Effects of different sterilization methods on the morphology, mechanical properties, and cytotoxicity of chitosan membranes used as wound dressings

The aim of this work was to compare the effects induced by two different sterilization methods (exposure to gamma radiation or ethylene oxide) and an antiseptic technique (immersion in 70% ethanol aqueous solution) on the morphology, tensile strength, percentage of strain at break, and in vitro cytotoxicity to Vero cells on chitosan membranes designed for wound healing. Four distinct membrane compositions were evaluated, with chitosan, glycerol, and chitin used as components. Gamma radiation, in spite of being one of the most commonly employed sterilizing agents, negatively affected the morphology of membranes composed solely by chitosan as well as the percentage of strain at break of the chitosan-membranes containing glycerol on their composition. Moreover, its use affected the color of the chitosan membranes. The use of 70% ethanol aqueous solution did not change the chitosan-membrane characteristics significantly, but its use has limitations concerning the process scale up. With ethylene oxide (EtO), chitosan-membrane morphology, percentage of strain at break, and in vitro cytotoxicity to Vero cells were not significantly affected. The tensile strength of the membranes containing chitin were reduced after the treatment with ethylene oxide; however, the obtained values were comprised in the range verified for normal human skin. Therefore, because the final characteristics of the membranes treated with ethylene oxide are appropriate when considering their use as wound healing devices, and because this sterilization process is easily adjusted to use on an industrial scale, EtO can be considered the most adequate sterilizing agent for chitosan membranes. However, it should be considered that this chemical is associated with toxicity, flammability, and environmental risks, as well as with possible material contamination with ethylene oxide residues.     

Preparation of water-soluble chitosan/heparin complex and its application as wound healing accelerator

To make effective wound healing accelerator, water-soluble chitosan (WSC)/heparin (CH) complex was prepared using WSC with wound healing ability and heparin with ability to attract or bind growth factor related to wound healing process. Water-soluble CH complex was prepared by the reaction between WSC and heparin, and then, by adding distilled water to it, ointment type with high viscosity was made. To evaluate the wound healing effect, full thickness skin excision was performed on the backs of the rat and then WSC and water-soluble CH complex ointments were applied in the wounds, respectively. After 15 days, gross and histologic examination was performed. Grossly, untreated control group revealed that the wound had well defined margin and was covered by crust. The second group treated with WSC ointment revealed small wound size with less amount of covering crust and ill-defined margin, which appeared to regenerate from margin. The third group treated with water-soluble CH complex ointment appeared to be nearly completely healed. Histology of each group was well correlated to gross findings. The third group shows nearly complete regeneration of appendage structure similar to normal in the dermis in contrast to control and second group with absence and less number of skin appendages, respectively.     

Fabrication and characterization of DTBP-crosslinked chitosan scaffolds for skin tissue engineering

Chitosan, the deacetylated derivative of chitin, is a promising scaffold material for skin tissue engineering applications. It is biocompatible and biodegradable, and the degradation products are resorbable. However, the rapid degradation of chitosan and its low mechanical strength are concerns that may limit its use. In this study, chitosan with 80%, 90% and 100% degree of deacetylation (DDA) was crosslinked with dimethyl 3-3, dithio bis' propionimidate (DTBP) and compared to uncrosslinked scaffolds. The scaffolds were characterized with respect to important tissue engineering properties. The tensile strength of scaffolds made from 100% DDA chitosan was significantly higher than for scaffolds made from 80% and 90% DDA chitosan. Crosslinking of scaffolds with DTBP increased the tensile strength. Crosslinking with DTBP had no significant effect on water vapour transmission rate (WVTR) or water absorption but had significant effect on the pore size and porosity of the samples. All samples showed a WVTR and pore size distribution suitable for skin tissue engineering; however, the water absorption and porosity were lower than the optimal values for skin tissue engineering. The biodegradation rate of scaffolds crosslinked with DTBP and glutaraldehyde (GTA) were reduced while no significant effect was observed in biodegradation of the samples made from 100% DDA chitosan whether crosslinked or uncrosslinked after 24 days of degradation.     

Growth factors bFGF and TGB beta accelerate the rate of wound repair in normal and in diabetic rats

The effects of basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGF beta) on the rate of wound repair in both normal and streptozotocin-induced diabetic rats were investigated using two model systems of wound repair, namely incisional wounding and subcutaneous implantation of polyvinyl alcohol (PVA) sponges. Both models showed the expected wound-healing defects of the diabetic rats. Granulation tissue collected from the implanted PVA sponges showed that the diabetic rats had reduced amounts of collagen, DNA and protein present at the wound site at two time points tested (7 and 9 days post-implantation). Fresh tensile strength of the incisional wounds, a measure of the collagen organization in a wound, was reduced to 53% of normal in diabetic rats on day 7 post-wounding, and was only 29% of normal by day 21. Formalin-fixed tensile strength, a measure of collagen content of the wound, was 41% of normal on day 7, and 78% of normal by day 21, giving evidence that while the collagen concentration of the diabetic wounds approached that of normal wounds, it did not undergo the normal maturation process. A single injection of 2 micrograms of TGF beta directly into the incision three days after wounding resulted in little difference in the fresh and fixed tensile strength of diabetic wounds when tested at 7, 14 and 21 days post-wounding. Normal rats, however, responded well, resulting in a greater than 30% increase in both fresh and fixed tensile strength.(ABSTRACT TRUNCATED AT 250 WORDS)     

Wound healing of 6.45-microm free electron laser skin incisions with heat-conducting templates

We have previously shown a reduction in lateral thermal damage with acute studies of skin incisions made in vitro using heat-conducting templates. Here we examined the wound-healing response to laser incisions with heat-conducting templates and explored the use of an optically transparent template with the free electron laser (FEL) at 6.45 microm. First we evaluated the effects of a sapphire heat-conducting template on the lateral thermal damage of FEL incisions using in vitro human skin samples. Next we compared wound tensile strength and histological scoring of the healing of incisions created on the dorsal pelts of live rats with the FEL utilizing metal and sapphire heat-conducting templates and scalpel incisions. The animals were euthanized and the wounds were analyzed at postoperative days 7, 14, and 21. The depth and lateral thermal damage of FEL incisions on in vitro human skin were significantly reduced with the sapphire heat-conducting template. Nonstatistically significant differences in wound tensile strengths and histological scoring of wound healing were noted at days 7 and 14. By day 21, all of the incisions appeared similar. When the data from days 7 and 14 were combined, statistically significant differences were found for each of the templates (except the histological evaluation with the aluminum template) and the scalpel compared with laser incisions made without using a template. The use of metal or sapphire heat-conducting templates reduced the wound-healing delay of laser incisions seen at postoperative days 7 and 14.     

The effects of ovariectomy on the mechanical properties of skin in rats

OBJECTIVE: After menopause, observable changes occur in the physical characteristics of the human skin. These changes and their responses to various treatments can be assessed with non-invasive in-vivo mechanical tests. However, tests measuring breaking strength and tensile strengths can only be done ex-vivo, they require relatively higher quantities of skin and thus have generally been performed on animals. Mechanical changes in the skin of ovariectomized rats, an appropriate model for the study of postmenopausal period, have not been dealt with in the literature. In this study mechanical characteristics of the skin, such as breaking strength and tensile strength have been tested and studied histologically in ovariectomized rats. METHODS: Sixteen rats were divided into two groups, one undergoing ovariectomy and one control group undergoing a sham operation. Three months later, the rats were sacrificed and tensile properties of their back skins were tested with a tensometer and evaluated histologically. RESULTS AND CONCLUSION: Breaking strength, tensile strength and the Young's modulus have increased and the thickness of the subcutis has decreased in ovariectomized rats. This study should be tested by others, because of existence of some conflicts between available knowledge and the results, relating to postmenopausal skin changes.     

Electrospinning of collagen nanofibers: effects on the behavior of normal human keratinocytes and early-stage wound healing

Electrospinning of type I collagen in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to fabricate a biomimetic nanofibrous extracellular matrix for tissue engineering was investigated. The average diameter of collagen nanofibers electrospun from 8% collagen solution in HFIP was 460 nm (range of 100-1200 nm). The as-spun collagen nanofibrous matrix was chemically cross-linked by glutaraldehyde vapor with a saturated aqueous solution and then treated with aqueous 0.1m glycine to block unreacted aldehyde groups. With vapor phase cross-linking for 12h, porosity of the collagen matrix decreased from 89% to 71%. The collagen nanofibrous matrix showed good tensile strength, even in aqueous solution. Effects on cytocompatibility, cell behavior, cell and collagen nanofiber interactions, and open wound healing in rats were examined. Relatively low cell adhesion was observed on uncoated collagen nanofibers, whereas collagen nanofibrous matrices treated with type I collagen or laminin were functionally active in responses in normal human keratinocytes. Collagen nanofibrous matrices were very effective as wound-healing accelerators in early-stage wound healing. Our results indicate that cross-linked collagen nanofibers coated with ECM proteins, particularly type I collagen, may be a good candidate for biomedical applications, such as wound dressing and scaffolds for tissue engineering.     

Photocrosslinkable chitosan as a dressing for wound occlusion and accelerator in healing process.

Application of ultraviolet light (UV-) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution resulted in an insoluble, flexible hydrogel like soft rubber within 60 s. The chitosan hydrogel could completely stop bleeding from a cut mouse tail within 30 s of UV-irradiation and could firmly adhere two pieces of sliced skins of mouse to each other. In order to evaluate its accelerating effect on wound healing, full thickness-skin incisions were made on the back of mice and subsequently an Az-CH-LA aqueous solution was added into the wound and irradiated with UV light for 90 s. Application of the chitosan hydrogel significantly induced wound contraction and accelerated wound closure and healing. Histological examinations also have demonstrated an advanced granulation tissue formation and epithelialization in the chitosan hydrogel treated wounds. The chitosan hydrogel due to its accelerating healing ability is considered to become an excellent dressing for wound occlusion and tissue adhesive in urgent hemostasis situations.     

Effect of high molecular weight water-soluble chitosan on the trabecular bone and thickness in ovariectomized rats

High molecular weight water-soluble chitosan(WSC), having an average molecular weight of 300,000 Da and a degree of deacethylation over 90%, can be produced using a simple multi-step membrane separation process. In this study, the trabecular bone area and thickness in ovariectomized(OVX) rats decreased by almost 50% from those in sham-operated rats. WSC was evaluated for inhibition of the progress of bone loss induced by OVX rats. We measured bone histomorphometry in sham, OVX or WSC-administered OVX rats. From light microscopic analyses, a porous or erosive appearances were observed on the surface of trabecular bone of tibia in OVX rats, whereas those of the same bone in sham-operated rats were composed of fine particles. The trabecular bone area and trabecular thickness in OVX rats decreased by 50% from those in sham rats, these decreases were completely inhibited by administration of WSC at a concentration of 15 mg/kg/daily for 7 weeks. In this study, the mechanical strength in femur neck was significantly enhanced by the treatment of WSC for 7 weeks. In OVX rats, free T(3) was normal in all cases, whereas free T(4) was significantly increased. Although there was no difference between OVX and WSC-administered rats in T(3) level, we have found significant difference between them in T(4) level. These results strongly suggest that WSC is effective in preventing the development of bone loss induced by OVX in rats.     

The chitosan prepared from crab tendon I: the characterization and the mechanical properties

Crystalline chitosan was prepared from crab tendon consisting mainly of chitin, including various proteins and calcium phosphates. The crab tendon has high mechanical properties due to its aligned molecular structure. Crab tendon components, i.e. proteins and calcium phosphates, were removed by deacetyl treatment using 50wt% NaOH aqueous solution at 100 degrees C, and a subsequent ethanol treatment. As judged from microscopic observations using an optical polarizer, the treated chitosan remained intact regarding its aligned molecular structure, and had a high tensile strength of 67.9+/-11.4MPa. The tensile strength was further enhanced to 235+/-30MPa by a thermal treatment at 120 degrees C, corresponding to the formation of the intermolecular hydrogen bonds.     

Physico-mechanical properties of wound dressing material and its biomedical application

A bioadhesive wound dressing material, based on gelatin, was prepared by solution casting, and its properties were evaluated. The tensile strength (TS) and percentage elongation at break (Eb) of the membranes were found to be 12.7 MPa and 40.4%, respectively. The buffer uptake and water uptake of the prepared membranes were found to be 298 and 312%, respectively, after 8 min. A scanning electron micrograph of the membrane revealed its uniform porosity, which is an essential criterion to be an ideal wound dressing. From microbial sensitivity analysis, it was found that the membrane had a significant resistance against infection. The wound-healing characteristics of the membrane were evaluated using a rat (Rattus norvegicus) model. Full-thickness wounds were created on the ventral side of the Rattus norvegicus and were dressed with the membrane; eco-plast was used as a control. The wound healing and bioadhesion were monitored at 3-day intervals by real-time imaging. The results revealed that the prepared membrane was more effective in healing the wound than conventional wound dressing.     

Flexible chitin films as potential wound-dressing materials: wound model studies

Chitin films possessing increased flexibility, softness, transparency, and conformability have been prepared. These attributes enable the potential application of chitin films as occlusive, semipermeable film wound dressings similar to commercial products such as Opsite trade mark. The chitin films are generally nonabsorbent, exhibiting a total weight gain of only up to 120-160% in physiological fluid. Dry chitin films transpire water vapor at a rate of about 600 g/m(2)/24 h, similar to commercial polyurethane-based film dressings, but rises to 2400 g/m(2)/24 h, when wet, which is higher than the water vapor transmission rate of intact skin. The chitin films are nontoxic to human skin fibroblasts, maintaining 70-80% cell viability. Wound studies using a rat model showed no signs of allergenicity or the high inflammatory response associated with biodegradable biomaterials. The chitin films displayed accelerated wound-healing properties. Based on histological examination, wound sites dressed with the chitin films stabilized and healed faster, and appeared stronger than those dressed with Opsite trade mark and gauze dressings after 7 days of healing.     

Effect of High Molecular Weight Water-Soluble Chitosan on the Trabecular Bone and Thickness in Ovariectomized Rats

High molecular weight water-soluble chitosan(WSC), having an average molecular weight of 300,000 Da and a degree of deacethylation over 90%, can be produced using a simple multi-step membrane separation process. In this study, the trabecular bone area and thickness in ovariectomized(OVX) rats decreased by almost 50% from those in sham-operated rats. WSC was evaluated for inhibition of the progress of bone loss induced by OVX rats. We measured bone histomorphometry in sham, OVX or WSC-administered OVX rats. From light microscopic analyses, a porous or erosive appearances were observed on the surface of trabecular bone of tibia in OVX rats, whereas those of the same bone in sham-operated rats were composed of fine particles. The trabecular bone area and trabecular thickness in OVX rats decreased by 50% from those in sham rats, these decreases were completely inhibited by administration of WSC at a concentration of 15 mg/kg/daily for 7 weeks. In this study, the mechanical strength in femur neck was significantly enhanced by the treatment of WSC for 7 weeks. In OVX rats, free T₃ was normal in all cases, whereas free T₄ was significantly increased. Although there was no difference between OVX and WSC-administered rats in T₃ level, we have found significant difference between them in T₄ level. These results strongly suggest that WSC is effective in preventing the development of bone loss induced by OVX in rats.     

Effect of High Molecular Weight Water-Soluble Chitosan on the Trabecular Bone and Thickness in Ovariectomized Rats

High molecular weight water-soluble chitosan(WSC), having an average molecular weight of 300,000 Da and a degree of deacethylation over 90%, can be produced using a simple multi-step membrane separation process. In this study, the trabecular bone area and thickness in ovariectomized(OVX) rats decreased by almost 50% from those in sham-operated rats. WSC was evaluated for inhibition of the progress of bone loss induced by OVX rats. We measured bone histomorphometry in sham, OVX or WSC-administered OVX rats. From light microscopic analyses, a porous or erosive appearances were observed on the surface of trabecular bone of tibia in OVX rats, whereas those of the same bone in sham-operated rats were composed of fine particles. The trabecular bone area and trabecular thickness in OVX rats decreased by 50% from those in sham rats, these decreases were completely inhibited by administration of WSC at a concentration of 15 mg/kg/daily for 7 weeks. In this study, the mechanical strength in femur neck was significantly enhanced by the treatment of WSC for 7 weeks. In OVX rats, free T₃ was normal in all cases, whereas free T₄ was significantly increased. Although there was no difference between OVX and WSC-administered rats in T₃ level, we have found significant difference between them in T₄ level. These results strongly suggest that WSC is effective in preventing the development of bone loss induced by OVX in rats.     

The effect of water-soluble chitosan on macrophage activation and the attenuation of mite allergen-induced airway inflammation

Chitin and chitosan have versatile anti-tumor, anti-fungal, and antimicrobial biological properties. Oral intakes and intranasal administration of chitin attenuated allergen-induced airway inflammation in sensitized mice, which may be due to its Th1 adjuvant properties. However, their mechanism of action is not entirely clear. In this report, we demonstrate that water-soluble chitosan (WSC) has specific immunomodulatory effects on dust mite allergen Dermatophagoides farinae (Der f)-stimulated, monocyte-derived macrophages (MDM). These effects include polarizing the cytokine balance towards Th1 cytokines, decreasing the production of the inflammatory cytokines IL-6 and TNF-alpha, down-regulating CD44 and TLR4 receptor expression, and inhibiting T cell proliferation. Scanning electron microscope (SEM) examination found that WSC reduced the rate of pseudopodia formation in Der f-stimulated MDM from allergic asthma patients. The effect of WSC on allergen-stimulated MDM may be mediated via inhibition of PKCzeta phosphorylation and NF-kappaB pathway activation. In a murine model of asthma, we found that intranasal application of WSC attenuates Der f-induced lung inflammation by reducing infiltration of inflammatory cells, epithelial damage, and goblet cell hyperplasia and markedly decreasing production of Arg I, iNOs, and thymic stromal lymphopoietin (TSLP) in the bronchial epithelium. Therefore, we believe that WSC may provide a new therapeutic modality for allergic asthma.     

CG创伤敷料的研制

以壳聚糖、明胶和甘油为基本原料，选用戊二醛为交联剂，探讨了交联反应的条件，避免了明胶蛋白与壳聚糖的静电沉淀反应。成膜后经再处理，制备出一种含水率高、透明的多孔网状纤维膜样的创伤敷料。该敷料的扯断强度为６．５ＭＰａ，扯断伸长率为２１０％，撕裂强度为５．３ＫＮ／ｍ，含水率为７６％。通过动物实验，证明该膜具有良好的组织相容性，对防止感染、促进伤口愈合起到良好的作用，适合作创伤敷料     

Chitin-based tubes for tissue engineering in the nervous system

The purpose of this study was to investigate chitin and chitosan as potential materials for biodegradable nerve guides. Transparent chitin hydrogel tubes were synthesized, for the first time, from chitosan solutions using acylation chemistry and mold casting techniques. Alkaline hydrolysis of chitin tubes resulted in chitosan tubes, with the extent of hydrolysis controlling the resulting amine content. This, in turn, impacted compressive strength and cell adhesion. Chitosan tubes were mechanically stronger than their chitin origins, as measured by the transverse compressive test, where tubes having degrees of acetylation of 1%, 3%, 18% (i.e. chitosan) and 94% (i.e. chitin) supported loads at a 30% displacement of 40.6 +/- 4.3, 25.3 +/- 4.5, 10.6 +/- 0.8, and 8.7 +/- 0.4 g, respectively. However, the chitin processing methodology could be optimized for compressive strength, by either incorporating reinforcing coils in the tube wall, or air-drying the hydrogel tubes. Chitin and chitosan supported adhesion and differentiation of primary chick dorsal root ganglion neurons in vitro. Chitosan films showed significantly enhanced neurite outgrowth relative to chitin films, reflecting the dependence of nerve cell affinity on the amine content in the polysaccharide: neurites extended 1794.7 +/- 392.0 microm/mm(2) on chitosan films vs. 140.5 +/- 41.6 microm/mm(2) on chitin films after 2 days of culture. This implies that cell adhesion and neurite extension can be adjusted by amine content, which is important for tissue engineering in the nervous system. The methods for easy processing and modification of chitin and chitosan described herein, allow the mechanical properties and cyto-compatibility to be controlled and provide a means for a broader investigation into their use in biomedical applications.     

Wound Contraction in Experimental Lathyrism

Lathyrism, which affects developing connective tissue, is produced by extracts of sweet-pea seeds (Lathyrus odoratus) and by various amino-nitriles. The basic lesion is a defect in the intra- and inter-molecular bonding of collagen, as a result of which wound tensile strength is diminished. Wounds involving skin loss in rats heal largely by a process known as contraction, in which the skin edges are brought together, leaving only a small defect to be closed by the migrating epithelium. Here, it has been found that a lathyrogenic agent (β-aminopropionitrile) has no effect on wound contraction and the implications of this are discussed. The result suggests that tensile strength and contraction reflect different aspects of healing.     

The biocompatibility of dibutyryl chitin in the context of wound dressings

Dibutyryl chitin (DBC) is a modified chitin carrying butyryl groups at 3 and 6 positions; its peculiarity is that it dissolves promptly in common solvents, while being insoluble in aqueous systems. The high biocompatibility of dibutyryl chitin in the form of films and non-wovens has been demonstrated for human, chick and mouse fibroblasts by the Viability/Cytotoxicity assay, In situ Cell Proliferation assay, Neutral Red Retention assay, Lactate Dehydrogenase Release assay, MTS cytotoxicity assay, and scanning electron microscopy. DBC was hardly degradable by lysozyme, amylase, collagenase, pectinase and cellulase over the observation period of 48 days at room temperature, during which no more than 1.33% by weight of the DBC filaments (0.3 mm diameter) was released to the aqueous medium. DBC non-wovens were incorporated into 5-methylpyrrolidinone chitosan solution and submitted to freeze-drying to produce a reinforced wound dressing material. The latter was tested in vivo in full thickness wounds in rats. The insertion of 4x4 mm pieces did not promote any adverse effect on the healing process, as shown histologically. DBC is therefore suitable for contacting intact and wounded human tissues.     

Chitosan: potential use as a bioactive coating for orthopaedic and craniofacial/dental implants

Chitosan is a biopolymer that exhibits osteoconductive, enhanced wound healing and antimicrobial properties which make it attractive for use as a bioactive coating to improve osseointegration of orthopaedic and craniofacial implant devices. Coatings made from 91.2% de-acetylated chitosan were chemically bonded to titanium coupons via silane-glutaraldehyde molecules. The bond strength of the coatings was evaluated in mechanical tensile tests, and their dissolution and cyto-compatibility were evaluated in vitro using cell-culture medium and UMR 106 osteoblastic cells, respectively. The results showed that the chitosan coatings were chemically bonded to the titanium substrate and that the bond strengths (1.5-1.8 MPa) were not affected by gas sterilization. However, the chitosan bond strengths were less than those reported for calcium-phosphate coatings. The gas-sterilized coatings exhibited little dissolution over 8 weeks in cell-culture solution, and the attachment and growth of the UMR 106 osteoblast cells was greater on the chitosan-coated samples than on the uncoated titanium. These results indicated that chitosan has the potential to be used as a biocompatible, bioactive coating for orthopaedic and craniofacial implant devices.