Healing-impaired Wounds Research Articles

Phylogenetic evaluation and genotypic identification of burn-related Pseudomonas aeruginosa strains isolated from post-burn human infections during hospitalization.

Cutaneous burn trauma, compromise of dermal layers and immune defense system is a physical and fiscal burden on healthcare systems. Burn-wound infections are a serious complication of thermal-injury and contribute significantly to care burden. After burn-induced trauma, sepsis by Pseudomonas aeruginosa impairs patient recovery and contributes to mortality and morbidity. Past studies show positive correlation between detection of Pseudomonas species and healing-impaired traumatic wounds. P. aeruginosa is a resilient opportunistic human pathogen and a nosocomial agent involved in pathology of healing-impaired wounds, especially in burn-patients. Expansive array of virulence determinants have resulted in gentamicin- and silver-resistant P. aeruginosa outbreaks. Knowledge of molecular dynamics and phylogeny of P. aeruginosa associated with burn-wounds is limited. Therefore, we conducted whole-genome sequencing for genotyping and phylogenetic analysis of P. aeruginosa burn-associated strains (n=19) isolated from 7 burn cases during hospitalization. Comparison of genetic features in P. aeruginosa strains in the core genome and mobilome detected genetic variations within some clonal infections overtime. Genetic variations were observed among different burn cases, with some features identified in severe lung infections. Polyclonal infections were also observed, with differing genotypes and virulence potentials, highlighting the importance of reasoned sampling of isolates for clinical testing.

Protective effect of FGF-2 and low-molecular-weight heparin/protamine nanoparticles on radiation-induced healing-impaired wound repair in rats.

We examined the effectiveness of localized administration of fibroblast growth factor-2 containing low-molecular-weight heparin/protamine nanoparticles (FGF-2&LMWH/P NPs) on apoptosis in vivo and on healing of radiation-induced skin injury in a rat model. FGF-2 binds onto LMWH/P NPs, which can significantly enhance and stabilize FGF-2 as a local carrier. X-irradiation at a dose of 25 Gy was administered to the lower part of the back (using a lead sheet with two holes) 1 h before the administration of FGF-2&LMWH/P NPs. Cutaneous full-thickness defect wounds were then formed in X-irradiated areas to examine the time-course of wound healing, and the wound tissues were microscopically and histologically compared and examined. Wound healing was significantly delayed by X-irradiation, but FGF-2&LMWH/P NPs administration prior to irradiation led to a significantly shorter delay compared with FGF-2 alone, LMWH/P NPs alone, and controls. Furthermore, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) staining showed that the proportions of apoptotic dermal fibroblasts in X-irradiated skin were significantly lower in rats administered FGF-2&LMWH/P NPs than in controls. However, 8-hydroxy-2’-deoxyguanosine (8-OHdG) staining showed no differences. Thus, localized administration of FGF-2&LMWH/P NPs prior to irradiation may alleviate X-irradiation-induced healing-impaired wound repair in normal tissue.

RNA-Seq Transcriptomic Responses of Full-Thickness Dermal Excision Wounds to Pseudomonas aeruginosa Acute and Biofilm Infection.

Pseudomonas aeruginosa infections of wounds in clinical settings are major complications whose outcomes are influenced by host responses that are not completely understood. Herein we evaluated transcriptomic changes of wounds as they counter P. aeruginosa infection—first active infection, and then chronic biofilm infection. We used the dermal full-thickness, rabbit ear excisional wound model. We studied the wound response: towards acute infection at 2, 6, and 24 hrs after inoculating 106 bacteria into day-3 wounds; and, towards more chronic biofilm infection of wounds similarly infected for 24 hrs but then treated with topical antibiotic to coerce biofilm growth and evaluated at day 5 and 9 post-infection. The wounds were analyzed for bacterial counts, expression of P. aeruginosa virulence and biofilm-synthesis genes, biofilm morphology, infiltrating immune cells, re-epithelialization, and genome-wide gene expression (RNA-Seq transcriptome). This analysis revealed that 2 hrs after bacterial inoculation into day-3 wounds, the down-regulated genes (infected vs. non-infected) of the wound edge were nearly all non-coding RNAs (ncRNAs), comprised of snoRNA, miRNA, and RNU6 pseudogenes, and their down-regulation preceded a general down-regulation of skin-enriched coding gene expression. As the active infection intensified, ncRNAs remained overrepresented among down-regulated genes; however, at 6 and 24 hrs they changed to a different set, which overlapped between these times, and excluded RNU6 pseudogenes but included snRNA components of the major and minor spliceosomes. Additionally, the raw counts of multiple types of differentially-expressed ncRNAs increased on post-wounding day 3 in control wounds, but infection suppressed this increase. After 5 and 9 days, these ncRNA counts in control wounds decreased, whereas they increased in the infected, healing-impaired wounds. These data suggest a sequential and coordinated change in the levels of transcripts of multiple major classes of ncRNAs in wound cells transitioning from inflammation to the proliferation phase of healing.

Cytotoxicity of Silver Nanoparticle and Chitin-Nanofiber Sheet Composites Caused by Oxidative Stress.

Size-controlled spherical silver nanoparticles (<10 nm) and chitin-nanofiber sheet composites (Ag NPs/CNFS) have previously been reported to have strong antimicrobial activity in vitro. Although Ag NPs/CNFS have strong antimicrobial activity, their cytotoxicity has not been investigated. This study was performed to evaluate the effects of Ag NPs/CNFS on cytotoxicity for fibroblasts in vitro and healing delay of wound repair in vivo, focused on oxidative stress. Cytotoxic activities of Ag NPs/CNFS were investigated using a fibroblast cell proliferation assay, nitric oxide/nitrogen dioxide (NO/NO2) measurement of the cell lysates in vitro, inhibitory effects of Ag NPs/CNFS on healing-impaired wound repair using diabetic mice in vivo, 8-hydroxy-2′-deoxyguanosine (8-OHdG) immunohistochemical staining of the skin sections, and generation of carbonyl protein in the wound was performed to evaluate cytotoxicity with oxidative stress. Ag NPs/CNFS exhibited cytotoxicity for fibroblasts and a significant increase of total NO/NO2 levels in the cell lysates in vitro and increased levels of 8-OHdG and carbonyl proteins in vivo. Although wound repair in the continuously Ag NPs/CNFS-treated group was delayed, it could be mitigated by washing the covered wound with saline. Thus, Ag NPs/CNFS may become accepted as an anti-infectious wound dressing.

Sodium carboxymethylation-functionalized chitosan fibers for cutaneous wound healing application

A water absorption biomaterial, sodium carboxymethylation-functionalized chitosan fibers (Na-NOCC fibers) were prepared, applied for cutaneous wound repair, and characterized by FTIR and NMR. The water absorption of Na-NOCC fibers increased significantly with substitution degree rising, from 3.2 to 6.8 g/g, and higher than that of chitosan fibers (2.2 g/g) confirmed by swelling behavior. In the antibacterial action, the high degree of substitution of Na-NOCC fibers exhibited stronger antibacterial activities against E. coli (from 66.54% up to 88.86%). The inhibition of Na-NOCC fibers against S. aureus were above 90%, and more effective than E. coli. The cytotoxicity assay demonstrated that Na-NOCC2 fibers were no obvious cytotoxicity to mouse fibroblasts. Wound healing test and histological examination showed that significantly advanced granulation tissue and capillary formation in the healing-impaired wounds treated with Na-NOCC fibers, as compared to those treated with gauze, which demonstrated that Na- NOCC fibers could promote skin repair and might have great application for wound healing.

Enhanced healing of mitomycin C-treated healing-impaired wounds in rats with PRP-containing fragmin/protamine microparticles (PRP&F/P MPs)

Purpose: The purpose of this study was to evaluate the accelerating effects of platelet-rich plasma-containing (PRP&) fragmin/protamine microparticles (F/P MPs) for repairing mitomycin C-treated healing-impaired wounds. Staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL-staining) showed that apoptosis of dermal fibroblast cells (DFCs) and epidermal keratinocyte cells (EKCs) were significantly induced in the skin of the mitomycin C-treated rats. Method: Full-thickness skin defects were made on the back of rats and mitomycin C was applied on the wounds to prepare a healing-impaired wound. After washing out the mitomycin C, saline (control), F/P MPs alone, PRP alone, and PRP&F/P MPs were injected around the wounds. The rats were later euthanised and histological sections of the wounds were then prepared at indicated time periods after the treatment. Results and conclusion: These results indicated the numbers of large, medium, and small capillary lumens 7 days after injection of PRP&F/P MPs were significantly higher than those after injection of PRP or F/P MPs alone. Furthermore, epithelium and granulation tissue formations were significantly stimulated in the healing-impaired wounds treated with PRP&F/P MPs 3, 7 and 14 days after injection of PRP&F/P MPs.

An injectable aldehyded 1-amino-3,3-diethoxy-propane hyaluronic acid-chitosan hydrogel as a carrier of adipose derived stem cells to enhance angiogenesis and promote skin regeneration.

We report an injectable aldehyded 1-amino-3,3-diethoxy-propane (ADEP)-hyaluronic acid (AHA)-chitosan (CS) hydrogel. This hydrogel can enhance wound healing by promoting cell migration, proliferation, granulation and angiogensis. During sol-gel transition, no chemical crosslinking agent was introduced and the structure of polysaccharide HA was grafted with a functional molecule, 1-amino-3,3-diethoxy-propane, but without using oxidation to break the HA structure as previously reported. Rheological tests showed the injectability and stability of this AHA-CS hydrogel. Full thickness skin defects (1 cm × 1 cm) were made on mice and the adipose derived stem cell loaded gel was tested in the healing-impaired wound. The AHA-CA hydrogel significantly accelerated wound closure, increased cell proliferation (Ki67 marker) and promoted keratinocyte migration (p63 marker). Histological examination demonstrated that the gel significantly advanced granulation tissue and capillary formation in the gel-treated wounds. mRNA expressions of angiogenesis (VEGF-A), chemotactic factors (SDF-1) and ECM-remodeling MMPs (MMP1 and MMP9) were also up-regulated in the hydrogel-treated wounds.

Protective effect of inhalation of hydrogen gas on radiation-induced dermatitis and skin injury in rats.

The effect of inhalation of hydrogen-containing gas (1.3% hydrogen + 20.8% oxygen + 77.9% nitrogen) (HCG) on radiation-induced dermatitis and on the healing of healing-impaired skin wounds in rats was examined using a rat model of radiation-induced skin injury. An X-ray dose of 20 Gy was irradiated onto the lower part of the back through two holes in a lead shield. Irradiation was performed before or after inhalation of HCG for 2 h. Inhalation of HCG significantly reduced the severity of radiodermatitis and accelerated healing-impaired wound repair. Staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) showed that the proportion of apoptotic keratinocytes and the level of staining in the X-irradiated skin of rats that pre-inhaled HCG were significantly lower than that of rats which did not pre-inhale HCG. Cutaneous full-thickness wounds were then created in the X-irradiated area to examine the time-course of wound healing. X-irradiation significantly increased the time required for wound healing, but the inhalation of HCG prior to the irradiation significantly decreased the delay in wound healing compared with the control and post-inhalation of HCG groups. Therefore, radiation-induced skin injury can potentially be alleviated by the pre-inhalation of HCG.

Effective Wound Healing in Streptozotocin-Induced Diabetic Rats by Adipose-Derived Stromal Cell Transplantation in Plasma-Gel Containing Fragmin/Protamine Microparticles

We investigated the effectiveness of the application of inbred adipose-derived stromal cells (IR-ASCs) in high inbred rat plasma (IRP) (6%)-Dulbecco modified Eagle medium (DMEM) gel with fragmin/protamine microparticles (F/P MPs) (IR-ASCs + IRP-DMEM gel + F/P MPs) on wound healing in streptozotocin-induced diabetic rats. F/P MPs have previously been used as a cell carrier for IR-ASCs in inbred Fisher 344 rats and for preservation and controlled release of various cytokines in IRP-DMEM gel. We applied IR-ASCs + IRP-DMEM gel + F/P MPs to full-thickness skin excisions on the backs of the diabetic rats. The statistical significance of wound closure was evaluated on postwounding days 3, 7, 10, and 14, and the skin area surrounding the wound was removed for histological examination on days 7 and 14. The wound closure rate and histological examination of wounds treated with IR-ASCs + IRP-DMEM gel + F/P MPs demonstrated significantly advanced epithelialization, capillary formation, and granulation tissue formation. When DiI-labeled IR-ASCs + IRP-DMEM gel + F/P MPs were applied to full-thickness skin wounds on the backs of the diabetic rats, histological observation at 2 weeks showed appearances of both DiI-labeled granulation tissue and CD31-immunostained microvessels in the transplant areas. A portion of the transplanted IR-ASCs + IRP-DMEM gel + F/P MPs had been taken up into the granulation tissues to promote wound healing. Thus, IR-ASCs + IRP-DMEM gel + F/P MPs were effective for repairing healing-impaired wounds such as those arising in the diabetic rats.

Comparison of collagen matrix treatment impregnated with platelet rich plasma vs bone marrow

This study has reported the efficacy of an autologous bone marrow-impregnated collagen matrix experimentally and clinically. Then, it reflected that platelet rich plasma (PRP) was as good a source of growth factors as bone marrow and available in a less invasive procedure. This study aimed to compare the efficacy of a PRP-impregnated collagen matrix with that of a bone marrow-impregnated collagen matrix by quantifying wound size and capillary density using genetically diabetic db/db mice. Bone marrow cells were obtained from femurs of ddy mice. Then, a small amount of collagen matrix was immersed in bone marrow suspension. This is called a bone marrow-impregnated collagen matrix. PRP was obtained from healthy human blood and a small amount of collagen matrix was immersed in PRP. This is called a PRP-impregnated collagen matrix. A bone marrow-impregnated collagen matrix and PRP-impregnated collagen matrix were applied to excisional skin wounds on a genetically healing-impaired mouse (n = 6) and wounds were evaluated 6 days after the procedure. Wounds were divided into two groups: PRP (n = 6), in which a PRP-impregnated collagen matrix was applied; and bone marrow (n = 6), in which collagen immersed in a bone marrow suspension was applied. There was no significant difference between the PRP and bone-marrow groups in the rate of vascular density increase or wound size decrease. The present study suggested that the PRP-impregnated collagen matrix promotes repair processes at least as strongly as the bone marrow-impregnated collagen matrix. Given lower invasiveness, the PRP-impregnated collagen matrix would have advantages in clinical use.

Attenuation of Limb Loss in an Experimentally Induced Hindlimb Ischemic Model by Fibroblast Growth Factor-2/Fragmin/Protamine Microparticles as a Delivery System

Fibroblast growth factor-2 (FGF-2) is a well-characterized protein that is used in the treatment of healing-impaired wounds. We previously reported that fragmin/protamine microparticles (F/P MPs) are useful as biodegradable carriers for the controlled release of cytokines. We examined the ability of FGF-2-containing (FGF-2/) F/P MPs to prevent limb loss in an experimentally induced ischemic hindlimb model using adult Balb/c-nu/nu male mice. One day after inducing ischemia, intramuscular injections of 100 μL of FGF-2/F/P MPs turbid suspension (10 μg/mL FGF-2 and 6 mg/mL F/P MPs) were administered into eight sites of the ischemic hindlimb. A 100-μL suspension of each of the following-10 μg/mL FGF-2, 6 mg/mL F/P MPs, and phosphate-buffered saline (PBS; the control)-was similarly injected into the hindlimb. From 5 days onward after the injections, recovery from ischemia was observed in the FGF-2/F/P MP-treated group, but only partial recovery occurred in the FGF-2-treated group. The F/P MP-treated and PBS-treated groups (i.e., control) exhibited no recovery from the ischemia. The histological evaluations of the hindlimbs also confirmed that the capillary (i.e., mature vessels) density was significantly higher in the FGF-2/F/P MP-treated group than in the other groups. The mice injected with FGF-2/F/P MPs also recovered hindlimb blood flow, as reflected by oxygen saturation and surface temperature evaluation. Our present approach using FGF-2/F/P MPs could be considered a valuable option for the therapeutic treatment of peripheral ischemic diseases.

Novel hydrocolloid-sheet as wound dressing to stimulate healing-impaired wound healing in diabetic db/db mice

To create a moist environment for wound healing, a hydrocolloid-sheet composed of alginate, chitin/chitosan and fucoidan (ACF-HS) has been developed as a functional wound dressing. ACF-HS gradually adsorbed medium without any maceration and the medium adsorption in vitro reached constant after 18 h. ACF-HS could effectively interact with and protect a healing-impaired wound in diabetic db/db mice, providing a good moist healing environment with exudate. Furthermore, the wound dressing could have other properties like ease of application and removal, and proper adherence. The aim of this study was to evaluate an accelerating effect of ACF-HS on wound healing for healing-impaired wounds in diabetic db/db mice. Round full-thickness skin defects (12 mm in diameter) were made on the back of db/db mice to prepare healing-impaired wounds. After applying ACF-HS to the wounds, the mice were later killed and histological sections of the wound were prepared. Histological examinations showed significantly advanced granulation tissue and capillary formations in the wounds treated with ACF-HS on days 4, 9 and 14 compared with those in commercially available hydrocolloid wound dressing and non-treatment (control). Thus, ACF-HS may serve as a new wound dressing for diabetic healing-impaired wounds.

Antimicrobial decapeptide KSL-W enhances neutrophil chemotaxis and function

Mammalian cationic antimicrobial peptides have received increased attention over the last decade, due to their prokaryotic selectivity and decreased risk of microbial resistance. In addition, antimicrobial peptides display differential biological effects on mammalian immune cell function, such as migration, adhesion, and modulation of respiratory burst, which make them even more attractive as therapeutic agents. Synthetic combinatorial libraries provide a time-efficient and cost-effective source for these diverse molecules. The novel synthetic antimicrobial peptide, KSLW (KKVVFWVKFK-NH2), has been shown to display a broad spectrum of antimicrobial activity against Gram (+) and Gram (−) bacteria, fungi and viruses. In this study, we evaluated the alternative biological activity of the decapeptide on neutrophil migration and function. KSLW was demonstrated to be chemotactic for neutrophils in micromolar amounts, and neutrophil treatment with KSLW, after 1min, resulted in significant increases in F-actin polymerization. KSLW was shown to inhibit oxygen radical production in PMA- and LPS-stimulated neutrophils. Future studies, to determine if KSLW regulates neutrophil phagocytosis, adhesion, and apoptosis, or examining the effect of KSLW on other mammalian cell types, such as cell populations of healing-impaired wounds, would provide significant insight for the potential therapeutic strategies offered by antimicrobial peptides.

Enhanced healing of mitomycin C-treated healing-impaired wounds in rats with hydrosheets composed of chitin/chitosan, fucoidan, and alginate as wound dressings

To create a moist environment for rapid wound healing, a hydrosheet composed of alginate, chitin/chitosan, and fucoidan (ACF-HS) has been developed as a functional wound dressing. The aim of this study was to evaluate the accelerating effect of ACF-HS on wound healing for rat mitomycin C-treated healing-impaired wounds. Full-thickness skin defects were made on the back of rats and mitomycin C was applied onto the wound for 10 minutes to prepare a healing-impaired wound. After thoroughly washing out the mitomycin C, ACF-HS was applied to the healing-impaired wounds. The rats were later euthanized and histological sections of the wounds were prepared. The histological examinations showed significantly advanced granulation tissue and capillary formations in the healing-impaired wounds treated with ACF-HS on days 7 and 14, in comparison with that in alginate fiber (Kaltostat), hydrogel wound dressing (DuoACTIVE), and nontreatment (negative control). Furthermore, in cell culture studies, ACF-HS-absorbed serum and fibroblast growth factor-2 was found to be proliferative for fibroblasts and endothelial cells, respectively, and ACF-HS-absorbed serum was found to be chemoattractive for fibroblasts. However, our results may not be strictly comparable with general healing-impaired wound models in humans because of the cell damage by mitomycin C. In addition, more biocompatibility studies of fucoidan are essential due to the possibility of renal toxicity.

Hydrogel blends of chitin/chitosan, fucoidan and alginate as healing-impaired wound dressings

In order to create a moist environment for rapid wound healing, a hydrogel sheet composed of a blended powder of alginate, chitin/chitosan and fucoidan (ACF-HS; 60:20:2:4 w/w) has been developed as a functional wound dressing. ACF-HS gradually absorbed DMEM without any maceration, and fluid absorption became constant within 18 h. On application, ACF-HS was expected to effectively interact with and protect the wound in rats, providing a good moist healing environment with exudates. In addition, the wound dressing has properties such as ease of application and removal and good adherence. Full-thickness skin defects were made on the backs of rats and mitomycin C solution (1 mg/ml in saline) was applied onto the wound for 10 min in order to prepare healing-impaired wounds. After thoroughly washing out the mitomycin C, ACF-HS was applied to the healing-impaired wounds. Although normal rat wound repair was not stimulated by the application of ACF-HS, healing-impaired wound repair was significantly stimulated. Histological examination demonstrated significantly advanced granulation tissue and capillary formation in the healing-impaired wounds treated with ACF-HS on day 7, as compared to those treated with calcium alginate fiber (Kaltostat ®; Convatec Ltd., Tokyo, Japan) and those left untreated.

Accelerated Wound Healing in Healing-Impaired db/db Mice by Autologous Adipose Tissue-Derived Stromal Cells Combined With Atelocollagen Matrix

Adipose tissue-derived stromal cells (ATSCs) have recently gained widespread attention as a potential alternate source to bone marrow-derived mesenchymal stem cells with a proliferative capacity and a similar ability to undergo multilineage differentiation. In this study, we evaluated the effectiveness of freshly isolated autologous ATSCs-containing atelocollagen matrix with silicon membrane (ACMS) on wound healing of diabetic (db/db) mice. Cultured ATSCs from (db/db) mice secreted significant amounts of growth factors and cytokines, which are suitable for wound repair. Two full thickness round skin defects were made on the backs of healing-impaired db/db mice. Freshly isolated autologous ATSCs-containing ACMS or ACMS alone were applied to the wounds. Twelve mice were treated and then killed at 1 or 2 weeks (n = 6 each). Histologic sections of the wounds were prepared at each time period after treatment. Histologic examination demonstrated significantly advanced granulation tissue formation, capillary formation, and epithelialization in diabetic healing-impaired wounds treated with autologous ATSCs-containing ACMS, compared with mice treated with ACMS alone. These results suggested that transplantation of autologous ATSCs-containing ACMS significantly accelerated wound healing in diabetic healing-impaired db/db mice.

Acceleration of diabetic wound healing with chitosan-crosslinked collagen sponge containing recombinant human acidic fibroblast growth factor in healing-impaired STZ diabetic rats

In order to develop a better wound-dressing to enhance diabetic wound healing, we have examined the biochemical and biophysical features of chitosan-crosslinked collagen sponge (CCCS) and pre-clinically evaluated the CCCS containing recombinant human acidic fibroblast growth factor (CCCS/FGF) in accelerating diabetic wound healing as compared to collagen sponge alone and FGF alone. Collagen crosslinked with chitosan showed several advantages required for wound dressing, including the uniform and porous ultrastructure, less water-imbibition, small interval porosity, high resistance to collagenase digestion and slow release of FGF from CCCS/FGF. Therapeutic effect of the new wound-dressing containing FGF (i.e.: CCCS/FGF) on diabetic wound healing was examined in type 1 diabetic rat model in which hyperglycemia was induced by single dose of streptozotocin (STZ) and persisted for two months. The CCCS/FGF provided the most efficiently therapeutic effect among various treatments, showing the shortest healing time (14 days in the CCCS/FGF-treated group as compared to 18 ∼ 21 days in other treatment groups), the quickest tissue collagen generation, the earliest and highest TGF-β1 expression and dermal cell proliferation (PCNA expression). All these results suggest that CCCS/FGF is an ideal wound-dressing to improve the recovery of healing-impaired wound such as diabetic skin wound, which provides a great potential use in clinics for diabetic patients in the future.

Enhanced healing of mitomycin C‐treated wounds in rats using inbred adipose tissue‐derived stromal cells within an atelocollagen matrix

The aim of this study was to evaluate the potential accelerating effects of an adipose tissue-derived stromal cells (ATSC)-containing atelocollagen matrix with silicone membrane (ACMS) for repairing mitomycin C-treated healing-impaired wounds. Mitomycin C was applied to full-thickness skin incisions in this study to create a healing-impaired wound model in rat. After thoroughly washing out the mitomycin C from the wound, ACMS alone or ATSC-containing ACMS was applied to the wounds. Histological sections of the wounds were then prepared at indicated time periods after the treatments. These results indicated significantly advanced granulation tissue and capillary formations in the healing-impaired wounds treated with ATSC-containing ACMS compared with those treated with ACMS alone. Thus, this study suggested that transplantation of inbred ATSC-containing ACMS is effective for repairing healing-impaired wounds.

Acceleration of wound healing in healing‐impaired db/db mice with a photocrosslinkable chitosan hydrogel containing fibroblast growth factor‐2

Application of ultraviolet light irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution including fibroblast growth factor-2 (FGF-2) results within 30 seconds in an insoluble, flexible hydrogel. The FGF-2 molecules retained in the chitosan hydrogel remain biologically active and are released from the chitosan hydrogel upon in vivo biodegradation of the hydrogel. To evaluate the accelerating effect on wound healing of this hydrogel, full-thickness skin incisions were made in the backs of healing-impaired diabetic (db/db) mice and their normal (db/+) littermates. The mice were later killed, and histological sections of the wound were prepared. The degree of wound healing was evaluated using several histological parameters such as the rate of contraction, epithelialization, and tissue filling. Application of the chitosan hydrogel significantly advanced the rate of contraction on Days 0 to 2 in db/db and db/+ mice. Although the addition of FGF-2 into the chitosan hydrogel in db/+ mice had little effect, application of the chitosan hydrogel-containing FGF-2 further accelerated the adjusted tissue filling rate (Days 2 to 4 and Days 4 to 8) in db/db mice. Furthermore, the chitosan hydrogel-containing FGF-2 markedly increased the number of CD-34-positive vessels in the wound areas of db/db mice on Day 4. Thus, the application of chitosan hydrogel-containing FGF-2 onto a healing-impaired wound induces significant wound contraction and accelerates wound closure and healing.

Expression of Secreted Platelet-Derived Growth Factor-B by Recombinant Nonreplicating and Noncytopathic Vaccinia Virus

The purpose of this study was to develop a noncytopathic vector for transient delivery of biologically active platelet-derived growth factor (PDGF) to wounds. Topical application of the protein PDGF-B has improved wound healing in experimental studies of healing-impaired wounds. However, use of PDGF-B has been limited by availability of recombinant protein, short half-life, and inability to reliably apply to the wounded area. One approach to supply local PDGF-B is through transient gene transfer and expression. Treatment of vaccinia virus with psoralen and long-wave ultraviolet irradiation makes it noncytopathic and nonreplicative. The authors inserted various transgenes encoding different forms of PDGF into recombinant vaccinia virus at the hemaglutinin locus by homologous recombination. Because the PDGF-B expressed from full length cDNA is not secreted because of the membrane retention sequence at the C-terminal end of the polypeptide, the authors inserted a 3'-truncated form of human PDGF-B cDNA in recombinant vaccinia virus to achieve secretion. To avoid interference in bioassays by a virally encoded epidermal growth factor homologue called vaccinia growth factor (VGF) in wild type vaccinia virus (CR-19), we used a VGF-negative strain to express PDGF-B (vSC20PDGF-B). Biologic activity of PDGF was tested by measuring proliferation of a 3T3 fibroblast cell line. Supernatant from CR-19-infected cells (VGF+) and from truncated vSC20PDGF infected cells caused mild and marked proliferation of 3T3 cells, respectively, whereas supernatant from full-length vSC20PDGF virally infected cells did not. Furthermore, in vitro infection of a confluent 3T3 monolayer with noncytopathic and nonreplicative vaccinia encoding either VGF or truncated PDGF also caused similar proliferation. These results provide important preliminary evidence for the ability to treat nonhealing wounds with nonreplicating and noncytopathic recombinant vaccinia viruses encoding cytokine growth factors.