Beta-caryophyllene as an antioxidant, anti-inflammatory and re-epithelialization activities in a rat skin wound excision model.

The skin is a critical organ for the maintenance of the integrity and protection of the organism. When a wound occurs, a sequence of healing mechanisms is triggered to reconstruct the wounded area. ß-caryophyllene is a sesquiterpene in Copaifera langsdorffii oleoresin with antioxidant and anti-inflammatory potential. On the basis of previous studies with C. langsdorffii, ß-caryophyllene was selected to evaluate its wound healing potential and pharmacological mechanisms. The excision wound model was used with male Wistar rats and macroscopic, histological, immunohistochemical and biochemical analyses were performed with skin samples, comparing the ß-caryophyllene-treated group with reference drugs. The results showed macroscopic retraction of the wounds treated with ß-caryophyllene. Biochemical assays revealed the antioxidant and anti-inflammatory mechanisms of the ß-caryophyllene-treated group with increasing levels of IL-10 and GPx and decreasing levels of pro-inflammatory molecules, including TNF-α, IFN-γ, IL-1ß and IL-6. After ß-caryophyllene treatment, immunohistochemical assays showed enhanced re-epithelialization, through the increase in laminin-γ2 and desmoglein-3 immunolabeling. ß-caryophyllene also act in the remodeling mechanism, increasing the collagen content in the Masson's trichrome staining. These findings indicated the wound-healing potential of ß-caryophyllene topical formulation in rat skin wounds, mediated by antioxidant, anti-inflammatory and re-epithelialization mechanisms.

Wound healing potential of licorice extract in rat model: Antioxidants, histopathological, immunohistochemical and gene expression evidences.

Wound healing is a public health concern. Licorice gained a great attention for its antioxidant and anti-inflammatory properties which expand its valuable effects as a herbal medicine. In this study, we pointed out to the wound healing potential and the mechanism by which licorice alcoholic extract can modulate cutaneous wound healing through immune, antioxidant, histopathological, immunohistochemical (IHC) and molecular studies. 24 Wister rats were assigned into 3 groups (n = 8 each); control group, topical and oral supplied groups. Licorice extract administration significantly increased total and differential leucocyte counts, phagocytic activity of neutrophils, antioxidant biomarkers as superoxide dismutase (SOD), glutathione peroxidase activities (GPx) and reduced glutathione (GSH) content with a notable reduction in oxidative stress marker malondialdehyde (MDA). Moreover, histopathological findings detected complete re-epithelialization with increasing collagen synthesis while IHC results revealed a significant enhancement in the expression of α-SMA, PDGFR-α, FGFR1 and Cytokeratin 14 in licorice treated groups compared with the control group. Licorice extract supplementation accelerated wound healing by increasing angiogenesis and collagen deposition through up-regulation of bFGF, VEGF and TGF-ß gene expression levels compared with the control group. UPLC-PDA-MS/MS aided to authenticate the studied Glycyrrihza species and recognized 101 potential constituents that may be responsible for licorice-exhibited potentials. Based on our observations we concluded that licorice enhanced cutaneous wound healing via its free radical-scavenging potential, potent antioxidant activities, and anti-inflammatory actions. Therefore, licorice could be used as a potential alternative therapy for wound injury which could overcome the associated limitations of modern therapeutic products.

Electrospun PLGA/SF/artemisinin composite nanofibrous membranes for wound dressing.

Combining biodegradable materials with natural plant components for wound dressing has been receiving significant attention. ART is a sesquiterpene lactone compound extracted from Artemisia annua L., possessing multiple pharmacological effects including antibacterial activity and anti-inflammatory property. Herein, the blended polylactic acid glycolic acid (PLGA)/silk fibroin (SF) membranes loaded with artemisinin (ART) are fabricated through electrospinning. With aid of SF, the fabricated membranes have a good sustained-release effect, and the accumulated ART release can reach 69% after three weeks. PLGA/SF/ART membranes exhibit favorable anti-inflammatory and cell compatibility in vitro evaluations. The in vivo experiment indicates that PLGA/SF/ART2 membranes can shorten the inflammation period and enhance skin regeneration in a full-thickness wound model through down-regulating the expressions of pro-inflammatory cytokines IL-1ß and TNF-α. To sum up, the fabricated PLGA/SF/ART2 composite membranes with anti-inflammatory properties can be a proposal wound dressing for chronic wound healing.

In-vivo wound healing activity of a novel composite sponge loaded with mucilage and lipoidal matter of Hibiscus species.

Many researches have been undergone to hasten the natural wound healing process. In this study, several Hibiscus species (leaves) were extracted with petroleum ether, methanol, and their mucilage was separated. All the tested species extracts were assessed for their viability percentage using the water-soluble tetrazolium. H.syriacus was the plant of choice to be incorporated in a new drug delivery system and evaluated for its wound healing activity. H.syriacus petroleum ether extract (PEE) showed a high percentage of palmitic and oleic acids while its mucilage demonstrated high glucosamine and galacturonic acid. It was selected to be formulated and pharmaceutically evaluated into three different composite sponges using chitosan in various ratios. Fourier-transformed infrared spectroscopy investigated the chemical interaction between the utilized sponges' ingredients. Morphological characteristics were evaluated using scanning electron microscopy. H.syriacus composite sponge of mucilage: chitosan (1:5) was loaded with three different concentrations of PEE. Medicated formulations were assessed in rat model of excision wound model. The wound healing ability was clearly proved by the clinical acceleration, histopathological examination, and modulation of correlated inflammatory parameters as tumor necrosis factor in addition to vascular endothelial growth factor suggesting a promising valuable candidate that supports the management of excision wounds using single-dose preparation.

Power Function Retention of Radionuclides in a Wound.

NCRP Report 156 describes soluble radionuclide retention kinetics in a wound, segregated into four retention categories: weak (W), moderate (M), strong (S), and avid (A). An alternate single-parameter model, the negative power function, t, is presented in this paper to describe the time behavior of radionuclide retention. With this mathematical description, γ is a single parameter that can be used to assign the wound retention category rapidly. Using the power function description of wound retention, the various wound categories present as straight lines on log scales with different slopes corresponding to the various retention categories. Regression analysis of average retention values in NCRP 156 shows γ = 0.735 ± 0.132, 0.514 ± 0.015, 0.242 ± 0.016, and 0.053 ± 0.023 for the weak, moderate, strong, and avid categories, respectively. A case study is presented (REAC/TS Registry case 1284) where a power function is shown to fit retention data in a Pu/Am hand wound up to 2,000 d (5.4 y) post-accident.

Advantages in Wound Healing Process in Female Mice Require Upregulation A2A-Mediated Angiogenesis under the Stimulation of 17ß-Estradiol.

Estrogenic steroids and adenosine A2A receptors promote the wound healing and angiogenesis processes. However, so far, it is unclear whether estrogen may regulate the expression and pro-angiogenic activity of A2A receptors. Using in vivo analyses, we showed that female wild type (WT) mice have a more rapid wound healing process than female or male A2A-deficient mice (A2AKO) mice. We also found that pulmonary endothelial cells (mPEC) isolated from female WT mice showed higher expression of A2A receptor than mPEC from male WT mice. mPEC from female WT mice were more sensitive to A2A-mediated pro-angiogenic response, suggesting an ER and A2A crosstalk, which was confirmed using cells isolated from A2AKO. In those female cells, 17ß-estradiol potentiated A2A-mediated cell proliferation, an effect that was inhibited by selective antagonists of estrogen receptors (ER), ERα, and ERß. Therefore, estrogen regulates the expression and/or pro-angiogenic activity of A2A adenosine receptors, likely involving activation of ERα and ERß receptors. Sexual dimorphism in wound healing observed in the A2AKO mice process reinforces the functional crosstalk between ER and A2A receptors.

Drupin, a cysteine protease from Ficus drupacea latex accelerates excision wound healing in mice.

Wound healing is a tightly regulated physiological process that restores tissue integrity after injury. Plant latex proteases (PLPs) are considered an integral part in herbal wound care as it interferes at different phases of the wound healing process. Although many studies have reported the involvement of PLPs in healing process, an in-depth investigation is required to understand the molecular mechanism. Hence, the effect of PLPs with fibrinolytic activity on wound healing was investigated systematically using mouse excision wound model. Among 29 latices from Ficus genus tested, Ficus drupacea exhibited potent fibrinolytic activity. Cysteine protease responsible for fibrinolysis was purified from the F. drupacea latex named it as drupin, tested for its wound healing efficacy. The accelerated wound healing was mediated by downregulation of matrix metalloprotease (MMP)-9 without altering MMP-8 expression. Besides, drupin enhanced the rate of collagen synthesis at the wound site by increasing arginase 1 activity. And also, drupin increased the expression of arginase 1 in macrophages and involved in cell proliferation, and migration via MAP kinase and PI3K/Akt pathways. Overall, the present study highlights the interference of drupin in wound healing by increased arginase 1 activity and collagen synthesis, and cell proliferation and migration.

Complex Tissue Regeneration in Mammals Is Associated With Reduced Inflammatory Cytokines and an Influx of T Cells.

While mammals tend to repair injuries, other adult vertebrates like salamanders and fish regenerate damaged tissue. One prominent hypothesis offered to explain an inability to regenerate complex tissue in mammals is a bias during healing toward strong adaptive immunity and inflammatory responses. Here we directly test this hypothesis by characterizing part of the immune response during regeneration in spiny mice (Acomys cahirinus and Acomys percivali) vs. fibrotic repair in Mus musculus. By directly quantifying cytokines during tissue healing, we found that fibrotic repair was associated with a greater release of pro-inflammatory cytokines (i.e., IL-6, CCL2, and CXCL1) during acute inflammation in the wound microenvironment. However, reducing inflammation via COX-2 inhibition was not sufficient to reduce fibrosis or induce a regenerative response, suggesting that inflammatory strength does not control how an injury heals. Although regeneration was associated with lower concentrations of many inflammatory markers, we measured a comparatively larger influx of T cells into regenerating ear tissue and detected a local increase in the T cell associated cytokines IL-12 and IL-17 during the proliferative phase of regeneration. Taken together, our data demonstrate that a strong adaptive immune response is not antagonistic to regeneration and that other mechanisms likely explain the distribution of regenerative ability in vertebrates.

Understanding the impact of fibroblast heterogeneity on skin fibrosis.

Tissue fibrosis is the deposition of excessive extracellular matrix and can occur as part of the body's natural wound healing process upon injury, or as a consequence of diseases such as systemic sclerosis. Skin fibrosis contributes to significant morbidity due to the prevalence of injuries resulting from trauma and burn. Fibroblasts, the principal cells of the dermis, synthesize extracellular matrix to maintain the skin during homeostasis and also play a pivotal role in all stages of wound healing. Although it was previously believed that fibroblasts are homogeneous and mostly quiescent cells, it has become increasingly recognized that numerous fibroblast subtypes with unique functions and morphologies exist. This Review provides an overview of fibroblast heterogeneity in the mammalian dermis. We explain how fibroblast identity relates to their developmental origin, anatomical site and precise location within the skin tissue architecture in both human and mouse dermis. We discuss current evidence for the varied functionality of fibroblasts within the dermis and the relationships between fibroblast subtypes, and explain the current understanding of how fibroblast subpopulations may be controlled through transcriptional regulatory networks and paracrine communications. We consider how fibroblast heterogeneity can influence wound healing and fibrosis, and how insight into fibroblast heterogeneity could lead to novel therapeutic developments and targets for skin fibrosis. Finally, we contemplate how future studies should be shaped to implement knowledge of fibroblast heterogeneity into clinical practice in order to lessen the burden of skin fibrosis.

Sorocea guilleminiana Gaudich.: Wound healing activity, action mechanisms, and chemical characterization of the leaf infusion.

ETHNOPHARMACOLOGICAL RELEVANCE: Sorocea guilleminina Gaudich. is a tree or shrub endemic to Brazil. Its leaves are used in Brazilian folk medicine for the healing of wounds, stomach problems, inflammation and as diuretic. The present study evaluates the activity and action mechanisms of the healing properties of the aqueous extract of S. guilleminiana leaves (AESg), in experimental models in vivo and in vitro, as well as performs a phytochemical analysis of the extract. MATERIALS AND METHODS: The AESg was prepared by infusion: Ten g of dry leaves powder in 1 L hot water, soaked for 15 min, filtered, lyophilized, and stored at -30 °C. Phytochemical analyses were realized by colorimetry and HPLC/ESI/MS. Its' in vitro cytotoxicity was evaluated on fibroblastic N3T3 cells. The potential of the wound healing activity in vivo was evaluated using excision and incision wound rat models, by histopathology of the injured skin along with the determination of nitric oxide, cytokines (IL-1ß, IL-10, and TNF-α), and antioxidant parameters (GSH, MPO and CAT). In vitro wound healing activity was also demonstrated in scratched N3T3 cells, by measuring the proliferation/migration rate. RESULTS: The phytochemical analysis of the AESg revealed a strong presence of polar compounds, especially flavonoids (4 majoritarian), as well as terpenes and/or sterols (2 majoritarian). The AESg showed no toxicity in the N3T3 cell line (IC50 > 800 µg/mL). Topical treatment with the AESg showed an increase (p < 0.05) in wound contraction with 2 mg/g cream on days 5 and 9 (43.56% and 6.70% increase, respectively), and with 50 mg/g on days 7 and 9 (10.88% and 7.91%, respectively), compared to the vehicle (non-ionic neutral cream). Topical application of AESg (2 or 50 mg/g non-ionic cream) in incised wounds caused an increase in the force necessary for the rupture of the wound when compared to the vehicle group. No changes in cytokines (IL-1ß, IL-10, or TNF-α) or NO accumulation was found with up to 50 mg/g AESg treatment. For antioxidant activity on the incision wound, an increase in GSH levels was denoted with the AESg use, at the lowest and highest dose (2 and 50 mg/g) by 75.86% and 61.20% respectively, when compared to the vehicle. Also, the CAT activity was accentuated by AESg at the highest dose (50 mg/g) by 85.87%. Finally, the AESg at all doses attenuated MPO activity significantly in the incision wound by 71.35%, 73.21%, 78.08%, respectively. In the scratch test on N3T3 cells, the treatment with AESg resulted also in an increase in fibroblast proliferation/migration rate, compared to the vehicle. CONCLUSION: AESg is not cytotoxic. The results confirm the popular use of the leaf infusion of S. guilleminiana for the treatment of cutaneous wounds, possibly by stimulating the proliferation of fibroblasts with a consequent deposition of collagen, fastening rearrangement of collagen fibers, and greater transformation into myofibroblasts, essential in the healing process. Preliminary chemical analyzes of AESg revealed the presence mainly of phenolic compounds, being salicylic acid, gallic acid, pinocembrin and isoquercitrin the majoritarian ones.

Incisional Injury Modulates Morphine Reward and Morphine-Primed Reinstatement: A Role of Kappa Opioid Receptor Activation.

BACKGROUND: Persistent use of prescription opioids beyond the period of surgical recovery is a large part of a public health problem linked to the current opioid crisis in the United States. However, few studies have been conducted to examine whether morphine reward is influenced by acute pain and injury. METHODS: In a mouse model of incisional injury and minor trauma, animals underwent conditioning, extinction, and drug-primed reinstatement with morphine to examine the rewarding properties of morphine in the presence of acute incisional injury and drug-induced relapse, respectively. In addition, we sought to determine whether these behaviors were influenced by kappa opioid receptor signaling and measured expression of prodynorphin messenger RNA in the nucleus accumbens and medial prefrontal cortex after conditioning and before reinstatement with morphine and incisional injury. RESULTS: In the presence of incisional injury, we observed enhancement of morphine reward with morphine-conditioned place preference but attenuated morphine-primed reinstatement to reward. This adaptation was not present in animals conditioned 12 days after incisional injury when nociceptive sensitization had resolved; however, they showed enhancement of morphine-primed reinstatement. Prodynorphin expression was greatly enhanced in the nucleus accumbens and medial prefrontal cortex of mice with incisional injury and morphine conditioning and remained elevated up to drug-primed reinstatement. These changes were not observed in mice conditioned 12 days after incisional injury. Further, kappa opioid receptor blockade with norbinaltorphimine before reinstatement reversed the attenuation induced by injury. CONCLUSIONS: These findings suggest enhancement of morphine reward as a result of incisional injury but paradoxically a protective adaptation with incisional injury from drug-induced relapse resulting from kappa opioid receptor activation in the reward circuitry. Remote injury conferred no such protection and appeared to enhance reinstatement.

Diabetes induces myeloid bias in bone marrow progenitors associated with enhanced wound macrophage accumulation and impaired healing.

Diabetes induces dysregulation throughout the spectrum of myeloid lineage cells from progenitors to terminally differentiated cells. Another complication of diabetes is persistent inflammation, including prolonged accumulation of macrophages, which contributes to impaired wound healing. However, it remains unclear whether diabetes disrupts the response of bone marrow progenitors to peripheral injury and whether such dysregulation leads to sustained inflammation and impaired healing. Here, we demonstrated that diabetic mice (db/db, referred to here as DB) exhibit myeloid lineage bias during homeostasis and following injury. In addition, cells in the LSK (Lin- Sca-1+ cKit+ ) population of DB mice are preprogrammed towards myeloid commitment at the transcriptional level, and cultured myeloid progenitors from DB mice produce more monocytes ex vivo than their non-diabetic counterparts. We also show via bone marrow transfer between interleukin-1 receptor 1 KO (Il1r1-/- ) and DB mice that IL-1R1 signaling is likely not involved in myeloid skewing in DB mice. Furthermore, in vitro experiments indicated that macrophage colony-stimulating factor receptor signaling is not likely involved in enhanced myeloid transcription factor expression in LSK cells of DB mice. Our findings indicate that myeloid lineage commitment in bone marrow may contribute to increased macrophage numbers observed in diabetic skin wounds, and that strategies to regulate monopoiesis during homeostasis or post-wounding may improve diabetic wound healing. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A novel target for the promotion of dermal wound healing: Ryanodine receptors.

Ryanodine receptors have an important role in the regulation of intracellular calcium levels in the nervous system and muscle. It has been described that ryanodine receptors influence keratinocyte differentiation and barrier homeostasis. Our goal was to examine the role of ryanodine receptors in the healing of full-thickness dermal wounds by means of in vitro and in vivo methods. The effect of ryanodine receptors on wound healing, microcirculation and inflammation was assessed in an in vivo mouse wound healing model, using skin fold chambers in the dorsal region, and in HaCaT cell scratch wound assay in vitro. SKH-1 mice were subjected to sterile saline (n = 36) or ryanodine receptor agonist 4-chloro-m-cresol (0.5 mM) (n = 42) or ryanodine receptor antagonist dantrolene (100 µM) (n = 42). Application of ryanodine receptor agonist 4-chloro-m-cresol did not influence the studied parameters significantly, whereas ryanodine receptor antagonist dantrolene accelerated the wound closure. Inhibition of the calcium channel also increased the vessel diameters in the wound edges during the process of healing and increased the blood flow in the capillaries at all times of measurement. Furthermore, application of dantrolene decreased xanthine-oxidoreductase activity during the inflammatory phase of wound healing. Inhibition of ryanodine receptor-mediated effects positively influence wound healing. Thus, dantrolene may be of therapeutic potential in the treatment of wounds.

Fibroblast growth factor-2 inhibits CD40-mediated periodontal inflammation.

Fibroblast growth factor-2 (FGF-2) stimulates periodontal regeneration by a broad spectrum of effects on periodontal ligament (PDL) cells, such as proliferation, migration, and production of extracellular matrix. A critical factor in the success of periodontal regeneration is the rapid resolution of inflammatory responses in the tissue. We explored an anti-inflammatory effect of FGF-2 during periodontal regeneration and healing. We found that FGF-2 on mouse periodontal ligament cells (MPDL22) markedly downregulated CD40 expression, a key player of inflammation. In addition, FGF-2 inhibited CD40 signaling by the non-canonical nuclear factor-kappa B2 (NFκB2) pathway, resulting in decreased production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which have the potential to recruit immune cells to inflamed sites. Furthermore, in vivo treatment of FGF-2 enhanced healing of skin wounds by counteracting the CD40-mediated inflammation. These results reveal that FGF-2 has an important function as a negative regulator of inflammation during periodontal regeneration and healing.

The role of TrkA in the promoting wounding-healing effect of CD271 on epidermal stem cells.

CD271, a receptor of nerve growth factor (NGF), affects the biological properties of epidermal stem cells (eSCs) which are essential for skin wound closure. Tropomyosin-receptor kinase A (TrkA), another receptor of NGF, combined with CD271 has been involved with nervous system and skin keratinocytes. However, the exact role of TrkA combined with CD271 in eSCs during skin wound closure is still unclear. This study aimed to reveal the role of TrkA in the promoting wounding-healing effect of CD271 on eSCs. We obtained CD271-vo (over-expression of CD271) eSCs by lentiviral infection. K252a was used to inhibit TrkA expression. Full-thickness skin mouse wound closure model (5 mm in diameter) was used to detect the ability of CD271 over-expressed/TrkA-deficient during wound healing. The biological characteristics of eSCs and their proliferation and apoptosis were detected using immunohistochemistry and western blot. The expressions of protein kinase B (pAkt)/Akt, phosphorylated extracellular-signal-related kinase (pERK)/ERK1/2, and c-Jun N-terminal kinase (pJNK)/JNK were also detected by western blot. We found that over-expression of CD271 promoted the biological functions of eSCs. Interestingly, over-expression of CD271 in the absence of TrkA neither promoted eSCs' migration and proliferation nor promoted wound healing in a mouse model. In addition, we observed the reduced expression of pAkt/Akt and pERK/ERK1/2 following TrkA inhibition in vitro. Our studies demonstrated that the role of TrkA in the promoting wounding-healing effect of CD271 on eSCs.

Different Trypanosoma cruzi calreticulin domains mediate migration and proliferation of fibroblasts in vitro and skin wound healing in vivo.

Calreticulin is an endoplasmic reticulum-resident, calcium-binding, stress-produced, chaperone protein that serves multiple functions and is widely distributed in eukaryotic cells. Exogenously applied recombinant calreticulin solution, markedly enhanced the rate and quality of skin wound healing. These modulatory effects are more efficient than commercially available topic platelet-derived growth factor ointments (Regranex®). Trypanosoma cruzi calreticulin is more effective in equimolar terms to human counterpart in accelerating skin wound healing. While the effect of externally added recombinant parasite calreticulin on wound healing has been reported, the domains responsible for these modulatory effects have not yet been established. Here, recombinant parasite calreticulin and some of its domains were tested to assess their influence in increasing proliferation and migration of fibroblasts in vitro and rat skin wound healing in vivo. Herein, we propose that Trypanosoma cruzi whole calreticulin or some of its domains are differentially involved in the modulation of wound-healing cell migration and proliferation, and cosmetic outcome. Therefore, precise combination of the parasite protein and its domains could allow us to tailor-specific desired effects during the skin wound-healing process.

The effect of estrogen on diabetic wound healing is mediated through increasing the function of various bone marrow-derived progenitor cells.

OBJECTIVE: Endothelial progenitor cells (EPCs) are the key cells of postnatal neovascularization, and mesenchymal stem cells (MSCs) possess pluripotent differentiation capacity and contribute to tissue regeneration and wound healing. Both EPCs and MSCs are critical to the wound repair process, which is hindered in diabetes mellitus. Diabetes has been shown to decrease the function of these progenitor cells, whereas estrogen has beneficial wound healing effects. However, the role of estrogen in modulating EPC and MSC biology in diabetes is unknown. We investigated the effect of estrogen on improving bone marrow (BM)-derived EPC and MSC function using a murine diabetic wound healing model. METHODS: Female diabetic db+/db+ and nondiabetic control mice were wounded cutaneously and treated with topical estrogen or placebo cream. On day 5 after wounding, BM cells were harvested to quantify EPC number and colony-forming units of EPCs and MSCs. Wound healing rate was concurrently studied. Vessel density and scar density were then quantified using whole body perfusion and laser confocal microscopy. EPC recruitment was documented by immunohistochemistry to identify CD34- and vascular endothelial growth factor receptor 2-positive cells in the vessel wall. Data were analyzed by analysis of variance. RESULTS: Topical estrogen significantly increased colony-forming units of both EPCs and MSCs compared with placebo treatment, indicating improved viability and proliferative ability of these cells. Consistently, increased recruitment of EPCs to diabetic wounds and higher vessel density were observed in estrogen-treated compared with placebo-treated mice. Consequently, topical estrogen significantly accelerated wound healing as early as day 6 after wounding. In addition, scar density resulting from collagen deposition was increased in the estrogen-treated group, reflecting increased MSC activity and differentiation. CONCLUSIONS: Estrogen treatment increases wound healing and wound neovascularization in diabetic mice. Our data implicate that these beneficial effects may be mediated through improving the function of BM-derived EPCs and MSCs.

Effect of herbal mixture composed of Alchemilla vulgaris and Mimosa on wound healing process.

Alchemilla vulgaris and Mimosa tenuiflora (Mimosa) have been used to treat cutaneous wounds as a traditional remedy due to their various biological activities. But, there are only a few studies about the effects of these herbs on wound healing. The purpose of this study is to investigate the wound healing effect of the herbal mixture, consisting of A. vulgaris and Mimosa, in mice and to determine the activity of the extract in vitro. In present study, application of an ointment containing the herbal mixture on the dorsal skin wounds of mice showed that the wound healing process was faster than treatment of Fusidic acid. Histological analysis demonstrated the herbal mixture promoted re-epithelialization, collagen synthesis, and especially the regeneration of skin appendages such as hair follicles. Immunohistochemical analysis revealed the herbal mixture improved angiogenesis and the stabilization of blood vessels, as well as accelerated the formation of granulation tissue. In addition, we demonstrated that herbal mixture enhanced the migration of HaCaT, fibroblasts, and HUVECs on a two-dimensional wound, and promoted the proliferation of macrophages and lymphatic vessels. Our results demonstrated that herbal mixture can promote the migration of keratinocytes, fibroblasts, and endothelial cells, and the proliferation of macrophages and lymphatic vessels. Furthermore, it showed that herbal mixture accelerates wound healing. Therefore, we suggest that herbal mixture may have a potential for therapeutic use for treatment and management of cutaneous wound.

The development of induced pluripotent stem cell-derived mesenchymal stem/stromal cells from normal human and RDEB epidermal keratinocytes.

BACKGROUND: Epidermolysis bullosa (EB) is a group of hereditary disorders caused by mutations in the genes encoding structural molecules of the dermal-epidermal junction (DEJ). Cell-based therapies such as allogeneic mesenchymal stem/stromal cell (MSC) transplantation have recently been explored for severe EB types, such as recessive dystrophic EB (RDEB). However, hurdles exist in current MSC-based therapies, such as limited proliferation from a single cell source and limited cell survival due to potential allogenic rejection. OBJECTIVES: We aimed to develop MSCs from keratinocyte-derived induced pluripotent stem cells (iPSCs). METHODS: Keratinocyte-derived iPSCs (KC-iPSCs) of a healthy human and an RDEB patient were cultured with activin A, 6-bromoindirubin-3'-oxime and bone morphogenetic protein 4 to induce mesodermal lineage formation. These induced cells were subjected to immunohistochemical analysis, flow cytometric analysis and RNA microarray analysis in vitro, and were injected subcutaneously and intravenously to wounded immunodeficient mice to assess their wound-healing efficacy. RESULTS: After their induction, KC-iPSC-induced cells were found to be compatible with MSCs. Furthermore, with the subcutaneous and intravenous injection of the KC-iPSC-induced cells into wounded immunodeficient mice, human type VII collagen was detected at the DEJ of epithelized areas. CONCLUSIONS: We successfully established iPSC-derived MSCs from keratinocytes (KC-iPSC-MSCs) of a normal human and an RDEB patient. KC-iPSC-MSCs may have potential in therapies for RDEB.

M2 macrophages promote wound-induced hair neogenesis.

BACKGROUND: De novo hair regeneration occurs in scars of normal adult mice. This interesting phenomenon is termed as wound-induced hair neogenesis (WIHN). We hypothesized that M2 macrophages are crucially involved in WIHN. OBJECTIVE: To clarify the contribution of M2 macrophages to WIHN. METHOD: We established a mouse model of WIHN. A full thickness skin excision was implemented on the back of C57BL/6 (B6) mice. Newly developing hair follicles were detected by a whole-mount assay. WIHN took place 2 weeks after wounding. RESULTS: At first, flow cytometry revealed increased infiltration of CD11b+/CD206+ M2 macrophages at the 2nd and 3rd week after wounding. Immunohistochemistry also showed the existence of CD206+ M2 macrophages in the vicinity of regenerated hair follicles. Secondly, the productions of growth factors were confirmed by culturing M2 macrophages isolated from the skin in a comparison with CD11b+ spleen cells. Array for 84 genes revealed increased expressions of various growth factors including Igf1 and Fgf2. Thirdly, we verified the effect of the growth factors on WIHN. WIHN was increased by 2 folds in mice treated with Fgf2 (p=0.05) or by 1.5 folds with Igf1 (p=0.05). Finally, we used B6.Tg(ITGAM-DTR) mice in which macrophages are ablated by diphtheria toxin. We depleted macrophages at one to 2 weeks after wounding when M2 macrophages were dominant. WIHN was attenuated to one third (P=0.05) by the ablation of macrophages. CONCLUSION: Our study suggests that M2 macrophages could promote WIHN through producing a panel of growth factors.