypha indica accelerates rat cutaneous wound healing by up-regulating the expression of Type I and III collagen

Ethnopharmacological relevanceAcalypha indica Linn. (Acalypha indica) vernacularly called Kuppaimeni in Tamil, has been used as a folklore medicine since ages for the treatment of wounds by tribal people of Tamil Nadu, Southern India. The present study investigates the biochemical and molecular rationale behind the healing potential of Acalypha indica on dermal wounds in rats. Material and methodsAcalypha indica extract (40mg/kg body weight) was applied topically once a day on full-thickness excision wounds created on rats. The wound tissue was removed and used for estimation of various biochemical and biophysical analyses and to observe histopathological changes with and with-out extract treatment. The serum levels of pro-inflammatory cytokine tumor necrosis factor (TNF-α) was measured at 12h, 24h, 48h and 72h post-wounding using ELISA. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed to study the expression pattern of transforming growth factor [TGF-β1], collagen 1 α (I) [Col 1 α (I)] and collagen 3 α (I) [Col 3 α (I)]. Likewise, linear incision wounds were created and treated with the extract and used for tensile strength measurements. ResultsWound healing in control rats was characterized by less inflammatory cell infiltration, lack of granulation tissue formation, deficit of collagen and significant decrease in biomechanical strength of wounds. Acalypha indica treatment mitigated the oxidative stress and decreased lipid peroxidation with concomitant increase in ascorbic acid levels. It also improved cellular proliferation, increased TNF-α levels during early stages of wound healing, up-regulated TGF-β1 and elevated collagen synthesis by markedly increasing the expression of Col 1 α (I) and Col 3 α (I). Increased rates of wound contraction, epithelialization, enhanced shrinkage temperature and high tensile strength were observed in the extract treated rats. ConclusionAcalypha indica extract was shown to augment the process of dermal wound healing by its ability to increase collagen synthesis through up-regulation of key players in different phases of wound healing and by its antioxidative potential.

Amniotic membrane reduces wound size in early stages of the healing process

To investigate the effects of dispase de-epithelialised, glycerol cryopreserved amniotic membrane (AM) on full-thickness skin defects, using a rat model. Skin defects of 15 mm diameter were surgically created and measured on the scalps of 53 male rats. Animals were divided into two groups and followed for 0, 3, 7, 14 or 21 days. AM group wounds were covered with de-epithelialised AM and sodium chloride-moistened Aquacel (ConvaTec Inc.); control group wounds were covered with sodium chloride-moistened Aquacel alone. After the follow-up, wounds were measured again, serum samples were taken and wound sites were harvested for histological analysis. Systemic interleukin-4 (IL-4) levels were analysed from serum. On day 3, a statistically significant difference (p < 0.01) was observed in mean wound size, with wound size in the AM group smaller than in the control group (60 ± 12% vs 81 ± 13% of the original size); other time points showed no significance difference in wound size between the two groups. We could not detect differences between the groups in histological parameters or serum IL -4 levels. According to this study, AM enhances early stage wound healing in terms of wound size but its effect decreases in later phases. The IL-4 results provide no clear evidence that IL-4 contributes to the effect of AM on wound healing. This study was financially supported by the Competitive Research Funding of the Tampere University Hospital (Grant 9H041, 9J047). The authors have no additional conflicts of interest to declare.

Flavonoids in propolis acting on mast cell-mediated wound healing

Barroso et al. have shown, on the latest issue of Inflammopharmacology, that the topical application of propolis on surgical wounds affected the number of mast cells recruited in these sites, and suggested two well-known antiinflammatory components present in propolis, namely caffeic acid and artepillin C, as possible active molecules (Borrelli et al. 2002; Paulino et al. 2008). It is widely acknowledged that propolis down-regulates type I allergy and inflammation by affecting mast cells, but the effective components of propolis, which cause these effects, remain still unknown. Propolis components vary depending on the area from which they are collected, mainly because of the genetic variability among wild plants in different geographic regions: variability in phenolics composition may result in different biochemical property of the propolis, depending on the main component active in raw propolis or in its extracts. In Chinese propolis, chrysin, kaempferol and its derivative, pinocembrin and galangin were identified as the main flavonoids able to act on mast cell-mediated inflammatory response, while chrysin was shown to inhibit IL-4 and MCP-1 production from antigen-stimulated RBL2H3 basophil/mast cell lines (Nakamura et al. 2010). On the other hand, Brazilian propolis extract contains only small amounts of these flavonoids, which might suggest that variation in propolis components could affect antiallergic and anti-inflammatory properties (Nakamura et al. 2010). Brazilian propolis contains, therefore, major percentage of phenolic acids, such as caffeic acid phenethyl ester (CAPE) and artepillin C (Park et al. 2002). As the activity of propolis, like many natural products, may be due to the synergic effect of several bioactive components, it will be necessary to distinguish between different types of propolis and analyse its complex compositions to guarantee specific biological activities of propolis diffused worldwide (Frankland Sawaya et al. 2011). Furthermore, inflammation by mast cells can be inhibited by several flavonoids present in propolis. Recent evidence was reported showing that chrysin decreased gene expression of pro-inflammatory cytokines, such as TNF-a, IL-1b, IL-4 and IL-6 in mast cells by a nuclear factor-jB (NF-jB) and caspase-1 dependent mechanism (Bae et al. 2011). Genistein modulates NF-jB and TNF-a expression during the early stage of wound healing (Park et al. 2011). CAPE accelerates cutaneous wound healing and it is arguable that propolis with a significant amount of this phenolic acid may exert a wound repairing property (Serarslan et al. 2007). The antiinflammatory property attributed to flavonoids in propolis might suggest that these polyphenols should exert their action toward other newly discovered function of mast cells (Ng 2010), even in association with CAPE or other phenolic acids. Wound healing is a complex process of lysis and reconstitution controlled by a series of cell signaling proteins and involved tissue regeneration and angiogenesis (Hiromatsu and Toda 2003; Nienartowicz et al. 2006). Mast cells have been shown to play a significant role in the early inflammatory stage of wound healing and also influence proliferation and tissue remodeling in skin; according to some authors in wound healing, mast cells are able to modulate only the inflammatory but not the proliferative response in healing and reconstituting damaged tissue (Egozi et al. 2003), while other reports showed that mast cells are involved in tissue remodeling in the late phase of wound healing (Iba et al. 2004), thus expanding S. Chirumbolo (&) Unit of Geriatry, Department of Medicine, University Laboratory of Medical Research, University of Verona, Policlinico GB Rossi piazzale AL Scuro 10, 37134 Verona, Italy e-mail: salvatore.chirumbolo@univr.it Inflammopharmacol (2012) 20:99–101 DOI 10.1007/s10787-012-0125-9 Inflammopharmacology

Keratinocyte-Targeted Overexpression of the Glucocorticoid Receptor Delays Cutaneous Wound Healing

Delayed wound healing is one of the most common secondary adverse effects associated to the therapeutic use of glucocorticoid (GC) analogs, which act through the ligand-dependent transcription factor GC-receptor (GR). GR function is exerted through DNA-binding-dependent and –independent mechanisms, classically referred to as transactivation (TA) and transrepression (TR). Currently both TA and TR are thought to contribute to the therapeutical effects mediated by GR; however their relative contribution to unwanted side effects such as delayed wound healing is unknown. We evaluated skin wound healing in transgenic mice with keratinocyte-restricted expression of either wild type GR or a mutant GR that is TA-defective but efficient in TR (K5-GR and K5-GR-TR mice, respectively). Our data show that at days (d) 4 and 8 following wounding, healing in K5-GR mice was delayed relative to WT, with reduced recruitment of granulocytes and macrophages and diminished TNF-α and IL-1β expression. TGF-β1 and Kgf expression was repressed in K5-GR skin whereas TGF-β3 was up-regulated. The re-epithelialization rate was reduced in K5-GR relative to WT, as was formation of granulation tissue. In contrast, K5-GR-TR mice showed delays in healing at d4 but re-established the skin breach at d8 concomitant with decreased repression of pro-inflammatory cytokines and growth factors relative to K5-GR mice. Keratinocytes from both transgenic mice closed in vitro wounds slower relative to WT, consistent with the in vivo defects in cell migration. Overall, the delay in the early stages of wound healing in both transgenic models is similar to that elicited by systemic treatment with dexamethasone. Wound responses in the transgenic keratinocytes correlated with reduced ERK activity both in vivo and in vitro. We conclude that the TR function of GR is sufficient for negatively regulating early stages of wound closure, while TA by GR is required for delaying later stages of healing.

Human Keratinocytes' Response to Injury Upregulates CCL20 and Other Genes Linking Innate and Adaptive Immunity

In the early stages of wound healing, keratinocytes become “activated” and release inflammatory molecules such as interleukin-1 and interleukin-8 that are linked to innate immune responses and neutrophil recruitment. It is unclear, however, whether keratinocytes release molecules linked to adaptive immune responses, e.g. CCL20, in their early state of activation without signals from infiltrating T cells. This study aims to isolate the immediate alterations in protective and inflammatory gene expression that occur in epidermal keratinocytes, with a particular focus on molecules associated with cell-mediated immunity. We used dispase-separated epidermis, followed by intercellular disassociation by trypsinization, as a model for epidermal injury. We obtained a pure population of keratinocytes using flow cytometry. As a control for uninjured epidermis, we performed laser capture microdissection on normal human skin. Sorted keratinocytes had an early burst of upregulated gene expression, which included CCL20, IL-15, IL-23A, IFN-κ, and several antimicrobial peptides. Our results provide insight into the potential role of keratinocytes as contributors to cell-mediated inflammation, and expand knowledge about gene modulation that occurs during early wound healing. Our findings may be relevant to cutaneous diseases such as psoriasis, where micro-injury can trigger the formation of psoriatic plaques at the site of trauma.

Chitosan liquid improves wound healing in rats

Objective To study the promotive effect of chitosan liquid on wound healing.Methods Wound model was established on the back of 60 rats,which were classified into 3 groups to be treated with topical chitosan liquid (group A),basic fibroblast growth factor (group B),and sodium chloride physiological solution (group C),respectively.The time required for the healing of wound was recorded.Regenerated tissues were resected from the rats on day 3,7 and 14 after the establishment of wound model,and observed with light microscopy.Results The time required for the healing of wound was 17.3 ± 1.35 days in group A,18.2 ± 1.15 days in group B,and 24.0 ± 1.37 days in group C.For the time required for the healing of wound,no significant difference was observed between the group A and B (P ＞ 0.05),but group C significantly differed from group A and B (both P ＜ 0.05).Chitosan liquid accelerated the generation of capillary sprouts and vascular endothelial cells at the early stage of wound healing,promoted the production of fibroblasts and collagen fibers at the middle stage,and improved the organization of collagen fibers at the late stage.Conclusion Chitosan liquid exerts a promising promotive effect on wound healing.