Increased nitric oxide formation followed by increased arginase activity induces relative lack of arginine at the wound site and alters whole nutritional status in rats almost within the early healing period

Nitric oxide (NO) production and free amino acid fluxes at the wound side during the first 3 days following cutaneous wound were investigated. Experiments were performed on Albino Oxford rats (n = 18) underwent cutaneous implantation of polyvinyl sponges. Intact animals (n = 6) were controls. Nitrites, nitrates, free amino acids and urea were measured both in plasma and wound fluids. Inducible nitric oxide synthase (iNOS) gene expressions at wound site were analyzed, too. The highest levels of both iNOS gene expression and its activity (increased wound fluid citrulline and nitrites) were at the first day. Wound fluid nitrates were significantly above plasma levels throughout the whole period, while molar nitrate to nitrite ratio steadily increased. It was associated with gradual increase of both ornithine and urea as well as steadily decreases of arginine and increases of phenylalanine at the wound site. Gradual decrease in glycine to branched-chain molar ratio was observed both in plasma and wound fluids. In conclusion, an early locally induced alterations in Arg metabolism, due to increased NO formation followed by increased arginase activity, produces relative lack of Arg at the wound site and disturbs nutritional status of the whole body almost within early healing period following cutaneous wound in rats. It is likely that NO autoxidation at the wound side is influenced by substrate availability.     

Improved cartilage integration and interfacial strength after enzymatic treatment in a cartilage transplantation model

The objective of the present study was to investigate whether treatment of articular cartilage with hyaluronidase and collagenase enhances histological and mechanical integration of a cartilage graft into a defect. Discs of 3 mm diameter were taken from 8-mm diameter bovine cartilage explants. Both discs and annulus were either treated for 24 hours with 0.1% hyaluronidase followed by 24 hours with 10 U/ml collagenase or left untreated (controls). Discs and annulus were reassembled and implanted subcutaneously in nude mice for 5 weeks. Integration of disc with surrounding cartilage was assessed histologically and tested biomechanically by performing a push-out test. After 5 weeks a significant increase in viable cell counts was seen in wound edges of the enzyme-treated group as compared with controls. Furthermore, matrix integration (expressed as a percentage of the total interface length that was connected; mean ± standard error) was 83 ± 15% in the treated samples versus 44 ± 40% in the untreated controls. In the enzyme-treated group only, picro-Sirius Red staining revealed collagen crossing the interface perpendicular to the wound surface. Immunohistochemical analyses demonstrated that the interface tissue contained cartilage-specific collagen type II. Collagen type I was found only in a small region of fibrous tissue at the level of the superficial layer, and collagen type III was completely absent in both groups. A significant difference in interfacial strength was found using the push-out test: 1.32 ± 0.15 MPa in the enzyme-treated group versus 0.84 ± 0.14 MPa in the untreated controls. The study shows that enzyme treatment of cartilage wounds increases histological integration and improves biomechanical bonding strength. Enzymatic treatment may represent a promising addition to current techniques for articular cartilage repair.     

Collagen polymorphism in experimental granulation tissue.

Dermal granulation tissues produced either in response to an acute inflammation by turpentine injection, or to a chronic inflammation by sponge implantation, have been shown to contain a high proportion of Type III collagen in marked contrast to the small amount normally present in mature skin. Although the acute granuloma is rapidly resorbed the synthesis of Type III is maintained in long-term sponge implants. The results demonstrate a reversion to embryonic collagen in proliferating granulation tissue, a fact of considerable importance in understanding wound healing.     

精氨酸和一氧化氮合成的关系及其在免疫调节中的作用

精氨酸 (Arginine,Arg)因其具有广泛的生物学作用而在所有氨基酸中占有重要地位。一定剂量的Arg可促进创伤愈合和胶原生成 ,刺激若干内分泌腺分泌激素 ,调节某些类型肿瘤生长 ,并增强细胞介导的免疫应答。因而常将Arg作为免疫增强膳食的一部分来对抗应激及严重疾病病人中常见的免疫抑制。Arg是高活性基团一氧化氮 (nitricoxide ,NO)的唯一供氮前体 ,已认定NO对越来越多的细胞过程有影响并存在于全身的许多组织。它可影响多种类型的免疫细胞。Arg自身或通过生成的NO参与了许多免疫调节过程 ,因此 ,对Arg体内合成、降解及外源性Arg转运在NO合成中作用的了解对其合理应用具有重要意义     

Nitric oxide produced by iNOS is associated with collagen synthesis in keloid scar formation

Nitric oxide (NO) has emerged as an important mediator of many physiological functions. Recent reports have shown that NO participates in the wound healing process, however, its role in keloid formation remains unclear. This study aimed to investigate the effect of NO on keloid fibroblasts (KF) and to determine the levels of inducible nitric oxide synthase (iNOS) expression in clinical specimens of keloid. Scar tissue from seven keloid patients with matched perilesion skin tissue controls was studied for inducible nitric oxide synthase expression and location. In addition, primary keloid and normal scar skin fibroblast cultures were set up to investigate the effects of NO in inducing collagen type I expression. Inducible nitric oxide synthase expression, and NO production were elevated in keloid scar tissues but not in matched perilesion skin tissues. Furthermore, exposure of KF to exogenous NO resulted in increased expression of collagen type I in a dose-dependent manner. NO exposure also induced time-course dependent collagen I expression that peaked at 24h in KF. Taken together, these results indicate that excess collagen formations in keloid lesion may be attributed to iNOS overexpression.     

The effects of arginine administration on the levels of arginine,other amino acids and related amino compounds in the plasma,heart, aorta,vena cava,bronchi and pancreas of the rat

Arginine (0.8g/kg, ip) or a vehicle was administered to rats and the levels of arginine and a large number of related amino compounds were measured in plasma, heart, aorta, vena cava, pancreas and bronchi at specified time intervals. Arginine levels (nmol/ml) increased in the plasma from 237 to 3172 at 15 min, 1236 at 30 min and 509 at 120 min. Peak concentrations (nmol/g) of arginine are reached in the tissues at 15 or 30 minutes with control and postinjection values of 500 and 1769 in the heart, 314 and 1563 in the aorta, 575 and 2976 in the vena cava, 760 and 1943 in the bronchi, and 234 and 3638 in the pancreas. Arginine injection also affects a number of amino acids and related compounds in the plasma and tissues most notably ornithine, isoleucine, phosphoserine, leucine and ethanolamine. However, plasma level changes do not predict tissue level changes which are highly specific for an individual compound and tissue. There was no general indication that arginine injection stimulates nitric oxide (NO) formation in any tissue. Thus, arginine is rapidly absorbed from the abdominal cavity into the blood stream, is quickly taken up by the tissues studied and disappears after about 2 to 3 hours. The effects seen after arginine administration could be caused by arginine per se and/or changes in one or more of the related amino compounds but not by NO.     

Tolerance and Long-Term MRI Imaging of Gadolinium-Modified Meshes Used in Soft Organ Repair

Background

Synthetic meshes are frequently used to reinforce soft tissues. The aim of this translational study is to evaluate tolerance and long-term MRI visibility of two recently developed Gadolinium-modified meshes in a rat animal model.

Materials and Methods

Gadolinium-poly-ε-caprolactone (Gd-PCL) and Gadolinium-polymethylacrylate (Gd-PMA) modified meshes were implanted in Wistar rats and their tolerance was assessed daily. Inflammation and biocompatibility of the implants were assessed by histology and immunohistochemistry after 30 days post implantation. Implants were visualised by 7T and 3T MRI at day 30 and at day 90. Diffusion of Gadolinium in the tissues of the implanted animals was assessed by Inductively Coupled Plasma Mass Spectrometry.

Results

Overall Gd-PMA coated implants were better tolerated as compared to those coated with Gd-PCL. In fact, Gd-PMA implants were characterised by a high ratio collagen I/III and good vascularisation of the integration tissues. High resolution images of the coated mesh were obtained in vivo with experimental 7T as well as 3T clinical MRI. Mass spectrometry analyses showed that levels of Gadolinium in animals implanted with coated mesh were similar to those of the control group.

Conclusions

Meshes coated with Gd-PMA are better tolerated as compared to those coated with Gd-PCL as no signs of erosion or significant inflammation were detected at 30 days post implantation. Also, Gd-PMA coated meshes were clearly visualised with both 7T and 3T MRI devices. This new technique of mesh optimisation may represent a valuable tool in soft tissue repair and management.     

Nitric oxide inhibits wound collagen synthesis

Nitric oxide (NO) is a messenger molecule which regulates many physiological functions like immunity, vascular tone and serves as a neurotransmitter. Although it is known to participate in healing process, its role in collagen synthesis is not clear. Therefore, the present investigation was done to study the role of NO in wound collagen synthesis. Rats received full thickness, circular (8 mm), transdermal wounds which were treated with NO releaser, sodium nitroprusside (SNP, 0.001 100 M) topically for 5 days. Wound collagen content estimated in terms of hydroxyproline (HP) and confirmed histochemically was decreased significantly by all SNP doses. L-Arginine, a substrate for nitric oxide synthase (NOS) when applied topically decreased collagen content of the wounded tissues. N-Nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NOS, increased wound collagen content significantly as compared to untreated and SNP treated animal wounds when administered intraperitoneally at the doses 3, 10 and 30 mg/kg. Furthermore, histological findings also demonstrated laying down of thick collagen bundles and proliferation of fibroblasts together with prominent angiogenesis in L-NAME treated wound tissues as compared to untreated and SNP treated tissues. N-nitro-D-arginine methyl ester, an inactive isomer, was found to have no effect on wound collagen levels. When L-arginine was administered in L-NAME pretreated rats, it significantly elevated wound HP content. The results indicate that NO plays an important role in regulating the collagen biosynthesis in skin model of a healing wound.     

Localization and synthesis of type III collagen and fibronectin in human reparative dentine

Summary The injury of dental pulp tissue, following caries, is accompanied by the deposit of a typical hard scar tissue known as reparative dentine which should be regarded as the mineralization of a new organic matrix. Highly purified antibodies were used in combination with immunoperoxidase or immunogold technique at the ultrastructural level to reveal the distribution and synthesis of types I and III collagen and fibronectin elaborated by typical matrix-forming cells in the new tissue.Specific immunoperoxidase labelling, on demineralized teeth, clearly demonstrated that type I collagen represents the main type of collagen (88%). It is associated with bundles of fine striated fibrils of type III collagen and in close vicinity with fibronectin and constituted, at least, the new organic matrix of reparative dentine.Immunogold staining gave precise localization mainly over Golgi apparatus for the 3 components, thus suggesting that the cells concerned should not be considered as new odontoblasts but rather as pulpal cells in the process of differentiation participating in the formation of new dentine. Moreover, these events are very similar to those observed during wound healing in other tissues.     

Localization and synthesis of type III collagen and fibronectin in human reparative dentine. Immunoperoxidase and immunogold staining

The injury of dental pulp tissue, following caries, is accompanied by the deposit of a typical hard scar tissue known as reparative dentine which should be regarded as the mineralization of a new organic matrix. Highly purified antibodies were used in combination with immunoperoxidase or immunogold technique at the ultrastructural level to reveal the distribution and synthesis of types I and III collagen and fibronectin elaborated by typical matrix-forming cells in the new tissue. Specific immunoperoxidase labelling, on demineralized teeth, clearly demonstrated that type I collagen represents the main type of collagen (88%). It is associated with bundles of fine striated fibrils of type III collagen and in close vicinity with fibronectin and constituted, at least, the new organic matrix of reparative dentine. Immunogold staining gave precise localization mainly over Golgi apparatus for the 3 components, thus suggesting that the cells concerned should not be considered as new odontoblasts but rather as pulpal cells in the process of differentiation participating in the formation of new dentine. Moreover, these events are very similar to those observed during wound healing in other tissues.     

Time course of nitric oxide production after endotoxin challenge in mice

Nitric oxide (NO) regulates numerous processes during endotoxemia and inflammation. However, the sequential changes in whole body (Wb) nitric oxide (NO) production during endotoxemia in vivo remain to be clarified. Male Swiss mice were injected intraperitoneally with saline (control group) or lipopolysaccharide (LPS group). After 0, 2, 4, 6, 9, 12, and 24 h, animals received a primed constant infusion of L-[guanidino-(15)N(2)-(2)H(2)]arginine, L-[ureido-(15)N]citrulline, L-[5-(15)N]glutamine, and L-[ring-(2)H(5)]phenylalanine in the jugular vein. Arterial blood was collected for plasma arginine (Arg), citrulline (Cit), glutamine (Gln), and phenylalanine (Phe) concentrations and tracer-to-tracee ratios. NO production was calculated as plasma Arg-to-Cit flux, Wb de novo Arg synthesis as plasma Cit-to-Arg flux, and Wb protein breakdown as plasma Phe flux. LPS reduced plasma Arg and Cit and increased Gln and Phe concentrations. Two peaks of NO production were observed at 4 and 12 h after LPS. Although LPS did not affect total Arg production, de novo Arg production decreased after 12 h. The second peak of NO production coincided with increased Wb Cit, Gln, and Phe production. In conclusion, the curve of NO production in both early and late phases of endotoxemia is not related to plasma Arg kinetics. However, because Wb Cit, Gln, and Phe fluxes increased concomitantly with the second peak of NO production, NO production is probably related to the catabolic phase of endotoxemia.     

Multiscale mechanobiology: Coupling models of adhesion kinetics and nonlinear tissue mechanics

The mechanical behavior of tissues at the macroscale is tightly coupled to cellular activity at the microscale. Dermal wound healing is a prominent example of a complex system in which multiscale mechanics regulate restoration of tissue form and function. In cutaneous wound healing, a fibrin matrix is populated by fibroblasts migrating in from a surrounding tissue made mostly out of collagen. Fibroblasts both respond to mechanical cues, such as fiber alignment and stiffness, as well as exert active stresses needed for wound closure.Here, we develop a multiscale model with a two-way coupling between a microscale cell adhesion model and a macroscale tissue mechanics model. Starting from the well-known model of adhesion kinetics proposed by Bell, we extend the formulation to account for nonlinear mechanics of fibrin and collagen and show how this nonlinear response naturally captures stretch-driven mechanosensing. We then embed the new nonlinear adhesion model into a custom finite element implementation of tissue mechanical equilibrium. Strains and stresses at the tissue level are coupled with the solution of the microscale adhesion model at each integration point of the finite element mesh. In addition, solution of the adhesion model is coupled with the active contractile stress of the cell population. The multiscale model successfully captures the mechanical response of biopolymer fibers and gels, contractile stresses generated by fibroblasts, and stress-strain contours observed during wound healing. We anticipate that this framework will not only increase our understanding of how mechanical cues guide cellular behavior in cutaneous wound healing, but will also be helpful in the study of mechanobiology, growth, and remodeling in other tissues.     

Role of arginine in superficial wound healing in man

Arginine supplementation has been identified as advantageous in experimental wound healing. However, the mechanisms underlying this beneficial effect in tissue repair remain unresolved. Animal studies suggest that the beneficial role of arginine supplementation is mediated, at least in part through NO. The latter component mediates processes involved in tissue repair, including angiogenesis, epithelialization and collagen formation. This prospective study is performed to investigate arginine metabolism in acute surgical wounds in man. Expression of enzymes, known to be involved in arginine metabolism, was studied in donor sites of skin grafts of 10 hospitalized patients undergoing skin transplantation. Plasma and wound fluid levels of arginine metabolites (ornithine, citrulline, nitrate and nitrite = NOx) were measured using High Performance Liquid Chromatography. Expression of iNOS, eNOS, arginase-1 and arginase-2 was studied by immunohistochemistry in paraffin sections of skin tissue. Arginase-1 concentration was measured in plasma and wound fluid using ELISA. Arginase-2 was determined using Western blot analysis. We observed increased levels of citrulline, ornithine, NOx and arginase-1 in wound fluid when compared with plasma. Arginase-2 was expressed in both plasma and wound fluid and seemed higher in plasma. iNOS was expressed by neutrophils, macrophages, fibroblasts, keratinocytes and endothelial cells upon wounding, whereas eNOS reactivity was observed in endothelial cells and fibroblasts. Arginase-1 was expressed in neutrophils post-wounding, while arginase-2 staining was observed in endothelial cells, keratinocytes, fibroblasts, macrophages and neutrophils. For the first time, human data support previous animal studies suggesting arginine metabolism for an NO- as well as arginase-mediated reparation of injured skin.     

Intrinsic fibroblast-mediated remodeling of damaged collagenous matrices in vivo

Numerous studies have examined wound healing and tissue repair after a complete tissue rupture and reported provisional matrix and scar tissue formation in the injury gap. The initial phases of the repair are largely mediated by the coagulation response and a principally extrinsic inflammatory response followed by type III collagen deposition to form scar tissue that may be later remodeled. In this study, we examine subfailure (Grade II sprain) damage to collagenous matrices in which no gross tissue gap is present and a localized concentration of provisional matrix or scar tissue does not form. This results in extracellular matrix remodeling that relies heavily upon type I collagen, and associated proteoglycans, and less heavily on type III scar tissue collagen. For instance, following subfailure tissue damage, collagen I and III expression was suppressed after 1 day, but by day 7 expression of both genes was significantly increased over controls, with collagen I expression significantly larger than type III expression. Concurrent with increased collagen expression were significantly increased expression of the collagen fibrillogenesis supporting proteoglycans fibromodulin, lumican, decorin, the large aggregating proteoglycan versican, and proteases cathepsin K and L. Interestingly, this remodeling process appears intrinsic with little or no inflammation response as damaged tissues show no changes in macrophage or neutrophils levels following injury and expression of the inflammatory markers, tumor necrosis factor-alpha and tartrate-resistant acid phosphatase were unchanged. Hence, since inflammation plays a large role in wound healing by inducing cell migration and proliferation, and controlling extracellular matrix scar formation, its absence leaves fibroblasts to principally direct tissue remodeling. Therefore, following a Grade II subfailure injury to the collagen matrix, we conclude that tissue remodeling is fibroblast-mediated and occurs without scar tissue formation, but instead with type I collagen fibrillogenesis to repair the tissue. As such, this system provides unique insight into acute tissue damage and offers a potentially powerful model to examine fibroblast behavior.     

Development of osteogenic tissue in diffusion chambers from early precursor cells in bone marrow of adult rats

Diffusion chambers containing bone marrow cells from adult rats were implanted intraperitoneally into rat hosts and cultured in vivo for up to 64 days. Biochemical and histological analyses of the contents of the chambers demonstrate that a connective tissue consisting of bone, cartilage and fibrous tissues is formed by precursor cells present in marrow stroma. The amounts of osteogenic tissue and DNA are directly correlated with time of implantation and with number of cells inoculated. In the chambers there is initial formation of fibrous tissue which is strongly reactive to collagen type III, laminin and fibronectin. In areas of osteogenesis which appear later within this fibrous anlage, expression of collagen type III, laminin and fibronectin decrease and collagen types I and II increase in association with bone and cartilage respectively. Where osteogenesis does not develop, fibrous tissue continues to express collagen type III. The sequential expression of the different extracellular matrix components is similar to that previously observed during osteogenic differentiation in embryonic and adult developmental systems. It is concluded that the formation of fibrous and osteogenic tissues in diffusion chambers by precursor cells present in adult marrow, resembles the normal developmental process.     

Curcumin improves wound healing by modulating collagen and decreasing reactive oxygen species

Wound healing consists of an orderly progression of events that re-establish the integrity of the damaged tissue. Several natural products have been shown to accelerate the healing process. The present investigation was undertaken to determine the role of curcumin on changes in collagen characteristics and antioxidant property during cutaneous wound healing in rats. Full-thickness excision wounds were made on the back of rat and curcumin was administered topically. The wound tissues removed on 4th, 8th and 12th day (post-wound) were used to analyse biochemical and pathological changes. Curcumin increased cellular proliferation and collagen synthesis at the wound site, as evidenced by increase in DNA, total protein and type III collagen content of wound tissues. Curcumin treated wounds were found to heal much faster as indicated by improved rates of epithelialisation, wound contraction and increased tensile strength which were also confirmed by histopathological examinations. Curcumin treatment was shown to decrease the levels of lipid peroxides (LPs), while the levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), activities were significantly increased exhibiting the antioxidant properties of curcumin in accelerating wound healing. Better maturation and cross linking of collagen were observed in the curcumin treated rats, by increased stability of acid-soluble collagen, aldehyde content, shrinkage temperature and tensile strength. The results clearly substantiate the beneficial effects of the topical application of curcumin in the acceleration of wound healing and its antioxidant effect. Both the authors have contributed equally towards this paper.     

A Preclinical Evaluation of Alternative Synthetic Biomaterials for Fascial Defect Repair Using a Rat Abdominal Hernia Model

Introduction

Fascial defects are a common problem in the abdominal wall and in the vagina leading to hernia or pelvic organ prolapse that requires mesh enhancement to reduce operation failure. However, the long-term outcome of synthetic mesh surgery may be unsatisfactory due to post-surgical complications. We hypothesized that mesh fabricated from alternative synthetic polymers may evoke a different tissue response, and provide more appropriate mechanical properties for hernia repair. Our aim was to compare the in vivo biocompatibility of new synthetic meshes with a commercial mesh.

Methods

We have fabricated 3 new warp-knitted synthetic meshes from different polymers with different tensile properties polyetheretherketone (PEEK), polyamide (PA) and a composite, gelatin coated PA (PA+G). The rat abdominal hernia model was used to implant the meshes (25×35 mm, n = 24/ group). After 7, 30, 60, 90 days tissues were explanted for immunohistochemical assessment of foreign body reaction and tissue integration, using CD31, CD45, CD68, alpha-SMA antibodies. The images were analysed using an image analysis software program. Biomechanical properties were uniaxially evaluated using an Instron Tensile® Tester.

Results

This study showed that the new meshes induced complex differences in the type of foreign body reaction over the time course of implantation. The PA, and particularly the composite PA+G meshes, evoked a milder early inflammatory response, and macrophages were apparent throughout the time course. Our meshes led to better tissue integration and new collagen deposition, particularly with the PA+G meshes, as well as greater and sustained neovascularisation compared with the PP meshes.

Conclusion

PA, PA+G and PEEK appear to be well tolerated and are biocompatible, evoking an overlapping and different host tissue response with time that might convey mechanical variations in the healing tissue. These new meshes comprising different polymers may provide an alternative option for future treatment of fascial defects.     

Role of Arginase 1 from Myeloid Cells in Th2-Dominated Lung Inflammation

Th2-driven lung inflammation increases Arginase 1 (Arg1) expression in alternatively-activated macrophages (AAMs). AAMs modulate T cell and wound healing responses and Arg1 might contribute to asthma pathogenesis by inhibiting nitric oxide production, regulating fibrosis, modulating arginine metabolism and restricting T cell proliferation. We used mice lacking Arg1 in myeloid cells to investigate the contribution of Arg1 to lung inflammation and pathophysiology. In six model systems encompassing acute and chronic Th2-mediated lung inflammation we observed neither a pathogenic nor protective role for myeloid-expressed Arg1. The number and composition of inflammatory cells in the airways and lungs, mucus secretion, collagen deposition, airway hyper-responsiveness, and T cell cytokine production were not altered if AAMs were deficient in Arg1 or simultaneously in both Arg1 and NOS2. Our results argue that Arg1 is a general feature of alternative activation but only selectively regulates Th2 responses. Therefore, attempts to experimentally or therapeutically inhibit arginase activity in the lung should be examined with caution.