Hyaluronic acid and chitosan-based nanosystems: a new dressing generation for wound care

ABSTRACT

Introduction: The main goal in the management of chronic wounds is the development of multifunctional dressings able to promote a rapid recovery of skin structure and function, improving patient compliance.

Areas covered: This review discusses the use of nanosystems, based on hyaluronic acid and chitosan or their derivatives for the local treatment of chronic wounds. The bioactive properties of both polysaccharides will be described, as well as the results obtained in the last decade by the in vitro and in vivo evaluation of the wound healing properties of nanosystems based on such polymers.

Expert opinion: In the last decades, there has been a progressive change in the local treatments of chronic wounds: traditional inert dressings have been replaced by more effective bioactive ones, based on biopolymers taking part in wound healing and able to release the loaded active agents in a controlled way. With the advance of nanotechnologies, the scenario has further changed: nanosystems, characterized by a large area-to-volume ratio, show an improved interaction with the biological substrates, amplifying the activity of the constituent biopolymers. In the coming years, a deeper insight into wound healing mechanisms and the development of new techniques for nanosystem manufacturing will results in the design of new scaffolds with improved performance.     

Topical Application of Keratinocyte Growth Factor Conjugated Gold Nanoparticles Accelerate Wound Healing

Keratinocyte growth factor (KGF) has been demonstrated to specifically stimulate the multiplication and migration of keratinocytes. However, due to rapid degradation, the results of topical application of growth factors on wounds are unsatisfactory. In this study, we cross-linked KGF to the surface of gold nanoparticles (GNPs) and explored their effects on wound healing. The as-synthesized nanocomposite (KGF-GNPs) displayed good colloidal stability, decent biocompatibility as well as negligible cellular cytotoxicity. The in vitro cellular experimental results demonstrated that KGF-GNPs could effectively promote the proliferation of keratinocytes in contrast to bare GNPs or KGF. Furthermore, in animal full-thickness wound model, KGF-GNPs are more conducive to wound healing than bare GNPs or KGF. KGF-GNPs enhanced wound healing by promoting wound re-epithelialization rather than granulation. The superior biocompatibility, colloidal depressiveness and biological activity of this nanocomposite indicate that it could be utilized as a promising wound healing drug for clinical application in the future.     

In vitro biocompatibility of chitosan porous skin regenerating templates (PSRTs) using primary human skin keratinocytes

Biopolymer chitosan (β-1,4-d-glucosamine) comprises the copolymer mixture of N-acetylglucosamine and glucosamine. The natural biocompatibility and biodegradability of chitosan have recently highlighted its potential use for applications in wound management. Chemical and physical modifications of chitosan influence its biocompatibility and biodegradability, but it is unknown as to what degree. Hence, the biocompatibility of the chitosan porous skin regenerating templates (PSRT 82, 87 and 108) was determined using an in vitro toxicology model at the cellular and molecular level on primary normal human epidermal keratinocytes (pNHEK). Cytocompatibility was accessed by using a 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl tetrazolium bromide (MTT) assay from 24 to 72 h. To assess the genotoxicity of the PSRTs, DNA damage to the pNHEK was evaluated by using the Comet assay following direct contact with the various PSRTs. Furthermore, the skin pro-inflammatory cytokines TNF-α and IL-8 were examined to evaluate the tendency of the PSRTs to provoke inflammatory responses. All PSRTs were found to be cytocompatible, but only PSRT 108 was capable of stimulating cell proliferation. While all of the PSRTs showed some DNA damage, PSRT 108 showed the least DNA damage followed by PSRT 87 and 82. PSRT 87 and 82 induced a higher secretion of TNF-α and IL-8 in the pNHEK cultures than did PSRT 108. Hence, based on our experiments, PSRT 108 is the most biocompatible wound dressing of the three tested.     

Potential of Ozonated Sesame Oil to Augment Wound Healing in Rats

The hypothesis that ozonated oil has wound healing property was investigated in an excision wound model using Sprague Dawley rats. The animals were divided into four groups, which were treated with sesame oil (vehicle), framycetin (standard), or two doses of ozonated sesame oil (peroxide values 500 and 700 mEq/1000 g, respectively). The formulations were topically applied on the excision wounds once daily for 11 consecutive days and the animals were euthanized on the 12^th day. Wound healing was assessed by measuring the wound contracture, tensile strength, collagen content and superoxide dismutase activity of skin of the excised wound area. On the terminal day, areas of the wounds of the group receiving high dose ozonated oil were significantly smaller than those of the group treated with vehicle. Ozonated oil treated wounds had significantly higher tensile strength, collagen content and superoxide dismutase activity than that of the vehicle treated wounds. Histopathological analysis of skin of the excised wound area treated with ozonated oil revealed better healing activity vis-à-vis vehicle-treated wounds. Thus, it can be concluded that ozonated oil can be of potential therapeutic use for healing wounds.     

Development and characterization of a superabsorbing hydrogel film containing Ulmus davidiana var. Japonica root bark and pullulan for skin wound healing

Ulmus davidiana var. japonica (UD) has widely been used in Korean traditional medicine for the treatment of various types of diseases including inflammation and skin wounds. The UD root bark powders possess gelling activity with an excellent capacity for absorbing water. This distinct property could make the UD root bark powders to be a great material for manufacturing a gel film specifically for the healing of large and highly exudating wounds (e.g., pressure sores and diabetic ulcers). In this research, we separated the UD root bark powder into 4 different samples based on their sizes and then tested their water absorption capacity and flowability. Based on these results, 75–150 μm sized and below 75 μm sized samples of UD root bark powders were chosen, and UD gel films were prepared. The UD gel films showed good thermal stability and mechanically improved properties compared with pullulan only gel film with excellent swelling capacity and favorable skin adhesiveness. Further, in the animal studies with the skin wound mice model, the UD gel films exhibited significant therapeutic effects on accelerating wound closure and dermal regeneration. Overall, this study demonstrated the applicability of UD root bark powders for hydrogel wound dressing materials, and the potential of UD gel films to be superior wound dressings to currently available ones.     

Wound Healing Potential of Formulated Extract from Hibiscus Sabdariffa Calyx

Wound healing agents support the natural healing process, reduce trauma and likelihood of secondary infections and hasten wound closure. The wound healing activities of water in oil cream of the methanol extract of Hibiscus sabdariffa L. (Malvaceae) was evaluated in rats with superficial skin excision wounds. Antibacterial activities against Pseudomonas aeroginosa, Staphylococcus aureus and Echerichia coli were determined. The total flavonoid content, antioxidant properties and thin layer chromatographic fingerprints of the extract were also evaluated. The extract demonstrated antioxidant properties with a total flavonoid content of 12.30±0.09 mg/g. Six reproducible spots were obtained using methanol:water (95:5) as the mobile phase. The extract showed no antimicrobial activity on the selected microorganisms, which are known to infect and retard wound healing. Creams containing H. sabdariffa extract showed significant (P<0.05) and concentration dependent wound healing activities. There was also evidence of synergism with creams containing a combination of gentamicin and H. sabdariffa extract. This study, thus, provides evidence of the wound healing potentials of the formulated extract of the calyces of H. sabdariffa and synergism when co-formulated with gentamicin.     

A comparison of topical Phenytoin with Silverex in the treatment of superficial dermal burn wounds

OBJECTIVE: To compare topical diphenylhydantion (Phenytoin) with silver sulphadiazine/chlorhexidine (Silverex) in terms of rate of wound healing, analgesic and antibacterial properties in small to moderate-sized (< 30% TBSA) superficial dermal (second degree) burn wounds. DESIGN: A prospective randomized controlled study. SETTING: Surgical wards, Muhimbili National Hospital from July 2000 to February 2001. SUBJECTS: Sixty four patients with acute burns, 32 in each group. INTERVENTIONS: Study group treated by sprinkling Phenytoin powder and control group by sprinkling Silverex powder on the wounds for 14 days or until the wound epithelialised or was ready for skin grafting. The data collected included demographic characteristics of patients, aetiology of burn injury, circumstances of injury, site and extent of burns, pus discharge and smell from the wound, pain and discomfort from the wound, bacterial cultures of wound swabs, rate of reduction in wound size and outcome of treatment. RESULTS: The study enrolled 33 male and 31 female patients, 69% being children under five years of age. Hot liquids (80%) and open flames (20%) were the only causes of burns. In 97% of patients injury was due to domestic accidents. In half of the patients burns involved the trunk, and 52% of all patients had less than 15% total body surface burnt. Pus discharge was recorded in 59% of Phenytoin-treated and 75% in Silverex-treated patients while foul smell was noted in 19% and 31% of cases respectively. There were more negative bacterial wound cultures in Phenytoin-than Silverex-treated wounds on day five and day 10 of treatment, the difference being statistically significant (p < 0.01 and 0.001 respectively). There was also a statistically significant difference in wound pain in favour of Phenytoin (p < 0.01). There was no statistically significant difference in the rate of healing in the two groups. CONCLUSION: Phenytoin is a cheap and easy-to-use medicament, effective in suppressing burn wound bacteria and relieving pain thereby promoting healing, and may be advocated for the purpose in resource-scarce environments.     

rhEGF凝胶促进兔Ⅲ度烧伤创面愈合的实验研究

目的:研究rhEGF凝胶对兔皮肤Ⅲ度烧伤创面愈合的影响。方法:铜棒烫伤法制备兔Ⅲ度烧伤创面,切痂后创面均匀涂抹rhEGF凝胶,观察创面愈合情况及创面痊愈所需时间。结果:与对照组相比,rhEGF凝胶组炎症持续时间短暂,创面痊愈所需时间更短(P<0.05)。结论:rhEGF凝胶促进兔Ⅲ度烧伤创面皮肤再生,缩短创面痊愈时间。     

Fresh and cryopreserved cultured keratinocyte allografts for wound healing

The use of cultured human epithelium for skin grafting has recently been developed and used successfully to treat burns and various smaller skin defects. Cultured epidermal allografts are replaced by the recipient's own skin. The use of cultured allografts has a healing effect in chronic ulcerations and in burn wounds, probably by releasing growth factors. We have recently demonstrated that cryopreserved allografts can also induce wound healing. Both an edge effect and complete healing were found in ulcers treated with fresh or cryopreserved cultured epidermal allografts. These findings could be important in patient care, since in wound-healing problems, cryopreserved allografts could be immediately available. The cryopreservation of human keratinocyte sheets may also open new perspectives in pharmacotoxicological research on topically applied substances.     

Keratinocyte wound healing activity of galactoglycerolipids from the fern Ophioglossum vulgatum L.

The genus Ophioglossum consists of ferns with different therapeutic properties, including vulnerary virtues. The species Ophioglossum vulgatum L. is traditionally used on wounds and burns as an ointment, suggesting the occurrence of lipophilic compounds with tissue repair properties. We isolated and characterized a galactosyldiacylglycerol mixture (1), composed mainly of 1,2-di-O-linolenoyl-3-O-β-d-galactopyranosyl-glycerol, from the frond dichloromethane extract. The wound healing properties of 1 were assessed in vitro on keratinocytes. Scratch wound assays showed increased wound closure rates in keratinocyte monolayers exposed to subtoxic doses, previously determined in cytotoxicity assays. The strongest effect, obtained at a dose of 5 μg/mL, approached that of a platelet lysate used in clinical settings. The use of inhibitors of the main cellular pathways involved in wound repair, revealed important roles for intracellular calcium and the ERK1/2 MAP kinase. Conversely, a PCR array of genes involved in wound healing showed an almost total absence of gene modulation. Taken together, the data suggest that 1 acts through a Ca²⁺-dependent, nongenomic mechanism involving the activation of ERK1/2 MAP kinase. Hence, 1 is a main candidate to explain the wound healing virtues of O. vulgatum ointment, and is proposed as a possible new drug in tissue repair and regenerative medicine.     

Skin substitutes to enhance wound healing

Biologically-based skin substitutes have developed as commercial products over the last 5 years. The first generation includes the collagen-based synthetic device, Integra, and Alloderm, which is based on devitalised and cross-linked human dermis. These are used as dermal replacements for third degree burns. Within the last year, the tissue-engineered product, Dermagraft-TC^®, has become available. While originally intended as a temporary covering for severe burns, Dermagraft-TC^® has proved to markedly improve the healing of deep second degree burns. The earliest living skin substitutes used autologous keratinocytes expanded in vitro. Two new products containing living cells, Dermagraft^® and Apligraf, are expected to be approved shortly for diabetic foot ulcers and venous stasis ulcers, respectively. Dermagraft^® is produced by growing human fibroblasts on a three-dimensional scaffold. The cells actively proliferate and lay down extracellular matrix to generate a papillary dermis-like device that shows a combination of angiogenic, growth factor and cell adhesion properties that enhance healing in diabetic foot ulcers. The production of Apligraf includes casting human fibroblasts in collagen, in order to generate a dermal equivalent on which is grown an epidermis. The structure is akin to a skin graft and is so applied. Despite Dermagraft^® and Apligraf being of allogeneic origin, rejection has not been an issue in clinical trials and possible contamination by pathogens has been eliminated as a concern through extensive testing. These developments represent a new concept and are expected to revolutionise wound care. They may also provide a platform for gene therapy applications.     

Evaluation of biomechanical and histological properties of corrosive chemical burns

This study was aimed to prepare a standard experimental chemical burns and evaluate biomechanical and histological properties. As a model corrosive acid burns, sulfuric acid burns were made on the peritoneal part of the rabbit ear with diameter 8?mm by varing sulfuric acid concentrations (1, 2, 18.8?M) and inflicted time (20, 40, 60 and 120?s). The progress of the chemical injury was evaluated for 3?weeks by macroscopic and microscopic observation. Degree of regeneration of damaged epidermis and dermis and presence of infected cells and arrangements of collagen were investigated. Histology of 18.8?M sulfuric acid for 20?s burns showed epidermal necrosis, diffuse vascular infiltrate and collagen degeneration at the level of the papillary dermis, which is a characteristic of a deep dermal burn. The stress?Cstrain curves of normal skin, 18.8?M sulfuric acid treated wound, 6?mm surgical biopsy punch wound and at 21?days post wound were evaluated. As tensile strength measures the ability of matrix to withstand rupture, as the damage from corrosive acid chemical involved wider and deep skin area with permanent damage, the healing could not be processed complete and showed less than 16?% of tensile strength (0.05?±?0.01?N/mm²) as compared with those of normal skin (0.31?±?0.08?N/mm²). The sulfuric acid inflicted wound showed flatter, reflecting the small magnitude of their moduli of elasticity. The elasticity constant for sulfuric acid treated skin (1.46?±?0.17?N/mm) is only 25?% of those of normal skin (5.87?±?2.14?N/mm). Surgical punch wound showed higher tensile strength (0.10?±?0.02?N/mm²) and elasticity constant (4.19?±?0.47?N/mm) than those of corrosive chemical acid burns. In conclusion, the proposed sulfuric acid burn condition (18.8?M, 20?s) could be employed as a useful corrosive chemical wound model. The degree of damage induced by corrosive acid chemicals is more significant than surgical full thickness wound and was dependent on the concentration and treatment time. By controlling the burn condition, the extent of damage could be designed. Biomechanical and histological study demonstrated that the corrosive acid burns induced full thickness permanent dermal injury and significant losses in tensile strength and elasticity modulus.     

Clinical Potential of a Silk Sericin-Releasing Bioactive Wound Dressing for the Treatment of Split-Thickness Skin Graft Donor Sites

Purpose

An ethyl alcohol-precipitated silk sericin/PVA scaffold that controlled the release of silk sericin was previously developed and applied for the treatment of full-thickness wounds in rats and demonstrated efficient healing. In this study, we aimed to further evaluate the clinical potential of this scaffold, hereafter called “silk sericin-releasing wound dressing”, for the treatment of split-thickness skin graft donor sites by comparison with the clinically available wound dressing known as “Bactigras®”.

Methods

In vitro characterization and in vivo evaluation for safety of the wound dressings were performed. A clinical trial of the wound dressings was conducted according to standard protocols.

Results

The sericin released from the wound dressing was not toxic to HaCat human keratinocytes. A peel test indicated that the silk sericin-releasing wound dressing was less adhesive than Bactigras®, potentially reducing trauma and the risk of repeated injury upon removal. There was no evidence of skin irritation upon treatment with either wound dressing. When tested in patients with split-thickness skin graft donor sites, the wounds treated with the silk sericin-releasing wound dressing exhibited complete healing at 12?±?5.0 days, whereas those treated with Bactigras® were completely healed at 14?±?5.2 days (p?=?1.99?×?10^?4). In addition, treatment with the silk sericin-releasing wound dressing significantly reduced pain compared with Bactigras® particularly during the first 4 postoperative days (p?=?2.70?×?10^?5 on day 1).

Conclusion

We introduce this novel silk sericin-releasing wound dressing as an alternative treatment for split-thickness skin graft donor sites.     

早期负压封闭吸引联合组织瓣序贯治疗深度电烧伤的疗效

目的 探讨早期负压封闭吸引（VSD）联合组织瓣序贯治疗深度电烧伤患者的疗效。方法 选取2013年4月至2017年4月安徽医科大学第一附属医院收治的40例深度电烧伤患者,按治疗方法不同将患者分为治疗组与对照组,每组20例。治疗组采用VSD联合组织瓣序贯治疗修复创面,对照组患者采用单纯创面扩创VSD吸引肉芽植皮治疗修复创面。近期（创面拆线前）及远期（出院后半年）进行随访,比较两组患者愈合时间、住院时间、治疗费用及瘢痕指数的差异。结果 治疗组愈合时间比对照组短,差异有统计学意义（t=-2.411,P=0.021）;治疗组住院时间比对照组短,差异有统计学意义（t=-2.102,P=0.046）;治疗组住院费用低于对照组,差异有统计学意义（t=-2.102,P=0.002）;治疗组瘢痕指数小于对照组,差异有统计学意义（t=-2.449,P=0.019）。结论 早期VSD联合组织瓣序贯治疗是深度电烧伤的有效修复方法,并能最大限度恢复电烧伤部位的功能和外观,其疗效明显好于使用单纯创面扩创VSD吸引肉芽创面植皮法。     

Caffeic Acid Phenethyl Ester Loaded Electrospun Nanofibers for Wound Dressing Application

Electrospinning is an advantageous method with a wide usage area, which enables the production of materials consisting of nano-thickness fibers. In this study, caffeic acid phenethyl ester (CAPE) molecule was loaded onto the poly(lactic-co-glycolic acid) (PLGA) nanofibers and obtained nanofibers were physicochemically and biologically investigated for the first time in the literature. The existence of CAPE molecules, loaded on PLGA membranes by dropping and spraying methods, was evaluated by a comparative investigation of Fourier-transform infrared (FTIR) spectra and X-Ray diffraction (XRD) patterns. Fiber morphology of the membranes was investigated by scanning electron microscope (SEM). CAPE release and swelling behaviors of the membranes were studied in vitro. The radical scavenging activity of CAPE-loaded wound dressing materials was determined by using an antioxidant assay. The antimicrobial properties of PLGA and CAPE-loaded PLGA membranes were evaluated against S. aureus, P. aeruginosa and C. albicans strains by the time-kill method. The biocompatibility study of the obtained CAPE-loaded fibers conducted on human fibroblast cell line and wound healing promoting effect of the fibers was investigated in vitro scratch assay.The results show that CAPE-loaded PLGA membranes are highly antimicrobial against all strains used in the experiment. Additionally, the results show that they are biocompatible and have wound healing properties on human fibroblasts.     

Novel Temperature Responsive Films Impregnated with Silver Nano Particles (Ag-NPs) as Potential Dressings for Wounds

Silver nanoparticles have attracted wide interest in medicine on account of their antibacterial activity. We report in this paper, the antibacterial activity and biocompatibility of a temperature responsive topical film fabricated from pullulan-g-pNIPAM and impregnated with two different concentrations (15 ppm and 30 ppm) of silver nanoparticles (Ag-NPs). The release of silver from the film under the influence of temperature above the LCST has been studied and the in vitro release profile of the films has been compared with a marketed silver nano formulation, ‘Meganano gel’. The release of silver from the films has a distinctive profile characterized by a sustained release over a period of 48 hrs, which is comparable to the marketed formulation. The films exhibit excellent swelling properties, making them ideal materials for absorption of exudates from wounds. The antibacterial activity of the films has been established at physiological temperature against gram-positive S. aureus and gram-negative E. coli and compared with the marketed formulation. A cytotoxicity evaluation on HeK293 cells has demonstrated their biocompatibility. The nanocomposite films are thus a new therapeutic device for management of non-healing wounds being constructed from temperature responsive polymers that release Ag-NPs when the temperature of the wound exudate is slightly higher than normal.     

Novel topical formulation for ischemic chronic wounds. Technological design,quality control and safety evaluation

Ulceration of the foot in diabetes is common and disabling, and frequently leads to amputation of the leg. The pathogenesis of foot ulceration is complex, clinical presentation variable and management requires early expert assessment. Despite treatment, ulcers readily become chronic wounds. Chronic wounds are those that remain in a chronic inflammatory state failing a normal healing process patterns. This is partially caused by inefficient eradication of opportunistic pathogens like Pseudomonas aeruginosa. We propose its control or eradication will promote wound healing. Lactobacillus plantarum cultures supernatants (LAPS) shows antipathogenic and pro-healing properties. The main objective was to design two pharmaceutical dosage forms by using LAPS as active pharmaceutical ingredient and to perform its quality control, in vitro activity conservation tests and human trials (safety evaluation). Both selected formulations reach the technological quality expected for 120 days, shows adequate occlusive characteristics and proper adhesion to human skin. From the in vitro release assays were found that LAPS shows adequate release from matrix and maintain its antimicrobial and anti-biofilm activity. First human trials were developed and neither edema nor erythema on healthy skin voluntaries was found. We conclude that C80 and C100 are adequate for their use in future clinical trials to demonstrate a comprehensive therapeutic effectiveness in ischemic chronic wounds.     

OM‐LV20, a novel peptide from odorous frog skin,accelerates wound healing in vitro and in vivo

The healing of chronic wounds remains a considerable challenge in clinical trials and imposes severe financial and physiological burdens on patients. Many works are being tried to find ideal clinical promoting wound healing biomaterials. Small bioactive peptides with low cost and easy production, store and transfer become excellent candidates. Here, we identified a novel peptide (named OM‐LV20) from skin secretions of odorous frog Odorrana margaretae. The peptide had an amino acid sequence of “LVGKLLKGAVGDVCGLLPIC,” contained an intramolecular disulfide bridge at the C‐terminus, and was produced by post‐translational processing of a 71‐residue prepropeptide. Our results showed that OM‐LV20 had no direct microbe‐killing effects, hemolytic activity, or acute toxicity, but did exhibit weak antioxidant activity. OM‐LV20 promoted wound healing against human keratinocytes (HaCaT) and human skin fibroblasts (HSF) in both time‐ and dose‐dependent manners. In addition, it induced the proliferation of HaCaT but not HSF cells. Of note, OM‐LV20 showed strong wound healing‐promoting activity in a mice model of full‐thickness skin wound. Our research indicates the cellular and animal level wound healing potential of OM‐LV20, and thus provides a novel bioactive peptide template for the development of wound healing agents and medicine.     

Exosome loaded genipin crosslinked hydrogel facilitates full thickness cutaneous wound healing in rat animal model

Full thickness cutaneous wound therapy and regeneration remains a critical challenge in clinical therapeutics. Recent reports have suggested that mesenchymal stem cells exosomes therapy is a promising technology with great potential to efficiently promote tissue regeneration. Multifunctional hydrogel composed of both synthetic materials and natural materials is an effective carrier for exosomes loading. Herein, we constructed a biodegradable, dual-sensitive hydrogel encapsulated human umbilical cord-mesenchymal stem cells (hUCMSCs) derived exosomes to facilitate wound healing and skin regeneration process. The materials characterization, exosomes identification, and in vivo full-thickness cutaneous wound healing effect of the hydrogels were performed and evaluated. The in vivo results demonstrated the exosomes loaded hydrogel had significantly improved wound closure, re-epithelialization rates, collagen deposition in the wound sites. More skin appendages were observed in exosomes loaded hydrogel treated wound, indicating the potential to achieve complete skin regeneration. This study provides a new access for complete cutaneous wound regeneration via a genipin crosslinked dual-sensitive hydrogel loading hUCMSCs derived exosomes.