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• W2053070963 abstract "Wound healing is a complex process that involves coordinated interactions between diverse immunological and biological systems. Long-term wounds remain a challenging clinical problem, affecting approximately 6 million patients per year, with a high economic impact. To exacerbate the problem, these wounds render the individual susceptible to life-threatening microbial infections. Because current therapeutic strategies have proved suboptimal, it is imperative to focus on new therapeutic approaches and the development of technologies for both short- and long-term wound management. In recent years, nitric oxide (NO) has emerged as a critical molecule in wound healing, with NO levels increasing rapidly after skin damage and gradually decreasing as the healing process progresses. In this study, we examined the effects of a novel NO-releasing nanoparticle technology on wound healing in mice. The results show that the NO nanoparticles (NO-np) significantly accelerated wound healing. NO-np modified leukocyte migration and increased tumor growth factor-β production in the wound area, which subsequently promoted angiogenesis to enhance the healing process. By using human dermal fibroblasts, we demonstrate that NO-np increased fibroblast migration and collagen deposition in wounded tissue. Together, these data show that NO-releasing nanoparticles have the ability to modulate and accelerate wound healing in a pleiotropic manner. Wound healing is a complex process that involves coordinated interactions between diverse immunological and biological systems. Long-term wounds remain a challenging clinical problem, affecting approximately 6 million patients per year, with a high economic impact. To exacerbate the problem, these wounds render the individual susceptible to life-threatening microbial infections. Because current therapeutic strategies have proved suboptimal, it is imperative to focus on new therapeutic approaches and the development of technologies for both short- and long-term wound management. In recent years, nitric oxide (NO) has emerged as a critical molecule in wound healing, with NO levels increasing rapidly after skin damage and gradually decreasing as the healing process progresses. In this study, we examined the effects of a novel NO-releasing nanoparticle technology on wound healing in mice. The results show that the NO nanoparticles (NO-np) significantly accelerated wound healing. NO-np modified leukocyte migration and increased tumor growth factor-β production in the wound area, which subsequently promoted angiogenesis to enhance the healing process. By using human dermal fibroblasts, we demonstrate that NO-np increased fibroblast migration and collagen deposition in wounded tissue. Together, these data show that NO-releasing nanoparticles have the ability to modulate and accelerate wound healing in a pleiotropic manner. Wound healing is a complex process mediated by a variety of factors responsible for the regeneration and reorganization of damaged tissue toward its normal architecture. Long-term wounds are a widespread problem, affecting nearly 6 million patients a year, with treatment costs approaching $20 billion annually.1Branski L.K. Gauglitz G.G. Herndon D.N. Jeschke M.G. A review of gene and stem cell therapy in cutaneous wound healing.Burns. 2009; 35: 171-180Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar To exacerbate the problem, these wounds are susceptible to life-threatening microbial infections. Because current therapeutic strategies have proved suboptimal, radically new approaches are urgently needed. In the past two decades, nitric oxide (NO) has emerged as a critical molecule in the wound-healing process.2Childress B.B. Stechmiller J.K. Role of nitric oxide in wound healing.Biol Res Nurs. 2002; 4: 5-15Crossref PubMed Scopus (38) Google Scholar, 3Diegelmann R.F. Evans M.C. Wound healing: an overview of acute, fibrotic and delayed healing.Front Biosci. 2004; 9: 283-289Crossref PubMed Scopus (1446) Google Scholar, 4Frank S. Kampfer H. Wetzler C. Pfeilschifter J. Nitric oxide drives skin repair: novel functions of an established mediator.Kidney Int. 2002; 61: 882-888Crossref PubMed Scopus (151) Google Scholar, 5Rizk M. Witte M.B. Barbul A. Nitric oxide and wound healing.World J Surg. 2004; 28: 301-306Crossref PubMed Scopus (120) Google Scholar After skin damage, NO levels increase rapidly, peaking 1 day after the initial injury during the inflammatory phase of wound healing and gradually decreasing as the healing process progresses toward proliferation and remodeling.5Rizk M. Witte M.B. Barbul A. Nitric oxide and wound healing.World J Surg. 2004; 28: 301-306Crossref PubMed Scopus (120) Google Scholar Furthermore, NO is released by a great diversity of immune and skin cells,6Bogdan C. Nitric oxide and the immune response.Nat Immunol. 2001; 2: 907-916Crossref PubMed Scopus (2540) Google Scholar causing pleiotropic effects. Among its many effects, NO plays a critical role in host defense by clearing the wound of infectious microbes and stimulating other healing factors to access the wound area by maintaining a constant blood flow.NO-based therapy for treatment of long-term wounds is challenging because maintaining an ideal concentration of the gas in the affected area is imperative; high or low levels may, in fact, serve to exacerbate the wound. For example, although down-regulation of NO production leads to delayed wound healing by decreasing collagen deposition and reducing wound tensile strength,7Schaffer M.R. Tantry U. Gross S.S. Wasserburg H.L. Barbul A. Nitric oxide regulates wound healing.J Surg Res. 1996; 63: 237-240Abstract Full Text PDF PubMed Scopus (231) Google Scholar, 8Schaffer M.R. Tantry U. Thornton F.J. Barbul A. Inhibition of nitric oxide synthesis in wounds: pharmacology and effect on accumulation of collagen in wounds in mice.Eur J Surg. 1999; 165: 262-267Crossref PubMed Scopus (120) Google Scholar sustained high concentrations of NO may needlessly prolong the inflammatory phase of wound healing, leading to keloid formation.9Cobbold C.A. Sherratt J.A. Mathematical modelling of nitric oxide activity in wound healing can explain keloid and hypertrophic scarring.J Theor Biol. 2000; 204: 257-288Crossref PubMed Scopus (38) Google Scholar, 10Hsu Y.C. Hsiao M. Wang L.F. Chien Y.W. Lee W.R. Nitric oxide produced by iNOS is associated with collagen synthesis in keloid scar formation.Nitric Oxide. 2006; 14: 327-334Crossref PubMed Scopus (44) Google Scholar To address this problem, we have developed an inexpensive, stable, and rapidly deployable NO-releasing platform using a hydrogel-based nanoparticle (NO-np) that can be conveniently topically applied.11Friedman A.J. Han G. Navati M.S. Chacko M. Gunther L. Alfieri A. Friedman J.M. Sustained release nitric oxide releasing nanoparticles: characterization of a novel delivery platform based on nitrite containing hydrogel/glass composites.Nitric Oxide. 2008; 19: 12-20Crossref PubMed Scopus (163) Google Scholar The sustained release rate and total concentration of NO can be modulated by altering the methods of nanoparticle production, with NO release rates in physiological concentrations that can be maintained for hours and applied in multiple doses. Moreover, we have shown that the NO-np has potent antimicrobial activity against drug-resistant Staphylococcus aureus and Acinetobacter baumannii and is therapeutic in experimental skin and abscess infections.12Han G. Martinez L.R. Mihu M.R. Friedman A.J. Friedman J.M. Nosanchuk J.D. Nitric oxide releasing nanoparticles are therapeutic for Staphylococcus aureus abscesses in a murine model of infection.PLoS One. 2009; 4: e7804Crossref PubMed Scopus (115) Google Scholar, 13Martinez L.R. Han G. Chacko M. Mihu M.R. Jacobson M. Gialanella P. Friedman A.J. Nosanchuk J.D. Friedman J.M. Antimicrobial and healing efficacy of sustained release nitric oxide nanoparticles against Staphylococcus aureus skin infection.J Invest Dermatol. 2009; 129: 2463-2469Crossref PubMed Scopus (199) Google Scholar, 14Mihu M.R. Sandkovsky U. Han G. Friedman J.M. Nosanchuk J.D. Martinez L.R. The use of nitric oxide releasing nanoparticles as a treatment against Acinetobacter baumannii in wound infections.Virulence. 2010; 1: 62-67Crossref PubMed Scopus (105) Google Scholar In the present study, we hypothesized that topically applied NO-np would enhance wound healing and investigated this possibility using in vitro and in vivo models.Materials and MethodsSynthesis of NO-npThe synthesis of NO-np was recently reported, along with its potential as a treatment for skin and soft tissue infections.11Friedman A.J. Han G. Navati M.S. Chacko M. Gunther L. Alfieri A. Friedman J.M. Sustained release nitric oxide releasing nanoparticles: characterization of a novel delivery platform based on nitrite containing hydrogel/glass composites.Nitric Oxide. 2008; 19: 12-20Crossref PubMed Scopus (163) Google Scholar, 13Martinez L.R. Han G. Chacko M. Mihu M.R. Jacobson M. Gialanella P. Friedman A.J. Nosanchuk J.D. Friedman J.M. Antimicrobial and healing efficacy of sustained release nitric oxide nanoparticles against Staphylococcus aureus skin infection.J Invest Dermatol. 2009; 129: 2463-2469Crossref PubMed Scopus (199) Google Scholar Briefly, a hydrogel-glass composite was synthesized using a mixture of tetramethylorthosilicate, polyethylene glycol, chitosan, glucose, and sodium nitrite in 0.5 mol/L sodium phosphate buffer (pH 7). The nitrite was reduced to NO within the matrix because of the glass properties of the composite affecting oxidation-reduction reactions initiated with thermally generated electrons from glucose. After the oxidation-reduction reaction, the ingredients were combined and dried with a lyophilizer, resulting in a fine powder composed of nanoparticles containing NO. Once exposed to an aqueous environment, the hydrogel properties of the composite allow for opening of water channels inside the particles, facilitating the release of the trapped NO over extended time periods. Control nanoparticles consist of the same formulation as NO-np, but without the addition of sodium nitrite.In Vivo Wound Model and NO-np TreatmentTo investigate the wound-healing efficacy of NO-np, female BALB/c mice (aged 6 to 8 weeks; National Cancer Institute, Bethesda, MD) were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine; the hair on their flanks was shaved; and the skin was disinfected with iodine. Then, sterile silicone rings were glued and stitched outside of a full-thickness wound generated by cutting out a template generated with a single 5-mm punch biopsy specimen on the back of mice. Next, a suspension with 5 mg/mL of NO-np or np dissolved in PBS was topically applied to the wounds. Mice were subsequently treated with NO-np or np every other day. Mice were housed separately, and their wounds were covered with an occlusive dressing (Tegaderm; 3M, St Paul, MN) to prevent microbial infections; the dressing was removed and replaced with a new dressing with each application of treatment (or at the same time the other groups were treated, for the control groups). All animal studies were conducted according to the experimental practices and standards approved by the Animal Welfare and Research Ethics Committee at the Albert Einstein College of Medicine, Bronx, NY.Measurement of NO ProductionTo distinguish the efficacy of NO delivered via nanoparticles from basal levels, NO production in the wounds of BALB/c mice, untreated or treated with aminoguanidine (AG), np, or NO-np was quantified 4 hours after treatment using the Griess method (Promega, Fitchburg, WI) according to the manufacturer's protocol. AG inhibits the expression of inducible NO synthase (iNOS) and was administered to the mice every other day by i.p. injection at a dose of 20 mg/kg. We chose the 4-hour time point after treatment to measure NO production because our previous work13Martinez L.R. Han G. Chacko M. Mihu M.R. Jacobson M. Gialanella P. Friedman A.J. Nosanchuk J.D. Friedman J.M. Antimicrobial and healing efficacy of sustained release nitric oxide nanoparticles against Staphylococcus aureus skin infection.J Invest Dermatol. 2009; 129: 2463-2469Crossref PubMed Scopus (199) Google Scholar has shown that this time point represents an intermediate between the initial burst and steady state of NO released from NO-np.Western Blot AnalysisTo further characterize the relationship between exogenous NO administration via nanoparticles and basal NO production, we assessed iNOS expression in the wounds of BALB/c mice by using Western blot analysis. On day 7 after wounding, tissue was excised, immediately frozen in liquid nitrogen, and stored at −80°C. The wound tissue was then homogenized in an ice-water bath with 8 vol of lysis buffer, containing 50 mmol/L HEPES, pH 7.4; 100 mmol/L 2-mercaptoethanol; 1 mmol/L EDTA; 1 mmol/L ethylene glycol bis (2-aminoethylether)-tetra acetic acid; 1% Tween 20; 1% Triton X-100; 1 mg/mL each of pepstatin, leupeptin, and aprotinin; 1 mmol/L phenylmethylsulfonyl fluoride; 1 mmol/L sodium orthovanadate; and 1 mmol/L sodium fluoride. The mixture was centrifuged at 10,000 × g for 10 minutes at 4°C, and the protein content of the supernatant was determined by the Bradford method using a protein assay kit (Bio-Rad Laboratories, Berkeley, CA). The supernatant was added with a sample buffer containing 1.6% sodium dodecyl sulfate, 5% glycerol, 0.1 mol/L dithiothreitol, 0.002% bromphenol blue, and 62.5 mmol/L Tris-HCl (pH 6.8), and the mixture was heated to 100°C for 5 minutes. Protein, 55 μg, was applied to each lane of a gradient gel (5% to 20%; Bio-Rad Laboratories). After electrophoresis at a constant 40 mA per gel, the proteins were transferred to a 0.2-mm polyvinylidene fluoride membrane (Bio-Rad Laboratories) and briefly stained with Ponceau S (Sigma-Aldrich, St. Louis, MO) to verify efficient transfer. The polyvinylidene fluoride membrane was incubated for 30 minutes at 37°C in a blocking solution containing 5% nonfatty dry milk, 0.04 mol/L Tris-HCl (pH 7.6), 0.8% NaCl, and 0.5% Tween 20. This was followed by a 30-minute incubation at 37°C with a rabbit polyclonal anti-iNOS antibody (Abcam, Boston, MA), with a subsequent peroxidase-linked anti-rabbit secondary antibody diluted in the blocking solution. Protein bands were detected and quantified with a Lumino image analyzer (LAS-1000; Fuji Film, Tokyo, Japan) after staining with electrochemiluminescence plus chemiluminescence detection reagents (Amersham Biosciences Co., Piscataway, NJ).Histological ExaminationsAt day 7 after wounding, wound tissues were excised from euthanized mice, fixed in 10% formalin for 24 hours, processed, and embedded in paraffin. Vertical sections (4 μm thick) were fixed to glass slides and subjected to H&E, Gomori's trichrome, CD34, Iba1, and myeloperoxidase (MPO) staining to examine tissue morphological characteristics, collagen deposition, vascularization, macrophagelike cell infiltration, and neutrophil infiltration, respectively. Slides were examined by light microscopy with an Olympus AX70 microscope (Olympus, Melville, NY), and images were obtained [QImaging Retiga 1300 digital camera (QImaging, Burnaby, BC, Canada)] with QCapture Suite V2.46 software (QImaging).In Vitro Wound Scratch Assay Using Human FibroblastsHuman dermal fibroblasts, a gift from Dr. Yuling Chi (Albert Einstein College of Medicine, Bronx, NY), were seeded in 6-well plates and grown until subconfluence in Dulbecco's modified Eagle's medium with supplementation of penicillin-streptomycin and 10% fetal calf serum (Mediatech, Inc., Manassas, VA). A sterile cell scraper, trimmed to 15 mm, was used to make a single-line wound across the middle of each plate, and the plates were subsequently washed with media to remove the detached cells. The remaining fibroblasts were incubated in the absence or presence of 5 mg of NO-np or np for 3 days. After 3 days, the plates were again washed with the basal media to remove any remaining NO-np or np. Blinded microscopic examinations by two investigators (G.H. and L.R.M.) were performed by light microscopy with an Axiovert 200 M inverted microscope (Carl Zeiss MicroImaging, Thornwood, NY), and measurements were taken by drawing lines through the leading edge of repopulation by the fibroblasts and quantifying the cleared, fibroblast-free space remaining.Real-Time RT-PCR for COL1 and COL3 Gene Expression Using Human FibroblastsHuman fibroblasts were grown to near confluence in Dulbecco's modified Eagle's medium/Ham's F-12 medium supplemented with 10% fetal calf serum, 1% HEPES (15 mmol/L), 1% nonessential amino acids, and 1% penicillin-streptomycin. Then, cells were incubated with or without 5 mg/mL NO-np or np at 37°C for 8 hours. After incubation, COL1 and COL3 gene expression levels were analyzed by quantitative RT-PCR. Briefly, cells were collected and washed, and then RNA was isolated according to the RNeasy kit protocol (Qiagen, Venlo, The Netherlands). For real-time RT-PCR detection of COL1 or COL3 transcripts, 10 μg of total RNA was treated with DNase at 37°C for 1 hour, precipitated with ethanol, and suspended in 100 μL of nuclease-free water. cDNA synthesis was performed from equal amounts of RNA in a cyclic Bio-Rad MyCycler (Bio-Rad Laboratories, Carlsbad, CA) using reagents from Invitrogen, according to the manufacturer's instructions. The expression of the COL1 gene was examined via RT-PCR with the following primers: 5′-ACGAAGACATCCCACCAATCA-3′ (forward) and 5′-TCACGTCATCGCACAACACC-3′ (reverse) for Col1A1 (access number NM_000088). The expression of the COL3 gene was examined via RT-PCR with the following primers: 5′-GGAAGCTGTTGAAGGAGGATG-3′ (forward) and 5′-CTGGGTTGGGGCAGTCTAAT-3′ (reverse) for Col3A1 (access number NM_000090). For an internal mRNA control, we used primers specific for glyceraldehyde-3-phosphate dehydrogenase (access number NM_002046), as follows: 5′-TTTGTCAAGCTCATTTCCTGG-3′ (forward) and 5′-TCTCTTCCTCTTGTGCTCTTGC-3′ (reverse). To confirm that similar concentrations of cDNA were achieved, the signals from glyceraldehyde-3-phosphate dehydrogenase PCR were compared. The COL1 and COL3 transcript levels were determined and quantitatively assessed using an iQ iCycler and Cycler iQ software (Bio-Rad Laboratories), respectively. The cycling conditions used were as follows: 95°C for 5 minutes and 40 cycles of 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. Next, the samples were cooled to 55°C, and a melting curve for temperatures between 55°C and 95°C, with 0.5°C increments, was recorded. Real-time expression measurements were normalized against the expression of the reference gene, ACT1. The relative RNA levels were calculated by using the threshold cycle (ΔΔCT) method; all primers resulted in amplification efficiencies of at least 95%. Experiments were performed twice in triplicate.TGF-β DeterminationsFive mice per group of wounded animals treated with or without NO-np or np were euthanized at day 7. Wound lesions were excised and homogenized in PBS with protease inhibitors (Complete Mini; Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT). Cell debris was removed from homogenates by centrifugation at 6000 × g for 10 minutes. Samples were stored at −80°C until tested. Supernatants were tested for transforming growth factor (TGF)-β by enzyme-linked immunosorbent assay (Becton Dickinson Biosciences Pharmingen, San Diego, CA). The limit of detection was 60 pg/mL for TGF-β.Statistical AnalysisAll data were subjected to statistical analysis using GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA). P values were calculated by analysis of variance and were adjusted by use of Bonferroni correction. P < 0.05 was considered significant.ResultsNO-np Enhances Wound Healing in VivoThe effect of NO-np on wound healing in BALB/c mice was investigated (Figure 1, A and B). After wounding, the wound closure progressed quickly in the NO-np mice (Figure 1A), reaching complete closure within 12 days after surgery (Figure 1B). In contrast, wound closure of untreated or np-only (np containing the same formulation as NO-np but without NO precursor or release) treated mice was significantly delayed (Figure 1B), and complete wound closure was not yet reached at day 15 after surgery when mice were euthanized. Thus, the rate of gross wound closure in NO-np–treated mice was improved by at least 3 days compared with np-treated mice. Application of NO-np ectopically onto murine wounds significantly accelerated closure at all time points (Figure 1B). At day 7, the amount of wound closure was approximately 5% for the untreated (P < 0.001) and approximately 20% for the np-treated (P < 0.001) groups, compared with the NO-np–treated group (Figure 1C).Histological examination findings revealed that wounds of untreated mice had intense localized inflammatory infiltrate in the epidermis, whereas wounds of np-treated mice displayed a diffuse and intense inflammatory infiltration reaching the dermal layer (Figure 1D). Tissue sections from the wounds of NO-np–treated mice revealed intact structural morphological characteristics and significantly less inflammation (Figure 1D).NO-np Enhances Wound Healing by Increasing Fibroblast Migration and Collagen DepositionThe mechanisms through which the NO-np accelerates wound healing were further explored by examining whether NO-np increased fibroblast migration and collagen deposition in wound tissue (Figure 2). Collagen content was highest in wounds treated with NO-np (Figure 2A). The dispersed blue stain indicated thicker and more mature tissue collagen formation in wounds treated with NO-np, suggesting that NO-np exposure maintained dermal architecture through fibroblast migration and ultimately collagen deposition. Figure 2B is a morphometric analysis of the data shown in Figure 2A.Figure 2NO-np enhances wound healing by increasing fibroblast migration and collagen deposition. A: Histological analysis of untreated, np-treated, and NO-np–treated BALB/c mice at day 7. The blue stain indicates collagen. Scale bar = 25 μm. B: Quantitative measurement of collagen intensity in 10 representative fields of the same size for untreated, np-treated, and NO-np–treated wounds. Data are given as the average of the results, and error bars denote SDs. *P < 0.05 in comparing the untreated group with the np- and NO-treated groups. C: Light microscopic images of human dermal fibroblast migration, as evaluated by scratch assay. NO-np–treated fibroblasts migrate faster toward the scratched area than untreated or np-treated cells. Dashed lines denote the scratched area. Scale bar = 25 μm. Fibroblast experiments were repeated three times, with similar results. D: Wound closure analysis of BALB/c mice skin lesions. Wounds were untreated or treated in the absence or presence of NO. Time points are the averages of the results for 10 independent measurements, and error bars denote SDs. *P < 0.05 (day 1), **P < 0.01 (day 3) in comparing the NO-treated groups with the untreated groups. Five animals per group were used. These experiments were performed twice, with similar results. E: Gene expression analysis of collagen types I and III in human dermal fibroblasts. ***P < 0.001 in comparing the np- and NO-treated groups with the untreated groups. This experiment was performed twice, with similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Human dermal fibroblasts were used to investigate whether NO-np stimulates migration and collagen deposition. To study fibroblast migration, an in vitro scratch test was performed. We demonstrated that NO-np–treated fibroblasts migrate faster toward the scratched area than untreated or np-treated cells (Figure 2C). One day after injury, the NO-np–treated fibroblasts significantly repopulated 55% of the scratched area compared with 40% by the untreated or np-treated cells (P < 0.05) (Figure 2D). Three days after injury, fibroblasts treated with NO-np displayed significantly improved 75% wound closure when compared with 60% and 40% observed in the untreated and np control groups, respectively (P < 0.01) (Figure 2D). In addition, NO-np accelerated fibroblast proliferation because a homogeneous monolayer of cells was formed by day 7 (data not shown).To determine whether NO-np enhances collagen deposition during wound healing, we assessed the gene expression of collagen types I and III in human fibroblasts (Figure 2E). NO-np and np significantly increased collagen type I expression when compared with untreated control cells (P < 0.001) (Figure 2E). However, the effect of NO-np on fibroblast collagen type III expression was >20 times higher than on untreated or np-treated cells (P < 0.001) (Figure 2E).Together, our data suggest that NO-np augments wound healing by facilitating cell migration and collagen deposition.NO-np Increases Levels of NO in VivoDuring wound healing, there is a well studied expression and up-regulation of NO synthase, leading to increased levels of NO. By using nanoparticle technology to provide exogenous NO, we showed significantly higher NO levels in wounds when compared with untreated or np-treated mice (P < 0.001) (Figure 3A). To differentiate the levels of exogenous and physiological NO in wound healing, we treated mice with AG, a specific inhibitor of iNOS. AG suppressed NO levels in wounds.Figure 3NO-np increases levels of NO in vivo. A: NO production in the wounds (day 7) of BALB/c mice untreated or treated with AG, np, or NO-np was quantified using the Griess method. Data are given as the means of 10 measurements, and error bars denote SDs. P values were calculated by analysis of variance and adjusted by use of Bonferroni correction. *P < 0.001 in comparing the NO-np–treated group with the untreated and np-treated groups; †P < 0.001 in comparing the AG-treated group with the untreated or np-or NO-np–treated groups. Five animals per group were used. These experiments were performed twice, with similar results. B: Western blot analysis of wounded dermal tissue from untreated or AG-, np-, or NO-np–treated BALB/c mice was performed to compare the expression of iNOS. These experiments were performed twice, with consistent results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further characterize the relationship between exogenous NO administration via nanoparticles and basal NO production, we assessed iNOS expression using Western blot analysis. We demonstrated a significant decrease in iNOS expression in animals treated with AG when compared with untreated, np-treated, or NO-np–treated mice (Figure 3B).Our results revealed that only topically applied NO, delivered via nanoparticles, supplements NO physiological levels and, therefore, accelerates wound healing.NO-np Modifies Leukocyte Infiltration to the Wounded Skin TissueWe investigated the effect of NO-np in leukocyte migration to the wound area (Figure 4, Figure 5). First, neutrophil infiltration was evaluated by measuring the production of MPO in the wound area. MPO is an enzyme most abundantly present in neutrophils. The localized brown stains indicated more neutrophil infiltration in untreated or np-treated compared with NO-np–treated wounds (Figure 4A). Our quantitative analysis confirmed the presence of fewer neutrophils in wounds of NO-np–treated mice per field when compared with wounds of untreated or np-treated mice (P < 0.01) (Figure 4B).Figure 4NO-np decreases neutrophil infiltration in wounded skin. A: Histological analysis of untreated, np-treated, and NO-np–treated wounded BALB/c mice at day 7. The brown staining indicates neutrophil infiltration. Representative MPO-immunostained sections of the skin lesions are shown. Scale bar = 25 μm. B: Number of neutrophils per field in wounded skin tissue of untreated and np- and NO-np–treated mice. Data are given as the average number of neutrophils in 15 different fields, and error bars denote SDs. **P < 0.01 in comparing the NO-treated group with the untreated and np-treated groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5NO-np increases macrophagelike cell infiltration to the wounded skin tissue. A: Histological analysis of untreated, np-treated, and NO-np–treated wounded BALB/c mice at day 7. The brown staining indicates macrophagelike cell infiltration. Representative Iba1–immunostained sections of the skin lesions are shown. Scale bar = 25 μm. B: Number of macrophages per field in wounded skin tissue of untreated and np- and NO-np–treated mice. Data are given as the average number of macrophages in 15 different fields, and error bars denote SDs. *P < 0.05, **P < 0.01 in comparing the NO-treated group with the np-treated and untreated groups, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Second, we identified macrophagelike infiltration by measuring the expression of Iba1, which is a general marker mostly expressed and up-regulated during the activation of these cells. Tissue sections from untreated murine wounds showed only scattered macrophagelike cells, whereas np- and NO-np–treated mice showed a massive macrophagelike cell infiltration to the wound area (Figure 5" @default.
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• W2053070963 title "Nitric Oxide–Releasing Nanoparticles Accelerate Wound Healing by Promoting Fibroblast Migration and Collagen Deposition" @default.
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