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.

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.

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.

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.

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.

Lucidone Promotes the Cutaneous Wound Healing Process via Activation of the PI3K/AKT, Wnt/ß-catenin and NF-κB Signaling Pathways.

Lucidone, which comprises a naturally occurring cyclopentenedione, has been investigated for its in vitro and in vivo wound healing properties, and the underlying molecular signaling cascades in the wound healing mechanism have been elucidated. We demonstrated the cell-/dose-specific responses of lucidone (0.5-8µM) on proliferation and migration/invasion of keratinocyte HaCaT and fibroblast Hs68 cells. In keratinocytes, lucidone-induced nuclear translocation of ß-catenin was accompanied by increased transcriptional target genes, including c-Myc and cyclin-D1, through GSK3ß-dependent pathway. Correspondingly, lucidone promoted the cell-cycle by increasing PCNA/CDK4 and decreasing p21/p27 expressions. Lucidone induced EMT through the downregulation of epithelial (E-cadherin/occludin) and upregulation of mesenchymal (vimentin/Twist/Snail) marker proteins. Activated MMP-9/-2 and uPA/uPAR as well as suppressed TIMP-1/-2 and PAI-1 expressions by lucidone may promote the migration/invasion of keratinocytes. Notably, lucidone activated NF-κB signaling via IKK-mediated-IκB degradation, and its inhibition abolished MMP-9 activation and keratinocyte migration. Inhibition of PI3K/AKT signaling impaired the lucidone-induced proliferation/migration with corresponding suppression of ß-catenin/c-Myc/cyclin-D1 and NF-κB/MMP-9 expressions. Results indicate that lucidone-induced PI3K/AKT signaling anchored the ß-catenin/NF-κB-mediated healing mechanism. ß-catenin knockdown substantially diminished lucidone-induced keratinocyte migration. Furthermore, lucidone increased endothelial cell proliferation/migration and triggered angiogenesis (MMP-9/uPA/ICAM-1). In macrophages, lucidone-activated NF-κB-mediated inflammation (COX-2/iNOS/NO) and VEGF, which may contribute to the growth of keratinocytes/fibroblasts and endothelial cells. Punched wounds on mice were rapidly healed with the topical application of lucidone (5mM) compared with control ointment-treated mice. Taken together, lucidone accelerates wound healing through the cooperation of keratinocyte/fibroblast/endothelial cell growth and migration and macrophage inflammation via PI3K/AKT, Wnt/ß-catenin and NF-κB signaling cascade activation.

Acceleration of diabetic wound healing with adipose-derived stem cells, endothelial-differentiated stem cells, and topical conditioned medium therapy in a swine model.

OBJECTIVE: The purpose of our study was to investigate the effect of adipose-derived stem cells (ASCs), endothelial-differentiated ASCs (EC/ASCs), and various conditioned media (CM) on wound healing in a diabetic swine model. We hypothesized that ASC-based therapies would accelerate wound healing. METHODS: Diabetes was induced in four Yorkshire swine through intravenous injection of streptozotocin. ASCs were harvested from flank fat and cultured in either M199 or EGM-2 medium. A duplicate series of seven full-thickness dorsal wounds were surgically created on each swine. The wounds in the cellular treatment group underwent injection of low-dose or high-dose ASCs or EC/ASCs on day 0, with a repeat injection of one half of the initial dose on day 15. Wounds assigned to the topical CM therapy were covered with 2 mL of either serum-free M199 primed by ASCs or human umbilical vein endothelial cells every 3 days. Wounds were assessed at day 0, 10, 15, 20, and 28. The swine were sacrificed on day 28. ImageJ software was used to evaluate the percentage of wound healing. The wounded skin underwent histologic, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay examinations to evaluate markers of angiogenesis and inflammation. RESULTS: We found an increase in the percentage of wound closure rates in cell-based treatments and topical therapies at various points compared with the untreated control wounds (P < .05). The results from the histologic, messenger RNA, and protein analyses suggested the treated wounds displayed increased angiogenesis and a diminished inflammatory response. CONCLUSIONS: Cellular therapy with ASCs, EC/ASCs, and topical CM accelerated diabetic wound healing in the swine model. Enhanced angiogenesis and immunomodulation might be key contributors to this 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.

Hyperbilirubinemia-induced pro-angiogenic activity of infantile endothelial progenitor cells.

OBJECTIVES: Bilirubin, a by-product of heme degradation, is suggested to have a role for vascular protection. There is increasing evidence that bilirubin may directly affect the function and secretory activity of endothelial cells. In this study, potential effect of hyperbilirubinemia on biological features of circulation endothelial progenitor cells (cEPCs) isolated from infants was investigated. METHODS: Circulation concentration, differentiation and migratory activity of cEPCs isolated from infants with (n = 111) or without (n = 73) hyperbilirubinemia were analyzed. Then, the potential beneficial effect of conditioned medium of cEPCs from infants with or without hyperbilirubinemia was examined on experimental mouse wounds. RESULTS: Our results revealed significantly higher percentages of cEPCs in infants with hyperbilirubinemia. Cell proliferation, and migratory properties of cEPCs isolated and expanded from infants with hyperbilirubinemia were significantly improved. Also, the conditioned medium of cEPCs from hyperbilirubinemic infants possessed a superior beneficial effect on wound healing, which was associated with increased protein levels of VEGF, IL-10, and Pho-ERK/ERK, and decreased TNF-α in the wound tissues. CONCLUSIONS: Our results showed that hyperbilirubinemia can activate migration, proliferating and angiogenic properties of cEPCs. Hyperbilirubinemia can promote the proangiogenic secretory activity of cEPCs, thereby resulting in enhancement of their regenerative wound healing properties.

IL-17A promotes neutrophilic inflammation and disturbs acute wound healing in skin.

In the wound healing process, neutrophils are the first inflammatory cells to move to the wound tissues. They sterilize wounds by killing microbes, and they stimulate other immune cells to protect the host from infection. In contrast, neutrophil-derived proteases cause damage to host tissues, so neutrophils play dual opposite roles in wound healing. Interleukin-17A (IL-17A) is a proinflammatory cytokine that promotes the recruitment of these cells. The role of this cytokine in the wound healing process is not fully clarified. In the present study, therefore, we examined how defect in IL-17A production affected the wound healing in skin. IL-17A-knockout (KO) mice showed promoted wound closure, myofibroblast differentiation and collagen deposition and decreased the neutrophil accumulation compared with wild-type (WT) mice. In contrast, the administration of recombinant IL-17A led to delayed wound closure, low collagen deposition and accelerated neutrophilic accumulation. In addition, the treatment of IL-17A-administered mice with a neutrophil elastase inhibitor improved the wound repair to the same level as that of WT mice. These results indicated that IL-17A hampered the wound healing process and suggested that neutrophilic inflammation caused by IL-17A may be associated with impaired wound healing in skin.

Dermal CD271+ Cells are Closely Associated with Regeneration of the Dermis in the Wound Healing Process.

Stem cells have recently been shown to play important roles in wound healing. The aim of this study was to investigate the role of dermal CD271+ cells in wound healing. Full-thickness wounds were produced on the backs of 5-year-old and 24-week-old mice, and time-course of wound closure, CD271+ cell counts, and gene expression levels were compared. Delayed wound healing was observed in 24-week-old mice. The peak of CD271+ cell increase was delayed in 24-week-old mice, and gene expression levels of growth factors in wounded tissue were significantly increased in 5-year-old mice. Dermal CD271+ cells purified by fluorescence-activated cell sorting (FACS) expressed higher growth factors than CD271- cells, suggesting that CD271+ cells play important roles by producing growth factors. This study also investigated dermal CD271+ cells in patients with chronic skin ulcers. Dermal CD271+ cells in patients were significantly reduced compared with in healthy controls. Thus, dermal CD271+ cells are closely associated with wound healing.

Antioxidant and wound healing potential of saponins extracted from the leaves of Algerian Urtica dioica L.

The Nettle is a herbaceous and vivace plant of Asian origin. It is integrated in several areas especially alimentary, agricultural, industrial and medicinal. The aim of this work is to demonstrate through pharmacological tests a possible antioxidant and wound healing effect of crude saponins of the leaves of Urtica dioica L. The extraction method is based on the degree of solubility of saponins in organic solvents. The antioxidant activity of the leaves extracts was evaluated by the diphenyl-picryl-hydrazyl test (DPPH). The wound healing effect is interpreted on the basis of the healing time and the evaluation of the surface of wounds. It appears from this study that the Nettle is rich in saponins, either 4.08% to 30 g of plant powder. The results also showed significant antioxidant effect similar to that of ascorbic acid (p> 0.05) with an IC50 of 0.159mg/ml. As regards the healing power, treatment of rats with the product based on crude saponins is achieved after 15 days, either 100% of wound reduction. This value is much higher than that obtained by the reference product (Madécassol®) on the same duration of treatment with 93.73% of wound reduction. The achievement of pharmacological tests has thus shown that crude saponins extracted from the leaves of Urtica dioica L. can be integrated into the pharmaceutical field or even in cosmetic.

In Vivo Wound Healing Studies.

Wound healing has emerged as a major treatment issue which has provoked the development of drugs that can improve the healing process. Studies using plant drugs have revealed many interesting results about existing commercial drugs. Effective wound healing leads to the restoration of tissue integrity and occurs through a highly organized multistage. Use of plant-derived medicines against excision, incision, and dead space models accelerates the wound healing process, which is briefly discussed in a manner to be followed easily during experimental sessions.

Effect of Ankaferd Blood Stopper on muscle healing.

PURPOSE: Ankaferd Blood Stopper (ABS), which is a standardized mixture of herbal extracts obtained from five plants, has been proven as an efficient hemostatic agent and is still used in emergency situations. It is not known exactly if decreased bleeding has positive or negative effects on muscle healing and fibrosis, so the purpose of this study was to test the effect of ABS on muscle healing and morphology. METHODS: A total of 66 outbred Wistar rats were divided into three control and three experimental subgroups. In the experimental groups, ABS was sprayed on the cut surface of the soleus. In the control groups, a saline solution was sprayed on the cut surface of the soleus. Subgroups were euthanized after 2 weeks, 3 weeks and 4 weeks, respectively. In each subgroup, eight rats were used for the biomechanical study to determine muscle healing and three rats were used for the histopathological investigation. RESULTS: Although muscle strength in the control groups was lower than that of the experimental groups in early weeks, no differences were found between the control and the experimental groups at 4 weeks. CONCLUSIONS: ABS has no negative effect on muscle healing. We also observed that ABS accelerated muscle healing compared to the control group. ABS could be used in hemostasis of open fractures and elective orthopedic surgeries.

Telocyte Behaviour During Inflammation, Repair and Tumour Stroma Formation.

In this chapter, we outline the role of human CD34+ stromal cells/telocytes (CD34+ SC/TCs) as progenitor cells during repair. The in vivo activation phenomena of CD34+ SC/TCs in this process include increased size; separation from the neighbouring structures (mainly of the vascular walls); association with inflammatory cells, predominantly macrophages; development of the organelles of synthesis (rough endoplasmic reticulum and Golgi apparatus); cell proliferation with presence of mitosis and high proliferative index (transit-amplifying cells); and fibroblastic and myofibroblastic differentiation. A procedure to study these tissue-resident cells, comparison of their behaviour in vivo and in vitro and different behaviour depending on location, time, type of injury (including tumour stroma) and greater or lesser proximity to the injury are also considered.

Wound healing activity of a collagen-derived cryptic peptide.

Wound healing involves a well-controlled series of interactions among cells and several mediators leading to the restoration of damaged tissue. Degradation of the extracellular matrix (ECM) protein collagen during remodelling of wound tissue leads to the release of bioactive peptides that can possibly influence the healing process. The RGD-containing, antioxidative collagen peptide E1 isolated in an earlier work was screened in this study for its ability to influence multiple steps of the wound healing process. E1 was assayed for and found to be chemotactic. Excision and incision wounds were created on separate groups of rats and E1 was administered topically. The wound tissues were isolated on the 4th and 8th days post-wound and subjected to biochemical and biophysical analysis. A significant decrease in lipid peroxides in the treatment group confirmed the in vivo antioxidant capacity of E1. The treatment group also displayed significant increase in total protein, collagen and amino sugar synthesis indicating faster ECM formation. The significantly increased rate of wound contraction and reepithelialisation along with higher tensile strength of the wound tissue corroborated the results of biochemical analysis. The results confirm the significant role played by collagen peptides in accelerating the healing process and justify their possible use as a pharmaceutical agent.

Mallotus philippinensis Muell. Arg fruit glandular hairs extract promotes wound healing on different wound model in rats.

BACKGROUND: Mallotus philippinensis Muell. Arg (MP, Euphorbiaceae) are widely distributed perennial shrub or small tree in tropical and subtropical region in outer Himalayas regions. Since, Mallotus philippinensis have been shown to have a number of medicinal values. Hence our present study was to investigate the healing potential of fruit extract in rat wound models. METHODS: The study includes acute toxicity and wound healing potential of 50% ethanol extract of MP fruit glandular hair (MPE). MPE (200 mg/kg) was administered orally, once daily for 10 days (incision and dead space wound) and 22 days (excision wound). MPE was found safe when given to rats upto 10 times of optimal effective dose. Wound breaking strength (WBS) in Incision wound and rate of contraction, period of epithelization and scar area in Excision wound were evaluated. Granulation tissue free radicals (nitric oxide and lipid peroxidation), antioxidants (catalase, superoxide dismutase, and reduced glutathione), acute inflammatory marker (myeloperoxidase), connective tissue markers (hydroxyproline, hexosamine, and hexuronic acid), and deep connective tissue histology were studied in Dead space wound. RESULTS: MPE significantly increased WBS and enhanced wound contraction, and decreased both epithelization period and scar area compared with control group. MPE was found to decrease free radicals (50.8 to 55.2%, P<0.001) and myeloperoxidase (44.0%, P<0.001) but enhanced antioxidants (41.1 to 54.5%, P<0.05 to P<0.001) and connective tissue markers (39.5 to 67.3%, P<0.05 to P<0.01). Histopathological evaluation revealed more density of collagen formation with minimal inflammatory cells in deeper tissues. CONCLUSION: Thus, the study revealed Mallotus philippinensis fruit hair extract, safe and effective in wound healing and the healing effects seemed to be due to decrease in free radical generated tissue damage, promoting effects on antioxidant status and faster collagen deposition as evidenced biochemically and histology.

Utility of a human-mouse xenograft model and in vivo near-infrared fluorescent imaging for studying wound healing.

To study the complex cellular interactions involved in wound healing, it is essential to have an animal model that adequately mimics the human wound microenvironment. Currently available murine models are limited because wound contraction introduces bias into wound surface area measurements. The purpose of this study was to demonstrate utility of a human-mouse xenograft model for studying human wound healing. Normal human skin was harvested from elective abdominoplasty surgery, xenografted onto athymic nude (nu/nu) mice, and allowed to engraft for 3 months. The graft was then wounded using a 2-mm punch biopsy. Wounds were harvested on sequential days to allow tissue-based markers of wound healing to be followed sequentially. On the day of wound harvest, mice were injected with XenoLight RediJect cyclooxygenase-2 (COX-2) probe and imaged according to package instructions. Immunohistochemistry confirms that this human-mouse xenograft model is effective for studying human wound healing in vivo. Additionally, in vivo fluorescent imaging for inducible COX-2 demonstrated upregulation from baseline to day 4 (P = 0·03) with return to baseline levels by day 10, paralleling the reepithelialisation of the wound. This human-mouse xenograft model, combined with in vivo fluorescent imaging provides a useful mechanism for studying molecular pathways of human wound healing.

Avocado/soybean unsaponifiables: a novel regulator of cutaneous wound healing, modelling and remodelling.

We investigated the effects of avocado/soybean unsaponifiables (ASU) on the healing response of cutaneous wound defect in rats. Sixty male rats were randomly divided into three groups including control, vehicle and treatment (n = 20 in each group). A 2 × 2 cm(2) wound defect was made on the dorsum. The control, vehicle and treatment groups were treated daily with topical application of saline, cream and cream/ASU for 10 days, respectively. The wounds were monitored daily. The animals were euthanised at 10, 20 and 30 days post injury (D). The dry matter, hydroxyproline, collagen, n-acetyl glucosamine (NAGLA) and n-acetyl galactosamine (NAGAA) contents of the skin samples were measured and the histopathological and biomechanical characteristics of the samples were investigated. Statistics of P < 0·05 was considered significant. Treatment significantly increased tissue glycosaminoglycans and collagen contents at various stages of wound healing compared to controls. Treatment modulated inflammation, improved fibroplasia and produced high amounts of scar tissue at short term. At long term, treatment reduced the scar tissue size and increased the quality and rate of wound contraction and reepithelisation compared to controls. The treated lesions were more cosmetically pleasing and had significantly higher biomechanical characteristics than controls. ASU was effective in rat wound healing.

Acute and chronic wound fluids influence keratinocyte function differently.

Wound healing requires a proper functioning of keratinocytes that migrate, proliferate and lead to a competent wound closure. Impaired wound healing might be due to a disturbed keratinocyte function caused by the wound environment. Basically, chronic wound fluid (CWF) differs from acute wound fluid (AWF). The aim of this study was to analyse the effects of AWF and CWF on keratinocyte function. We therefore investigated keratinocyte migration and proliferation under the influence of AWF and CWF using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test and scratch assay. We further measured the gene expression by qRT-PCR regarding growth factors and matrixmetalloproteinases (MMPs) involved in regeneration processes. AWF had a positive impact on keratinocyte proliferation over time, whereas CWF had an anti-proliferative effect. Keratinocyte migration was significantly impaired by CWF in contrast to an undisturbed wound closure under the influence of AWF. MMP-9 expression was strongly upregulated by CWF compared with AWF. Keratinocyte function was significantly impaired by CWF. An excessive induction of MMP-9 by CWF might lead to a permanent degradation of extracellular matrix and thereby prevent wounds from healing.