Wound Repair Activity Research Articles

An investigation on the antimicrobial activity of shrimp exoskeleton extracted glycosaminoglycans (GAGs) against Escherichia coli and Staphylococcus aureus

The present study was aimed to extract the Glycosaminoglycans (GAGs) from the shrimp head waste and carry out analysis for any antimicrobial activity against Escherichia coli and Staphylococcus aureus. Glycosaminoglycans (GAGs) are linear acidic polysaccharides with repeating disaccharide units. GAGs are important for cell signaling, which controls a wide range of metabolic activities including regulation of cell growth, promotion of cell adhesion, wound repair, proliferation, anti-coagulation and antimicrobial activity. In the present study, the shrimp heads were washed, dried and pulverized. From this pulverized shrimp head waste, extraction of GAG protein was achieved. The total protein content in the biological sample was estimated. Antimicrobial study of GAGs complex was accomplished by using Minimal Inhibitory Concentration, Minimal Bactericidal Concentration, Well Diffusion Method against E. coli and Staphylococcus aureus. It was observed that Staphylococcus aureus has the highest antibacterial activity when compared to Escherichia coli.

Molecular modeling, expression and wound-repair activity in vivo of platelet-derived growth factor from Periplaneta americana

Platelet-derived growth factors (PDGFs) promote the repair of the wounded tissue by stimulating macrophages to produce and secrete growth factors. Active constituents extracted from Periplaneta americana were primary for treatment of wounds, pressure sores, and burns. In this study, PDGF from Periplaneta americana (PAPDGF) was identified, and the sequence analysis, physical properties, 3D structure of PAPDGF and the interfaces of PAPDGF-PDGFRβ were predicted by bioinformatics. In addition, to explore the relationship between the key amino acid residues and activity of PAPDGF, recombinant mutant of PAPDGF (rmPAPDGF, D164H) was constructed. The sequence of PAPDGF and mutant of PAPDGF (D164H) were expressed and purified by Ni-affinity chromatography. The wound-repair activity of recombinant PAPDGF (rPAPDGF) and rmPAPDGF were tested in vivo. The results showed that rPAPDGF and rmPAPDGF were successfully obtained and exhibited a single band on SDS-PAGE at 49.87 kDa. The rPAPDGF showed potent repair activity by stimulating the expression of collagen and PDGFRβ, whereas the rmPAPDGF exhibited low repair activity in vivo, which indicated that the residue D164 in PAPDGF was the key amino acid to maintain activity. Taken together, PAPDGF may be served as a promising candidate agent for the treatment of wound in the future.

Stimulatory Effects of Paederia foetida Flower Absolute on the Skin Wound and Barrier Repair Activities of Keratinocytes

Paederia foetida (PF) has antidiarrheal, antidiabetic, and anti-inflammatory activities. However, its biological activities on skin remain unclear. In this study, we examined the effect of PF flower absolute (PFFA) on skin wound healing- and skin barrier-linked responses in human epidermal keratinocytes (HaCaT cells). PFFA contained 23 components and increased the proliferation and sprout outgrowth of HaCaT cells and modestly increased migration. PFFA enhanced the phosphorylation levels of extracellular signal-regulated kinase1/2, serine/threonine-specific protein kinase (AKT), and p38 mitogen-activated protein kinase (MAPK) in HaCaT cells, and upregulated type I and IV collagen synthesis and filaggrin (an epidermal barrier protein) expression in HaCaT cells. These findings suggest PFFA may promote skin wound repair by stimulating migratory and proliferative activities (probably through the AKT/MAPK pathway), collagen synthesis, and skin barrier repair by upregulating the expressions of filaggrin in epidermal keratinocytes. Therefore, PFFA may be useful for developing agents that enhance skin wound and barrier-repair functions.

Effects of Agricultural Organic Dusts on Human Lung-Resident Mesenchymal Stem (Stromal) Cell Function.

Agricultural organic dust exposures trigger harmful airway inflammation, and workers experiencing repetitive dust exposures are at increased risk for lung disease. Mesenchymal stem/stromal cells (MSCs) regulate wound repair processes in the lung, and may contribute to either proresolution or -fibrotic lung responses. It is unknown how organic dust exposures alter lung-resident MSC activation and proinflammatory versus prorepair programs in the lung. To address this gap in knowledge, we isolated human lung-resident MSC from lung tissue. Cells were stimulated with aqueous extracts of organic dusts (DE) derived from swine confinement facilities and were assessed for changes in proliferative and migratory capacities, and production of proinflammatory and prorepair mediators. Through these investigations, we found that DE induces significant release of proinflammatory mediators TNF-α, IL-6, IL-8, and matrix metalloproteases, while also inducing the production of prorepair mediators amphiregulin, FGF-10, and resolvin D1. In addition, DE significantly reduced the growth and migratory capacities of lung-resident MSC. Together, these investigations indicate lung-resident MSC activation and wound repair activities are altered by organic dust exposures. These findings warrant future investigations to assess how organic dusts affect lung-resident mesenchymal stem/stromal cell function and impact airway inflammation, injury, and repair during agricultural aerosol exposures.

Anti-pseudomonal and anti-endotoxic effects of surfactin-stabilized biogenic silver nanocubes ameliorated wound repair in streptozotocin-induced diabetic mice

Pseudomonas aeruginosa and its lipopolysaccharides play a key role in the pathogenesis of diabetic foot infection, for which, currently no effective therapeutic agents are available. Hence, newer forms of therapeutic agents are required for treating Pseudomonas aeruginosa infection. In this present study, nanocrystalline silver nanoparticles (AgNPs) were synthesized using culture filtrate of Brevibacillus brevis KN8(2) followed by an investigation of its in vivo anti-pseudomonal and anti-endotoxic properties. Biosynthesized AgNPs was predominantly cubical in shape with an average particle size of 15.40 nm as observed through field emission scanning electron microscopy (FESEM) and X-ray diffraction analysis. The LC-ESI-MS/MS analysis indicates the presence of surfactin in culture filtrate of B. brevis KN8(2). The MIC of surfactin-stabilized AgNPs against P. aeruginosa was 10 μg ml−1 and its wound repair activity was observed in P. aeruginosa-infected wounds of diabetic mice by measuring wound area closure, bacterial counts, mRNA expressions, and histopathology. Further, surfactin-stabilized AgNPs suppressed the transcription of LPS-triggered expression of the TNF-α in wounds that LPS-assisted extension of wound repair in diabetes mellitus conditions was circumvented quite well. Results gathered in this study established that surfactin-stabilized AgNPs could effectively offer to the novel treatment of Gram-negative bacilli infection in diabetic wounds.

Intact wound repair activity of human mesenchymal stem cells after YM155 mediated selective ablation of undifferentiated human embryonic stem cells

BackgroundRisk of teratoma formation during human pluripotent stem cell (hPSC)-based cell therapy is one of the technical hurdles that must be resolved before their wider clinical application. To this end, selective ablation of undifferentiated hPSCs has been achieved using small molecules whose application should be safe for differentiated cells derived from the hPSCs. ObjectiveHowever, the functional safety of such small molecules in the cells differentiated from hPSCs has not yet been extensively validated. MethodWe used the survivin inhibitor YM155, which induced highly selective cell death of hPSCs for ablating undifferentiated hESCs after differentiation to human mesenchymal stem cells (hMSCs) and examined whether hMSCs remained fully functional after being exposed by YM155. ResultsWe demonstrated that human mesenchymal stem cells (hMSCs) derived from human embryonic stem cells (hESCs) remained fully functional in vitro and in vivo, while hESCs were selectively ablated. ConclusionThese results suggest that a single treatment with YM155 after differentiation of hMSCs would be a valid approach for teratoma-free cell therapy.

Growth Factor-Reinforced ECM Fabricated from Chemically Hypoxic MSC Sheet with Improved In Vivo Wound Repair Activity.

MSC treatment can promote cutaneous wound repair through multiple mechanisms, and paracrine mediators secreted by MSC are responsible for most of its therapeutic benefits. Recently, MSC sheet composed of live MSCs and their secreted ECMs was reported to promote wound healing; however, whether its ECM alone could accelerate wound closure remained unknown. In this study, Nc-ECM and Cc-ECM were prepared from nonconditioned and CoCl2-conditioned MSC sheets, respectively, and their wound healing properties were evaluated in a mouse model of full-thickness skin defect. Our results showed that Nc-ECM can significantly promote wound repair through early adipocyte recruitment, rapid reepithelialization, enhanced granulation tissue growth, and augmented angiogenesis. Moreover, conditioning of MSC sheet with CoCl2 dramatically enriched its ECM with collagen I, collagen III, TGF-β1, VEGF, and bFGF via activation of HIF-1α and hence remarkably improved its ECM's in vivo wound healing potency. All the Cc-ECM-treated wounds completely healed on day 7, while Nc-ECM-treated wounds healed about 85.0% ± 8.6%, and no-treatment wounds only healed 69.8% ± 9.6% (p < 0.05). Therefore, we believe that such growth factor-reinforced ECM fabricated from chemically hypoxic MSC sheet has the potential for clinical translation and will lead to a MSC-derived, cost-effective, bankable biomaterial for wound management.

Podoplanin is involved in the prognosis of head and neck squamous cell carcinoma through interaction with VEGF-C.

We investigated the clinical significance of podoplanin expression in relation to clinicopathological variables in head and neck squamous cell carcinoma (HNSCC), to determine its effectiveness as a marker for high-risk HNSCC patients. Upregulation of podoplanin in HNSCC tissues was examined using immunohistochemistry and clinicopathological analyses. Wound healing and invasion assays were performed using HNSCC cells that were transfected with podoplanin siRNA, podoplanin vector and cotransfection with both the podoplanin vector and VEGF-C siRNA. High podoplanin expression was significantly associated with ~3- and 5-fold increases in the presence of positive lymph node metastasis and poor histological grade, respectively (P<0.05). High levels of podoplanin expression were significantly associated with decreased overall and disease-specific survival rates (P<0.05). Furthermore, upregulation of podoplanin induced cell wound repair activity and invasiveness in the FaDu and SNU-1041 cells, respectively, while downregulation of podoplanin expression through VEGF-C silencing using co-transfection of both the podoplanin vector and VEGF-C siRNA suppressed cell wound healing and invasion abilities in the HNSCC cells. Our findings suggest that high podoplanin expression is associated with aggressive tumor behavior, poor prognosis and metastatic regulation through interaction with VEGF-C, suggesting that podoplanin may be used as a potential prognostic biomarker for HNSCC.

Critical Role of the GM-CSF Signaling Pathway in Macrophage Pro-Repair Activities

Objective: Macrophages play a critical role in intestinal wound repair. However, the molecular pathways that regulate macrophage wound repair activities remain poorly understood. The aim of this study was to evaluate the role of GM-CSF receptor signaling in the wound repair activities of macrophages. Methods: Murine macrophages were differentiated from bone marrow cells and human macrophages from monocytes isolated from peripheral blood mononuclear cells of Crohn's disease (CD) patients. In vitro models were used to study the repair activities of macrophages. Results: We provide evidence that GM-CSF receptor signaling is required for murine macrophages to promote epithelial repair. In addition, we demonstrate that the deficient repair properties of macrophages from CD patients with active disease can be recovered via GM-CSF therapy. Conclusion: Our data support a critical role of the GM-CSF signaling pathway in the pro-repair activities of mouse and human macrophages.

Polysaccharides of Aloe vera induce MMP-3 and TIMP-2 gene expression during the skin wound repair of rat

Polysaccharides are the main macromolecules of Aloe vera gel but no data about their effect on extracellular matrix (ECM) elements are available. Here, mannose rich Aloe vera polysaccharides (AVP) with molecular weight between 50 and 250kDa were isolated and characterized. Open cutaneous wounds on the back of 45 rats (control and treated) were daily treated with 25mg (n=15) and 50mg (n=15) AVP for 30 days. The levels of MMP-3 and TIMP-2 gene expression were analyzed using real time PCR. The levels of n-acetyl glucosamine (NAGA), n-acetyl galactosamine (NAGLA) and collagen contents were also measured using standard biochemical methods. Faster wound closure was observed at day 15 post wounding in AVP treated animals in comparison with untreated group. At day 10 post wounding, AVP inhibited MMP-3 gene expression, while afterwards MMP-3 gene expression was upregulated. AVP enhanced TIMP-2 gene expression, collagen, NAGLA and NAGA synthesis in relation to untreated wounds. Our results suggest that AVP has positive effects on the regulation of ECM factor synthesis, which open up new perspectives for the wound repair activity of Aloe vera polysaccharide at molecular level.

In vitro and in vivo Repair Activities of Undifferentiated and Classically and Alternatively Activated Macrophages

Objective: Macrophages play a critical role in wound repair. However, the specific role of the different macrophage subtypes in wound repair remains incompletely understood. The aim of this study was to compare the wound repair activities of undifferentiated macrophages (M0), classically activated macrophages (M1) and alternatively activated (M2) macrophages. Methods: The macrophage repair activities of intestinal wounds were evaluated using in vitro and in vivo models. Results: All three macrophage subtypes enhanced wound closure in vitro, with the M2 macrophages demonstrating greater repair activities than the M0 and M1 macrophages. Injection of M0 and M2 macrophages into mice with experimental dextran sodium sulfate-induced colitis significantly enhanced ulcer repair when compared to control mice. In contrast, injection of M1 macrophages did not affect ulcer repair. Conclusions: These results underscore the wound repair capacity of different macrophage subsets. Notably, wound repair activity is not restricted to M2 macrophages, as the current literature suggests.

Characterization of activin A in the culture of primitive human umbilical cord blood hematopoietic cells

Transforming growth factor-β (TGF-β) superfamily controls many physiological processes such as cell proliferation and differentiation, immune responses, wound repair and various endocrine activities. As a member of TGF-β, activin A can maintain the pluripotency of embryonic stem cells. We report here that activin A exhibited cell type-dependent function of expanding the human primitive hematopoietic cells isolated from umbilical cord blood (UCB). However, the multipotency of the cells pretreated with activin A was exhausted in the sequential dilution culture. In conclusion, activin A may not be a key factor, but a regulator, in the multipotency maintenance of primitive hematopoietic cells and the application of activin A in the hematopoietic stem/progenitor cells (HS/PCs) culture expansion remains a significant challenge.

The n-Hexane, ethylacetate, and butanol fractions from Hydnocarpi Semen enhanced wound healing in a mice ulcer model

Our previous report showed that Hydnocarpi Semen (HS) extract has wound repair activity at ulcer lesion in diabetic mice. In this study, fractions of n-Hexane, ethylacetate (EtOAc), and butanol (BuOH) from HS crude extract were evaluated for their wound healing activity by using in vivo diabetic ulcer models and in vitro acute inflammation model. Although n-Hexane and EtOAc fractions promote wound healing in mice with ulcer, the BuOH fraction exhibited the most potent wound healing activity and the wound area score significantly decreased after treatment of BuOH fraction even at dose of 2 mg/kg. BuOH fraction stimulated macrophages to increase the production of nitric oxide (NO) and TNF-α. The BuOH fraction also enhanced the production of TGF-β and VEGF, which were involved in fibroblast activation and angiogenesis. The mRNA expression and activation of MMP-9 were increased by three fractions and the activity was higher in BuOH fraction-treated group compared to the other groups. The mechanism that the HS helps to promote healing of diabetic ulcer is possibly associated with the production of TNF-α, a proinflammatory cytokine, as well as the secretion of VEGF, TGF-β, and MMP-9, which were involved in proliferation of capillaries and fibroblasts. These results suggest that HS can be a new candidate material for the treatment of wound in skin ulcer.

Wound repair and proliferation of bronchial epithelial cells regulated by CTNNAL1

Adhesion molecules play vital roles in airway hyperresponsiveness (AHR) or airway inflammation. Our previous study indicated that adhesion molecule catenin alpha-like 1 (CTNNAL1) is relevant closely to asthma susceptibility, but its biological function or significance is still unclear. In the present study, we observed the temporal and spatial distribution of CTNNAL1 expression in mouse lung tissue with the OVA-sensitized asthma model and found that the level of CTNNAL1 mRNA showed a prominent negative correlation with pulmonary resistance (R(L)). To study the function of CTNNAL1 in airway, effects of CTNNAL1 on proliferation and wound repair activity of human bronchial epithelial cells (HBEC) was investigated with antisense oligonucleotide (ASO) technique. The results showed that: (1) CTNNAL1 ASO could decelerate the repairing velocity and proliferation of HBEC; (2) CTNNAL1 expression was increased on the edge cells of mechanic wounded area in culture; (3) extracellular matrix component fibronectin (Fn) obviously promoted wound repair activity and proliferation of HBEC, which could be blocked by CTNNAL1 ASO; (4) Western blot showed that Fn could promote FAK phosphorylation, which also be inhibited by CTNNAL1 ASO. In conclusion, the level of CTNNAL1 mRNA expression is highly correlated to airway resistance; CTNNAL1 may contribute to the wound repair and proliferation of HBEC. Furthermore, it may serve to Fn mediated cell-extracellular adhesion and its signal transduction.

Endothelial Nitric Oxide Synthase Interaction with Actin Contributes to Mild Hyperoxia‐induced Endothelial Wound Repair.

We previously reported that endothelial nitric oxide synthase (eNOS) association with actin increases eNOS activity in pulmonary artery endothelial cells (PAEC). In the present study, PAEC were exposed to 40% O2 for 1-24 h after which eNOS activity and endothelial monolayer wound repair was measured, and eNOS/actin association was evaluated by using confocal microscopy and immunoprecipitation. We found that exposure of PAEC to 40% O2 for 24 h resulted in increases in endothelial monolayer wound repair and eNOS activity and caused partial translocation of eNOS to plasma membrane where it co-localizes with cortical F-actin. We did not detect eNOS co-localization with stress fibers in normoxic or hyperoxic PAEC. Immunoprecipitation study revealed that mild hyperoxia increased association of eNOS with actin in both Triton X-100-soluble and Triton X-100-insoluble fractions. Knockdown of actin in PAEC by actin siRNA reduced the actin level and eNOS/actin association, and thus attenuated hyperoxia-induced increase in eNOS activity and in endothelial monolayer wound repair. These results indicate that mild hyperoxia increases eNOS activity and eNOS/actin association and that increased eNOS activity due to eNOS/actin association may contribute to mild hyperoxia-induced wound repair of PAEC. Supported by Grants from: Florida DOH, FAMRI, AHAFL, ALAF, NIH, and VA

Platelet-Derived Growth Factor and Epidermal Growth Factor Play a Major Role in Human Colonic Fibroblast Repair Activities

Background and Aims: Ulceration is a common feature of inflammatory bowel diseases, where subepithelial cell growth is frequently necessary for resolution. In order to further understand the role of colonic fibroblasts in this process, we have used an in vitro model of wound repair to study the response of human colonic fibroblasts to several growth factors expressed in colonic tissues. Methods: Proliferation was determined by [³H]thymidine incorporation into DNA in subconfluent fibroblast cultures. In vitro wound repair was determined in confluent fibroblast monolayers after mechanical denudation. The presence of growth factors secreted by fibroblasts was studied in conditioned medium by heparin affinity chromatography and immunodetection with specific antibodies. Results: Serum and platelet-derived growth factor (PDGF-BB) induced a dramatic increase in both colonic fibroblast proliferation and closure of wounded cell monolayers. Epidermal growth factor (EGF) stimulated both fibroblast activities, but the effect was less potent. However, colonic fibroblasts did not respond to transforming growth factor-β₁. Conditioned medium stimulated fibroblast proliferation and wound repair activity, which was reverted by the addition of suramin. Furthermore, a PDGF-like factor was isolated from colonic fibroblast-conditioned medium. Conclusions: EGF and PDGF-BB promote human colonic fibroblast-dependent wound repair activities. Human colonic fibroblasts may exert an autocrine regulation via the production of growth factors.

Mitogenic whey extract stimulates wound repair activity in vitro and promotes healing of rat incisional wounds

The ability of single growth factors to promote healing of normal and compromised wounds has been well described, but wound healing is a process requiring the coordinated action of multiple growth factors. Only the synergistic effect on wound healing of combinations containing at most two individual growth factors has been reported. We sought to assess the ability of a novel milk-derived growth factor-enriched preparation ¿mitogenic bovine whey extract (MBWE), which contains six known growth factors, to promote repair processes in organotypic in vitro models and incisional wounds in vivo. MBWE stimulated the contraction of fibroblast-populated collagen lattices in a dose-dependent fashion and promoted the closure of excisional wounds in embryonic day 17 fetal rat skin. Application of MBWE increased incisional wound strength in normal animals on days 3, 5, 7, and 10 and reversed the decrease in wound strength observed following steroid treatment. Wound histology showed increased fibroblast numbers in wounds from normal and steroid-compromised animals. These data suggest the mixture of factors present in bovine milk exerts a direct action on the cells of cutaneous wound repair to enhance both normal and compromised healing.

Effects of extracellular pH on restitution and proliferation of rabbit oesophageal epithelial cells.

Exposure to luminal acid induces mucosal damage in the oesophagus. The extent and degree of interaction of acid with wound repair mechanisms of the oesophagus have not been explored. Rabbit oesophageal cells were isolated, cultured and exposed to different extracellular pHs for several periods of time. Cell mortality was studied in both isolated and primary cultures of these cells by cell flow cytometry using fluorochrome propidium iodide and LDH release. Subconfluent cell cultures were used to study proliferative responses determined by [3H]-thymidine incorporation into DNA. Restitution was studied in confluent monolayers wounded by mechanical denudation. Isolated cells were resistant to acid when exposed to pH > 2 for 15 min. Cell proliferation increased with small pH decreases (7-6.5) if the period of exposure was less than 6 h, but was inhibited with lower pHs and/or longer periods of acid exposure. However, restitution was gradually inhibited with further pH decreases (6.5-3) and time of acid exposure (0.5-24 h). The wound repair activities of oesophageal epithelial cells are deeply affected by even small decreases in pH. These findings may help to explain the need for profound acid inhibition in acid-related reflux oesophagitis.

In vitro wound repair by human gastric fibroblasts: implications for ulcer healing.

Fibroblasts modulate epithelial biological activities and play a key role in the ulcer healing process. There is no information regarding the biological response of human gastric fibroblasts to regulatory compounds. The aim of this study was to assess the effects of growth factors and prostaglandins on an in vitro model of human gastric fibroblast wound repair. Subconfluent fibroblast cultures were used to study proliferative responses, determined by [3H]thymidine incorporation into DNA. In vitro wound repair was determined in confluent fibroblast monolayers after mechanical denudation. The presence of putative growth factors secreted by fibroblasts was studied in conditioned medium by heparin-affinity chromatography and immunodetection with specific antibodies. Serum and platelet-derived growth factor (PDGF) -BB induced a dramatic increase in both gastric fibroblast proliferation and closure of wounded cell monolayers, whereas these activities were inhibited by both transforming growth factor (TGF) -beta1 and prostaglandin E1. Basal activities in unstimulated gastric fibroblasts were lower than those obtained in skin fibroblasts. Conditioned medium stimulated fibroblast proliferation and wound repair activity, which was inhibited by the addition of suramin, and was partially dependent on the presence of PDGF-like factor. PDGF is a major, autocrine promotor of human gastric fibroblast-dependent wound repair activities, which are inhibited by prostaglandins and TGF-beta. These findings might be important for future therapeutic ulcer healing approaches.

Effect of growth factors and prostaglandin E2 on restitution and proliferation of rabbit esophageal epithelial cells.

Factors and mechanisms involved in esophageal mucosal injury and repair are not well known. The aim of this study was to assess the effects of growth factors and prostaglandins on esophageal mucosal cell repair activities. Rabbit esophageal cells were isolated, cultured, and exposed to different growth factors and prostaglandin E2. Subconfluent cell cultures were used to study proliferative responses determined by [3H]thymidine incorporation into DNA. Restitution was studied in confluent monolayers wounded by mechanical denudation. Restitution was the main mechanism involved in wound repair within the first 24 hr. HGF, IGF-I, and EGF dose-dependently stimulated cell proliferation but did not affect restitution. TGF-beta1 inhibited both proliferation and restitution while PDGF-BB and prostaglandin E2 had no effect. Esophageal epithelial cell restitution and proliferation are affected by growth factors. HGF, IGF-I, EGF (stimulation), and TGF-beta1 (inhibition) are major growth factors affecting in vitro esophageal wound repair activities, which, unlike those of other areas of the digestive tract, are not affected by prostaglandins.