Investigations on the Wound Healing Potential of Tilapia Piscidin (TP)2-5 and TP2-6

Wound healing is a highly orchestrated process involving many cell types, such as keratinocytes, fibroblasts and endothelial cells. This study aimed to evaluate the potential application of synthetic peptides derived from tilapia piscidin (TP)2, TP2-5 and TP2-6 in skin wound healing. The treatment of HaCaT keratinocytes with TP2-5 and TP2-6 did not cause cytotoxicity, but did enhance cell proliferation and migration, which could be attributed to the activation of epidermal growth factor receptor signaling. In CCD-966SK fibroblasts, although TP2-5 (31.25 μg/mL) and TP2-6 (125 μg/mL) showed cytotoxic effects, we observed the significant promotion of cell proliferation and migration at low concentrations. In addition, collagen I, collagen III, and keratinocyte growth factor were upregulated by the peptides. We further found that TP2-5 and TP2-6 showed pro-angiogenic properties, including the enhancement of human umbilical vein endothelial cell (HUVEC) migration and the promotion of neovascularization. In a murine model, wounds treated topically with TP2-5 and TP2-6 were reduced by day 2 post-injury and healed significantly faster than untreated wounds. Taken together, these findings demonstrate that both TP2-5 and TP2-6 have multifaceted effects when used as topical agents for accelerating wound healing.     

Development and Characterization of VEGF165-Chitosan Nanoparticles for the Treatment of Radiation-Induced Skin Injury in Rats

Radiation-induced skin injury, which remains a serious concern in radiation therapy, is currently believed to be the result of vascular endothelial cell injury and apoptosis. Here, we established a model of acute radiation-induced skin injury and compared the effect of different vascular growth factors on skin healing by observing the changes of microcirculation and cell apoptosis. Vascular endothelial growth factor (VEGF) was more effective at inhibiting apoptosis and preventing injury progression than other factors. A new strategy for improving the bioavailability of vascular growth factors was developed by loading VEGF with chitosan nanoparticles. The VEGF-chitosan nanoparticles showed a protective effect on vascular endothelial cells, improved the local microcirculation, and delayed the development of radioactive skin damage.     

An Electrospun Scaffold Loaded with an Enteromorpha Polysaccharide for Accelerated Wound Healing in Diabetic Mice

The design and development of innovative multifunctional wound dressing materials in engineered biomaterials is essential for promoting tissue repair. In this study, nanofibrous wound dressing materials loaded with anti-inflammatory ingredients were manufactured by a promising electrospinning strategy, and their capability for treating diabetic wounds was also investigated. A scaffold blend consisting of an Enteromorpha polysaccharide and polyvinyl alcohol (PVA) was fabricated. The in vitro and in vivo studies confirmed the efficacy of PVA/EPP1 fiber. We found that PVA/EPP1 fiber accelerated the repair of a full-thickness skin wound in diabetic mice. The results suggest that this scaffold could effectively shorten the wound healing time by inhibiting inflammatory activity, which makes it a promising candidate for the treatment of hard-to-heal wounds caused by diabetes.     

Proteomic analysis of anti-tumor effects of 11-dehydrosinulariolide on CAL-27 cells

The anti-tumor effects of 11-dehydrosinulariolide, an active ingredient isolated from soft coral Sinularia leptoclados, on CAL-27 cells were investigated in this study. In the MTT assay for cell proliferation, increasing concentrations of 11-dehydrosinulariolide decreased CAL-27 cell viability. When a concentration of 1.5 μg/mL of 11-dehydrosinulariolide was applied, the CAL-27 cells viability was reduced to a level of 70% of the control sample. The wound healing function decreased as the concentration of 11-dehydrosinulariolide increased. The results in this study indicated that treatment with 11-dehydrosinulariolide for 6 h significantly induced both early and late apoptosis of CAL-27 cells, observed by flow cytometric measurement and microscopic fluorescent observation. A comparative proteomic analysis was conducted to investigate the effects of 11-dehydrosinulariolide on CAL-27 cells at the molecular level by comparison between the protein profiling (revealed on a 2-DE map) of CAL-27 cells treated with 11-dehydrosinulariolide and that of CAL-27 cells without the treatment. A total of 28 differential proteins (12 up-regulated and 16 down-regulated) in CAL-27 cells treated with 11-dehydrosinulariolide have been identified by LC-MS/MS analysis. Some of the differential proteins are associated with cell proliferation, apoptosis, protein synthesis, protein folding, and energy metabolism. The results of this study provided clues for the investigation of biochemical mechanisms of the anti-tumor effects of 11-dehydrosinulariolide on CAL-27 cells and could be valuable information for drug development and progression monitoring of oral squamous cell carcinoma (OSCC).     

Enzyme-Digested Peptides Derived from Lates calcarifer Enhance Wound Healing after Surgical Incision in a Murine Model

Surgical wounds are common injuries of skin and tissues and usually become a clinical problem. Until now, various synthetic and natural peptides have been widely explored as potential drug candidates for wound healing. Inhibition of the TNF-α signaling pathway and promotion of angiogenesis are suggested to be involved in their effects. Angiogenesis at the wound site is one of the essential requisites for rapid healing. In the present study, a novel peptide extract derived from the natural source Lates calcarifer, commonly known as sea bass or barramundi, was evaluated for its wound healing property. The specific acidic and enzymatic approaches were employed for producing sea bass extract containing small size peptides (molecular weight ranging from 1 kD to 5 kD). The cytotoxicity of the extract was examined in HaCaT and NIH3T3. After this, the effects of enzyme digested peptide extracts of sea bass on wound healing in mice were investigated. The peptide extracts (660 and 1320 mg/kg/day) and control protein (1320 mg/kg/day) was orally given to the wounded mice, respectively, for 12 days. The surgical method was improved by implanting a silicone ring at the wound site. The ring avoided the contracting effect in murine wounds, making it more closely related to a clinical condition. The results showed promising improvement at the wound site in mice. Sea bass peptide extracts accelerated the wound healing process and enhanced the microvessel formation at the wound site. The remarkable effects of this novel sea bass peptide extract in healing traumatic injuries revealed a new option for developing wound management.     

Collagen-Containing Fish Sidestream-Derived Protein Hydrolysates Support Skin Repair via Chemokine Induction

Restoring homeostasis following tissue damage requires a dynamic and tightly orchestrated sequence of molecular and cellular events that ensure repair and healing. It is well established that nutrition directly affects skin homeostasis, while malnutrition causes impaired tissue healing. In this study, we utilized fish sidestream-derived protein hydrolysates including fish collagen as dietary supplements, and investigated their effect on the skin repair process using a murine model of cutaneous wound healing. We explored potential differences in wound closure and histological morphology between diet groups, and analyzed the expression and production of factors that participate in different stages of the repair process. Dietary supplementation with fish sidestream-derived collagen alone (Collagen), or in combination with a protein hydrolysate derived from salmon heads (HSH), resulted in accelerated healing. Chemical analysis of the tested extracts revealed that Collagen had the highest protein content and that HSH contained the great amount of zinc, known to support immune responses. Indeed, tissues from mice fed with collagen-containing supplements exhibited an increase in the expression levels of chemokines, important for the recruitment of immune cells into the damaged wound region. Moreover, expression of a potent angiogenic factor, vascular endothelial growth factor-A (VEGF-A), was elevated followed by enhanced collagen deposition. Our findings suggest that a 5%-supplemented diet with marine collagen-enriched supplements promotes tissue repair in the model of cutaneous wound healing, proposing a novel health-promoting use of fish sidestreams.     

A Marine-Derived Anti-Inflammatory Scaffold for Accelerating Skin Repair in Diabetic Mice

Reconstructing the typical analogue of extracellular matrix (ECM) in engineered biomaterials is essential for promoting tissue repair. Here, we report an ECM-mimetic scaffold that successfully accelerated wound healing through enhancing vascularization and regulating inflammation. We prepared an electrospun fiber comprising a brown alga-derived polysaccharide (BAP) and polyvinyl alcohol (PVA). The two polymers in concert exerted the function upon the application of PVA/BAP2 fiber in vivo; it started to reduce the inflammation and promote angiogenesis at the wound site. Our serial in vitro and in vivo tests validated the efficacy of PVA/BAP2 fiber. Particularly, PVA/BAP2 fiber accelerated the repair of a full-thickness skin wound in diabetic mice and induced optimal neo-tissue formation. Generally, our results suggest that, by mimicking the function of ECM, this fiber as an engineered biomaterial can effectively promote the healing efficiency of diabetic wounds. Our investigation may inspire the development of new, effective, and safer marine-derived scaffold for tissue regeneration.     

Mycosporine-Like Amino Acids Promote Wound Healing through Focal Adhesion Kinase (FAK) and Mitogen-Activated Protein Kinases (MAP Kinases) Signaling Pathway in Keratinocytes

Mycosporine-like amino acids (MAAs) are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS). In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH), Mycosporine-glycine (M-Gly), and Porphyra (P334) were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK), extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK). These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies.     

Oral administration of skin gelatin isolated from Chum salmon (Oncorhynchus keta) enhances wound healing in diabetic rats

Care for diabetic wounds remains a significant clinical problem. The present study was aimed at investigating the effect of skin gelatin from Chum Salmon on defective wound repair in the skin of diabetic rats. Full-thickness excisional skin wounds were made in 48 rats, of which 32 were diabetes. The diabetic rats were orally treated daily for 14 days with skin gelatin from Chum Salmon (2 g/kg) or its vehicle. Sixteen non-diabetic control rats received the same amount of water as vehicle-treated non-diabetic rats. Rats were killed to assess the rate of wound closure, microvessel density (MVD), vascular endothelial growth factor (VEGF), hydroxyproline (HP) contents in wound tissues and nitrate in plasma and wound tissue at 7 and 14 days after wounding. Skin gelatin-treated diabetic rats showed a better wound closure, increased MVD, VEGF, hydroxyproline and NO contents and a reduced extent of inflammatory response. All parameters were significant (P < 0.05) in comparison to vehicle-treated diabetic group. In light of our finding that skin gelatin of Chum Salmon promotes skin wound repair in diabetic rats, we propose that oral administration of Chum Salmon skin gelatin might be a beneficial method for treating wound disorders associated with diabetes.     

Topical formulations of water-soluble chitin as a wound healing assistant

Water-soluble chitin (WSC) was prepared by carefully deacetylating chitins to about 50% of N-acetyl content. Topical formulations based on WSC were prepared and their effects on wound healing were evaluated on a rabbit ear model. Full-thickness, open skin wounds were made on the ears of rabbits and WSC ointments were embedded in the open wounds. The application of WSC ointments significantly accelerated wound healing and wound contraction. The areas of epithelialization and granulation tissues in WSC ointment group are remarkably larger than those in control group (no treatment) and in placebo group (treated with ointment-base materials). A large number of grown granulation tissues including dense fibroblast deposition were observed under the thickened epithelium of the wound treated with WSC ointments. The number of inflammatory cells in WSC ointment group was significantly decreased compared with those in control and placebo groups, indicating that WSC would give low stimuli to wounds and prevent excessive scar formation. Neovascularization was the most prominent in WSC ointment group. Wound contraction in WSC ointment group was much larger than those in control and placebo groups. Overall results demonstrate that the topical formulation based on WSC is considered to become an excellent dressing as a wound healing assistant.     

Tilapia Piscidin 4 (TP4) Stimulates Cell Proliferation and Wound Closure in MRSA-Infected Wounds in Mice

Antimicrobial peptides (AMPs) are endogenous antibiotics that directly affect microorganisms, and also have a variety of receptor-mediated functions. One such AMP, Tilapia piscidin 4 (TP4), was isolated from Nile tilapia (Oreochromis niloticus); TP4 has antibacterial effects and regulates the innate immune system. The aim of the present study was to characterize the role of TP4 in the regulation of wound closure in mice and proliferation of a keratinocyte cell line (HaCaT) and fibroblast cell line (Hs-68). In vitro, TP4 stimulated cell proliferation and activated collagen I, collagen III, and keratinocyte growth factor (KGF) gene expression in Hs-68 cells, which induces keratin production by HaCaT cells. This effect was detectable at TP4 concentrations of 6.25 µg/mL in both cell lines. In vivo, TP4 was found to be highly effective at combating peritonitis and wound infection caused by MRSA in mouse models, without inducing adverse behavioral effects or liver or kidney toxicity. Taken together, our results indicate that TP4 enhances the survival rate of mice infected with the bacterial pathogen MRSA through both antimicrobial and wound closure activities mediated by epidermal growth factor (EGF), transforming growth factor (TGF), and vascular endothelial growth factor (VEGF). The peptide is likely involved in antibacterial processes and regulation of tissue homeostasis in infected wounds in mice. Overall, these results suggest that TP4 may be suitable for development as a novel topical agent for wound dressing.     

Characterization of Isoforms of the Lectin Isolated from the Red Algae Bryothamnion seaforthii and Its Pro-Healing Effect

Lectins are a structurally heterogeneous group of proteins that have specific binding sites for carbohydrates and glycoconjugates. Because of their biotechnological potential, lectins are widely used in biomedical research. The present study aimed to evaluate the healing potential of the lectin isolated from the marine red alga Bryothamnion seaforthii (BSL). The lectin was purified using ion exchange chromatography with DEAE cellulose and characterized using tandem mass spectrometry. For healing tests, skin wounds were induced in the dorsal thoracic region of mice. These animals were randomly divided into three groups and subjected to topical treatment for 12 days with BSL, bovine serum albumin and 150 mM NaCl. To evaluate the potential of each treatment, the animals were anesthetized and sacrificed on days 2, 7 and 12, respectively. The parameters evaluated included the wound area, the proportion of wound closure and the histological diagnosis. The wound closure was more effective with BSL (Postoperative Day 7 and 12) than controls. The luminal epithelium was completely restructured; the presence of collagen in the dermis and the strongly active presence of young skin annexes demonstrate the potential of treatment with BSL compared with controls. Our findings suggest that BSL has pro-healing properties and can be a potential medical process in the treatment of acute wounds.     

Effect of fertilisation, development stage, mechanical impact and heating during processing on subsurface hardening in pre-peeled potatoes (Solarium tuberosum L.)

Summary Studies of the effect of fertilisation stress, harvest time, machine sorting, storage time, peeling and blanching of potatoes (Solarium tuberosum L.) were carried out. Mechanical impact by sorting before storage did not have a significant effect on hardness of pre-peeled potatoes. Potato hardness decreased significantly with the storage time for raw potatoes following an exponential function. Treatment of potatoes in a damage drum for 5 minutes (36 drops from 35 cm) increased the subsurface hardening, production of brick-like cells and deposition of suberin. Potato hardness throughout the whole potatoes increased with blanching time by blanching at 85 and 95 °C. It was concluded that development of potato hardiness occur because of two mechanisms. That is: 1) wound healing by periderm formation with deposition of suberin and: 2) activation of methyl esterase activity by blanching and subsequent crosslinking of demethoxylated pectin by calcium ions, respectively.     

Preparation of Ion2+-COS/SA Multifunctional Gel Films for Skin Wound Healing by an In Situ Spray Method

The rapid preparation of safe and efficient wound dressings that meet the needs of the entire repair process remains a major challenge for effective therapeutic wound healing. Natural, sprayable Ion²⁺-COS/SA multifunctional dual-network gel films created by the in situ coordination of chitooligosaccharide (COS), metal ions and sodium alginate (SA) using casting and an in-situ spray method were synthesized. The gel films exhibited excellent physicochemical properties such as swelling, porosity and plasticity at a COS mass fraction of 3%. Furthermore, at this mass fraction, the addition of bimetallic ions led to the display of multifunctional properties, including significant antioxidant, antibacterial and cytocompatibility properties. In addition, experiments in a total skin defect model showed that this multifunctional gel film accelerates wound healing and promotes skin regeneration. These results suggest that the sprayable Ion²⁺-COS/SA multifunctional pro-healing gel film may be a promising candidate for the clinical treatment of allodermic wounds.     

Effect of Bone Marrow Mesenchymal Stem Cell Sheets on Skin Capillary Parameters in a diabetic wound model: A Novel Preliminary Study

Background:Treatment with BMMSCs has anti-inflammatory, tissue regenerative, angiogenic, and immune-stimulating effects. When using as sheets or accumulate, BMMSCs causes the development of neoangiogenesis in damaged skin tissue. Diabetes, a metabolic disorder, can negatively affect many physiological functions, including the process of skin injury repair. This adverse impact may increase the risk of skin surgery. RSF is commonly used in reconstructive surgery. The terminal part of the RSF is often affected by necrosis because of impaired blood flow, which is exacerbated in diabetes. This study investigated the effect of stem cells, applied as accumulated or cell sheets, along with RSF surgery on skin capillaries in STZ-induced diabetic rats. Methods:Thirty male Wistar rats were divided into three groups (n = 10): diabetes-RSF control, diabetes-RSF local applied stem cells (loc-BMMSCs), diabetes-RSF applied stem cells as accumulated or cell sheets (ac-BMMSCs). Two weeks after the STZ injection, RSF surgery and stem cell therapy (6 × 10⁹) were carried out (day zero). Furthermore, stereological methods were used to investigate the capillary patterns among the groups. Anti-CD31/PCAM1 immunohistochemistry was also used for further confirmation of changes in capillary parameters. Results:The results demonstrated that capillaries were protected by MSC sheets in the flap tissue, and the thickness of the epidermal layer was improved, indicationg the possible beneficial effects of MSC sheets on diabetic wound treatment. Conclusion:Stem cells, as ac-BMMSCs, may decrease the levels of wound healing complications in diabetes and can be considered as a cell therapy option in such conditions.Key Words: Neoangiogenesis, Skin flap, Transplantation, Wound healing     

Immunocytochemical study on microtubule reorganization in HL-60 leukemia cells undergoing apoptosis

BACKGROUND: Microtubules (MT) are important components of cell cytoskeleton and play key roles in cell motility mitosis and meiosis. They are also the targets of several anticancer agents which indicating their importance in maintaining cell viability. Microtubular reorganization contributing to apoptotic morphology occurs in normal and neoplastic cells undergoing apoptosis induced by cytotoxic drugs. The aim of this study was to correlate the changes in the MT with behavior of the gamma-tubulin in apoptotic cell, and to see if apoptitic MT showed biochemical characteristics of stable MT. METHODS: Apoptosis was induced in the human leukemia cells (HL-60) by treatment with 1 microM of all-trans retinoic acid over a 5-day period. The time course of changes was assessed using flow cytometry, DNA fragmentation and immunocytochemistry in cells labeled for alpha-tubulins, acetylated alpha-tubulin and gamma-tubulin. RESULTS: The results indicated that gamma-tubulin content is increased after cells have gone through the apoptosis with a diffuse cytoplasmic pattern. Alpha-tubulin did not reveal any specific pattern of polymerization in apoptotic cells and acetylated alpha-tubulin content was also decreased in comparison with non-apoptotic cells. CONCLUSION: Our results support the idea that microtubule reorganization is an important factor of the mammalian cells response to apoptosis, and the altered properties of the MT did not reflect changes in function as apoptosis progresses.     

Xyloketal B Suppresses Glioblastoma Cell Proliferation and Migration in Vitro through Inhibiting TRPM7-Regulated PI3K/Akt and MEK/ERK Signaling Pathways

Glioblastoma, the most common and aggressive type of brain tumors, has devastatingly proliferative and invasive characteristics. The need for finding a novel and specific drug target is urgent as the current approaches have limited therapeutic effects in treating glioblastoma. Xyloketal B is a marine compound obtained from mangrove fungus Xylaria sp. (No. 2508) from the South China Sea, and has displayed antioxidant activity and protective effects on endothelial and neuronal oxidative injuries. In this study, we used a glioblastoma U251 cell line to (1) explore the effects of xyloketal B on cell viability, proliferation, and migration; and (2) investigate the underlying molecular mechanisms and signaling pathways. MTT assay, colony formation, wound healing, western blot, and patch clamp techniques were employed. We found that xyloketal B reduced cell viability, proliferation, and migration of U251 cells. In addition, xyloketal B decreased p-Akt and p-ERK1/2 protein expressions. Furthermore, xyloketal B blocked TRPM7 currents in HEK-293 cells overexpressing TRPM7. These effects were confirmed by using a TRPM7 inhibitor, carvacrol, in a parallel experiment. Our findings indicate that TRPM7-regulated PI3K/Akt and MEK/ERK signaling is involved in anti-proliferation and migration effects of xyloketal B on U251 cells, providing in vitro evidence for the marine compound xyloketal B to be a potential drug for treating glioblastoma.     

Induction of Apoptosis and Antitumor Activity of Eel Skin Mucus,Containing Lactose-Binding Molecules,on Human Leukemic K562 Cells

For innate immune defense, lower animals such as fish and amphibian are covered with skin mucus, which acts as both a mechanical and biochemical barrier. Although several mucus sources have been isolated and studied for their biochemical and immunological functions, the precise mechanism(s) of action remains unknown. In the present study, we additionally found the eel skin mucus (ESM) to be a promising candidate for use in anti-tumor therapy. Our results showed that the viability of K562 cells was decreased in a dose-dependent manner by treatment with the isolated ESM. The cleaved forms of caspase-9, caspase-3 and poly adenosine diphosphate-ribose polymerase were increased by ESM. The levels of Bax expression and released cytochrome C were also increased after treatment with ESM. Furthermore, during the ESM mediated-apoptosis, phosphorylation levels of ERK1/2 and p38 but not JNK were increased and cell viabilities of the co-treated cells with ESM and inhibitors of ERK 1/2 or p38 were also increased. In addition, treatment with lactose rescued the ESM-mediated decrease in cell viability, indicating lactose-containing glycans in the leukemia cells acted as a counterpart of the ESM for interaction. Taken together, these results suggest that ESM could induce mitochondria-mediated apoptosis through membrane interaction of the K562 human leukemia cells. To the best of our knowledge, this is the first observation that ESM has anti-tumor activity in human cells.     

Surface modification of silk fibroin nanofibrous mat with dextran for wound dressing

In this study, we examined the effects of a dextran-modified silk fibroin nanofibrous mat (D-SFNM) on wound healing. To increase the hydrophilicity of silk fibroin (SF), the SF nanofibrous mat (SFNM) was modified with oxidized dextran. The D-SFNM absorbed water faster than the SFNM, and the swelling ratio was increased by approximately 80 % compared with the SFNM. An in vitro cell (NIH3T3) test revealed that fewer cells attached to the D-SFNM than the SFNM, but the proliferation of cells was not significantly affected by the presence of dextran. An in vivo wound healing test with mice indicated that the D-SFNM resulted in a good wound recovery effect similar to a commercial wound dressing material. The increased hydrophilicity of the D-SFNM might balance the moist environment at the wound site, which improves the wound healing compared with the SFNM.     

Research Progress of Chitosan-Based Biomimetic Materials

Chitosan is a linear polysaccharide produced by deacetylation of natural biopolymer chitin. Owing to its good biocompatibility and biodegradability, non-toxicity, and easy processing, it has been widely used in many fields. After billions of years of survival of the fittest, many organisms have already evolved a nearly perfect structure. This paper reviews the research status of biomimetic functional materials that use chitosan as a matrix material to mimic the biological characteristics of bivalves, biological cell matrices, desert beetles, and honeycomb structure of bees. In addition, the application of biomimetic materials in wound healing, hemostasis, drug delivery, and smart materials is briefly overviewed according to their characteristics of adhesion, hemostasis, release, and adsorption. It also discusses prospects for their application and provides a reference for further research and development.