Local NO synthase inhibition produces histological and functional recovery in Achilles tendon of rats after tenotomy

Repair of injured tendon is a very slow process and involves the release of many molecules, including nitric oxide. We investigate the influence of local nitrergic inhibition in histological and functional recovery of injured Achilles tendon. A standard murine model of tendon injury by rupture was used. The animals were divided into three experimental groups: control, injury + vehicle (normal saline) and injury + Nω-nitro-L-arginine methyl ester (L-NAME). The products were injected into the paratendinous region every 2 days and body weight gain and Achilles functional index (AFI) were evaluated on days 0, 7, 14 and 21 after tendon injury. On day 21 post-injury, the animals were killed to evaluate nitric oxide production and tissue organization. We observed that tendon surgical division led to increased tissue nitrite levels, which were reduced in L-NAME-treated rats. The AFI revealed functional recovery of L-NAME-treated animals on day 21 post-injury, which was not observed in the saline-treated group. Microscopic analysis of hematoxylin-eosin staining and collagen autofluorescence showed that L-NAME-treated rats had more aligned areas of collagen fibers and that the diameter of newly organized collagen in this group was also greater than that in the vehicle-treated one. We demonstrate that local treatment with L-NAME significantly improves the functional parameters and accelerates histomorphological recovery.     

Early voluntary exercise does not promote healing in a rat model of Achilles tendon injury.

Mechanical stress is an important modulator of connective tissue repair. However, the effects on tendon healing are very poorly defined, preventing optimal use of mechanical stress. We hypothesized that early voluntary exercise initially retards tendon repair but results in a faster recovery rate at longer term. Male Wistar rats were injured by a collagenase injection in the Achilles tendon, and exercise was voluntarily performed on a running wheel. We observed the persistent presence of neutrophils in injured tendons of rats that began exercise immediately after the trauma [injured + early exercise (Inj+EEx)]. Early exercise also increased the concentration of ED1(+) macrophages in injured tendons after 3 and 7 days compared with ambulatory injured rats (Inj). Similar results were obtained with the subset of ED2(+) macrophages in the tendon core 3 days after the collagenase injection. Furthermore, collagen content returned to normal values more rapidly in the Inj+EEx tendons than in the Inj group, but this was not associated with an increase in cell proliferation. Surprisingly, Inj+EEx tendons roughly displayed lower stiffness and force at rupture point relative to Inj tendons at day 28. Injured tendons of rats that began exercise only from day 7 had better mechanical properties than those of early-exercised rats 28 days postinjury. We speculate that the persistence of the inflammatory response and undue mechanical loading in the Inj+EEx tendons led to fibrosis and a loss of tendon function.     

Chronic low-dose L-NAME treatment increases nitric oxide production and vasorelaxation in normotensive rats

N(G)-nitro-L-arginine methyl ester (L-NAME) is a non-specific nitric oxide (NO) synthase inhibitor, commonly used for the induction of NO-deficient hypertension. The aim of this study was to investigate the effect of chronic low-dose administration of L-NAME on NO production, vascular function and structure of the heart and selected arteries of rats. Adult male Wistar rats were treated with L-NAME in the dose of approximately 1.5 mg/kg/day in drinking water for 8 weeks. Basal blood pressure (BP) of rats (determined by tail-cuff) was 112+/-3 mm Hg. The low-dose administration of L-NAME significantly elevated BP measured on the third and sixth week of treatment vs. controls by approximately 9 % and 12 %, respectively. After this period, BP of L-NAME-treated rats returned to the control values. The relative left ventricular mass, heart fibrosis and collagen III/collagen I ratio were not affected by L-NAME. Similarly, there were no alterations in the cross-sectional area and wall thickness/diameter ratio of the aorta and the femoral artery of L-NAME-treated rats. NO synthase activity (determined by conversion of [(3)H]-L-arginine to [(3)H]-L-citrulline) was not altered in the hypothalamus of L-NAME-treated rats. Interestingly, chronic low-dose L-NAME treatment significantly elevated NO synthase activity in the left ventricle and aorta, increased endothelium-dependent acetylcholine-induced vasorelaxation and reduced serotonin-induced vasoconstriction of the femoral artery. The data suggest that chronic low-dose L-NAME treatment can increase NO production and vasorelaxation in normotensive rats without negative structural changes in the cardiovascular system.     

Programmable mechanical stimulation influences tendon homeostasis in a bioreactor system

Identification of functional programmable mechanical stimulation (PMS) on tendon not only provides the insight of the tendon homeostasis under physical/pathological condition, but also guides a better engineering strategy for tendon regeneration. The aims of the study are to design a bioreactor system with PMS to mimic the in vivo loading conditions, and to define the impact of different cyclic tensile strain on tendon. Rabbit Achilles tendons were loaded in the bioreactor with/without cyclic tensile loading (0.25 Hz for 8 h/day, 0–9% for 6 days). Tendons without loading lost its structure integrity as evidenced by disorientated collagen fiber, increased type III collagen expression, and increased cell apoptosis. Tendons with 3% of cyclic tensile loading had moderate matrix deterioration and elevated expression levels of MMP‐1, 3, and 12, whilst exceeded loading regime of 9% caused massive rupture of collagen bundle. However, 6% of cyclic tensile strain was able to maintain the structural integrity and cellular function. Our data indicated that an optimal PMS is required to maintain the tendon homeostasis and there is only a narrow range of tensile strain that can induce the anabolic action. The clinical impact of this study is that optimized eccentric training program is needed to achieve maximum beneficial effects on chronic tendinopathy management. Biotechnol. Bioeng. 2013; 110: 1495–1507. © 2012 Wiley Periodicals, Inc.     

Tendon crimps and peritendinous tissues responding to tensional forces

Tendons transmit forces generated from muscle to bone making joint movements possible. Tendon collagen has a complex supramolecular structure forming many hierarchical levels of association; its main functional unit is the collagen fibril forming fibers and fascicles. Since tendons are enclosed by loose connective sheaths in continuity with muscle sheaths, it is likely that tendon sheaths could play a role in absorbing/transmitting the forces created by muscle contraction. In this study rat Achilles tendons were passively stretched in vivo to be observed at polarized light microscope (PLM), scanning electron microscope (SEM) and transmission electron microscope (TEM). At PLM tendon collagen fibers in relaxed rat Achilles tendons ran straight and parallel, showing a periodic crimp pattern. Similarly tendon sheaths showed apparent crimps. At higher magnification SEM and TEM revealed that in each tendon crimp large and heterogeneous collagen fibrils running straight and parallel suddenly changed their direction undergoing localized and variable modifications. These fibril modifications were named fibrillar crimps. Tendon sheaths displayed small and uniform fibrils running parallel with a wavy course without any ultrastructural aspects of crimp. Since in passively stretched Achilles tendons fibrillar crimps were still observed, it is likely that during the tendon stretching, and presumably during the tendon elongation in muscle contraction, the fibrillar crimp may be the real structural component of the tendon crimp acting as shock absorber. The peritendinous sheath can be stretched as tendon, but is not actively involved in the mechanism of shock absorber as the fibrillar crimp. The different functional behaviour of tendons and sheaths may be due to the different structural and molecular arrangement of their fibrils.     

Uphill running improves rat Achilles tendon tissue mechanical properties and alters gene expression without inducing pathological changes

Overuse Achilles tendinopathy is a common and challenging problem in sports medicine. Little is known about the etiology of this disorder, and the development of a good animal model for overuse tendinopathy is essential for advancing insight into the disease mechanisms. Our aim was to test a previously proposed rat model for Achilles tendon overuse. Ten adult male Sprague-Dawley rats ran on a treadmill with 10° incline, 1 h/day, 5 days/wk (17-20 m/min) for 12 wk and were compared with 12 control rats. Histological, mechanical, and gene-expression changes were measured on the Achilles tendons after the intervention, and local tendon glucose-uptake was measured before and after the intervention with positron emission tomography. No differences were detected between runners and controls in tissue histology or in glucose uptake, indicating that tendon pathology was not induced. Greater tendon tissue modulus (P < 0.005) and failure stress/body weight (P < 0.02) in runners compared with controls further supported that tendons successfully adapted to uphill running. Several genes of interest were regulated after 12 wk of running. Expression of collagen III and insulin-like growth factor I was increased, while collagen I was unchanged, and decreases were seen in noncollagen matrix components (fibromodulin and biglycan), matrix degrading enzymes, transforming growth factor-β1, and connective tissue growth factor. In conclusion, the tested model could not be validated as a model for Achilles tendinopathy, as the rats were able to adapt to 12 wk of uphill running without any signs of tendinopathy. Improved mechanical properties were observed, as well as changes in gene-expression that were distinctly different from what is seen in tendinopathy and in response to short-term tendon loading.     

Biomechanical and structural response of healing Achilles tendon to fatigue loading following acute injury

Achilles tendon injuries affect both athletes and the general population, and their incidence is rising. In particular, the Achilles tendon is subject to dynamic loading at or near failure loads during activity, and fatigue induced damage is likely a contributing factor to ultimate tendon failure. Unfortunately, little is known about how injured Achilles tendons respond mechanically and structurally to fatigue loading during healing. Knowledge of these properties remains critical to best evaluate tendon damage induction and the ability of the tendon to maintain mechanical properties with repeated loading. Thus, this study investigated the mechanical and structural changes in healing mouse Achilles tendons during fatigue loading. Twenty four mice received bilateral full thickness, partial width excisional injuries to their Achilles tendons (IACUC approved) and twelve tendons from six uninjured mice were used as controls. Tendons were fatigue loaded to assess mechanical and structural properties simultaneously after 0, 1, 3, and 6 weeks of healing using an integrated polarized light system. Results showed that the number of cycles to failure decreased dramatically (37-fold, p<0.005) due to injury, but increased throughout healing, ultimately recovering after 6 weeks. The tangent stiffness, hysteresis, and dynamic modulus did not improve with healing (p<0.005). Linear regression analysis was used to determine relationships between mechanical and structural properties. Of tendon structural properties, the apparent birefringence was able to best predict dynamic modulus (R²=0.88–0.92) throughout healing and fatigue life. This study reinforces the concept that fatigue loading is a sensitive metric to assess tendon healing and demonstrates potential structural metrics to predict mechanical properties.     

Onset of neonatal locomotor behavior and the mechanical development of Achilles and tail tendons

Tendon tissue engineering approaches are challenged by a limited understanding of the role mechanical loading plays in normal tendon development. We propose that the increased loading that developing postnatal tendons experience with the onset of locomotor behavior impacts tendon formation. The objective of this study was to assess the onset of spontaneous weight-bearing locomotion in postnatal day (P) 1, 5, and 10 rats, and characterize the relationship between locomotion and the mechanical development of weight-bearing and non-weight-bearing tendons. Movement was video recorded and scored to determine non-weight-bearing, partial weight-bearing, and full weight-bearing locomotor behavior at P1, P5, and P10. Achilles tendons, as weight-bearing tendons, and tail tendons, as non-weight-bearing tendons, were mechanically evaluated. We observed a significant increase in locomotor behavior in P10 rats, compared to P1 and P5. We also found corresponding significant differences in the maximum force, stiffness, displacement at maximum force, and cross-sectional area in Achilles tendons, as a function of postnatal age. However, the maximum stress, strain at maximum stress, and elastic modulus remained constant. Tail tendons of P10 rats had significantly higher maximum force, maximum stress, elastic modulus, and stiffness compared to P5. Our results suggest that the onset of locomotor behavior may be providing the mechanical cues regulating postnatal tendon growth, and their mechanical development may proceed differently in weight-bearing and non-weight-bearing tendons. Further analysis of how this loading affects developing tendons in vivo may inform future engineering approaches aiming to apply such mechanical cues to regulate engineered tendon formation in vitro.     

Functionally distinct tendons have different biomechanical,biochemical and histological responses to in vitro unloading

Tendons with different in vivo functions are known to have different baseline biomechanics, biochemistry and ultrastructure, and these can be affected by changes in loading. However it is not know whether different tendon types respond in the same, or different ways, to changes in loading.This study performed in vitro un-loading (stress deprivation) in culture on ovine medial extensor tendons (MET, a positional tendon), and superficial and deep digital flexor tendons (SDFTs and DDFTs, with energy-storing and intermediate functions respectively), for 21 days (n = 14 each). Tensile strength and elastic modulus were then measured, followed by biochemical assays for sulphated glycosaminoglycan (sGAG) and hydroxyproline content. Histological inspection for cell morphology, cell density and collagen alignment was also performed.The positional tendon (MET) had a significant reduction (∼50%) in modulus and strength (P < 0.001) after in vitro stress-deprivation, however there were no significant effects on the energy-storing tendons (SDFT and DDFT). In contrast, sGAG was not affected in the MET, but was reduced in the SDFT and DDFT (P < 0.001). All tendons lost compactness and collagen organisation, and had reduced cell density, but these were more rapid in the MET than the SDFT and DDFT.These results suggest that different tendon types respond to identical stimuli in different ways, thus; (i) the results from an experiment in one tendon type may not be as applicable to other tendon types as previously thought, (ii) positional tendons may be particularly vulnerable to clinical stress-deprivation, and (iii) graft tendon source may affect the biological response to loading in ligament and tendon reconstruction.     

Matrix metabolism rate differs in functionally distinct tendons.

Tendon matrix integrity is vital to ensure adequate mechanical properties for efficient function. Although historically tendon was considered to be relatively inert, recent studies have shown that tendon matrix turnover is active. During normal physiological activities some tendons are subjected to stress and strains much closer to their failure properties than others. Tendons with low safety margins are those which function as energy stores such as the equine superficial digital flexor tendon (SDFT) and human Achilles tendon (AT). We postulate therefore that energy storing tendons suffer a higher degree of micro-damage and thus have a higher rate of matrix turnover than positional tendons. The hypothesis was tested using tissue from the equine SDFT and common digital extensor tendon (CDET). Matrix turnover was assessed indirectly by a combination of measurements for matrix age, markers of degradation, potential for degradation and protein expression. Results show that despite higher cellularity, the SDFT has lower relative levels of mRNA for collagen types I and III. Non-collagenous proteins, although expressed at different levels per cell, do not appear to differ between tendon types. Relative levels of mRNA for MMP1, MMP13 and both pro-MMP3 and MMP13 protein activity were significantly higher in the CDET. Correspondingly levels of cross-linked carboxyterminal telopeptide of type I collagen (ICTP) were higher in the CDET and tissue fluorescence lower suggesting more rapid turnover of the collagenous component. Reduced or inhibited collagen turnover in the SDFT may account for the high level of degeneration and subsequent injury compared to the CDET.     

Single dose of inducible nitric oxide synthase inhibitor induces prolonged inflammatory cell accumulation and fibrosis around injured tendon and synovium

The aim of the current study was to investigate the effect of inhibition of nitric oxide (NO) production after injury on inflammatory cell accumulation and fibrosis around digital flexor tendon and synovium. A standard crush injury was applied to the flexor tendons of the middle digit of the hindpaw and the overlying muscle and synovium of female Wistar rats. Thirty animals received an intraperitoneal injection of either isotonic saline or N(G)-nitro-l-arginine methyl ester (L-NAME; 5 mg/kg) immediately following the crush injury, and five animals were then sacrificed at various intervals and the paws processed for histology. Another group of five animals was sacrificed after 3 days for nitrite determinations. The results showed that nitrite production and hence NO synthase activity is doubled at the acute phase of tendon wound healing, and we can prevent this by administering a single dose of L-NAME immediately after injury. The incidence and severity of fibrocellular adhesions between tendon and synovium was much more marked in animals treated with L-NAME. Treatment with L-NAME elicited a chronic inflammatory response characterised by a persistent and extraordinarily severe accumulation of large numbers of inflammatory cells in the subcutaneous tissues, in muscle and in tendon. These findings indicate that in the case of injured tendon and synovium, NO could act to protect the healing tissue from an uncontrolled inflammatory response.     

Mechanical,histological, and functional properties remain inferior in conservatively treated Achilles tendons in rodents: Long term evaluation

Conservative treatment (non-operative) of Achilles tendon ruptures is suggested to produce equivalent capacity for return to function; however, long term results and the role of return to activity (RTA) for this treatment paradigm remain unclear. Therefore, the objective of this study was to evaluate the long term response of conservatively treated Achilles tendons in rodents with varied RTA. Sprague Dawley rats (n = 32) received unilateral blunt transection of the Achilles tendon followed by randomization into groups that returned to activity after 1-week (RTA1) or 3-weeks (RTA3) of limb casting in plantarflexion, before being euthanized at 16-weeks post-injury. Uninjured age-matched control animals were used as a control group (n = 10). Limb function, passive joint mechanics, tendon properties (mechanical, histological), and muscle properties (histological, immunohistochemical) were evaluated. Results showed that although hindlimb ground reaction forces and range of motion returned to baseline levels by 16-weeks post-injury regardless of RTA, ankle joint stiffness remained altered. RTA1 and RTA3 groups both exhibited no differences in fatigue properties; however, the secant modulus, hysteresis, and laxity were inferior compared to uninjured age-matched control tendons. Despite these changes, tendons 16-weeks post-injury achieved secant stiffness levels of uninjured tendons. RTA1 and RTA3 groups had no differences in histological properties, but had higher cell numbers compared to control tendons. No changes in gastrocnemius fiber size or type in the superficial or deep regions were detected, except for type 2x fiber fraction. Together, this work highlights RTA-dependent deficits in limb function and tissue-level properties in long-term Achilles tendon and muscle healing.     

Influence of acetaminophen consumption and exercise on Achilles tendon structural properties in male Wistar rats

Chronic consumption of acetaminophen (APAP) during exercise training leads to a reduction in tendon stiffness and modulus compared with a placebo. We explored whether this effect could be due to a reduction in tendon collagen content or cross-linking. Ten-week-old male Wistar rats (n = 50) were divided into placebo or APAP groups and into sedentary or treadmill-exercised groups. APAP (200 mg/kg) or saline was administered once daily by oral gavage. Rats in the exercise groups ran on a treadmill 5 days per week for 8 wk with progression to 60 min per day, 20 m/min, and 8° incline. After 8 wk, lyophilized Achilles tendon samples were assayed for the collagen-specific amino acid hydroxyproline and cross-linking [hydroxylyslpyridinoline (HP)] content by high-performance liquid chromatrography. Collagen content was not influenced by exercise or APAP (P > 0.05). Compared with placebo, tendon water content was 7% (P = 0.006, main effect) lower in animals consuming APAP (placebo: 54.79 ± 0.8%, APAP: 50.89 ± 1.2%). HP in the Achilles tendon was 36% greater (sedentary: 141 ± 15, exercise: 204 ± 26 mmol/mol collagen) in the exercise-trained rats independent of drug treatment (P = 0.020, main effect). Independent of exercise, HP content was 33% lower (P = 0.032, main effect) in the animals consuming APAP (placebo: 195 ± 21, APAP: 140 ± 19 mmol/mol collagen). Our data suggests that chronic consumption of APAP results in a reduction in collagen cross-linking and a loss of tissue water independent of chronic exercise. This reduction in cross-linking and water content could contribute to the decrease in tendon stiffness noted in humans chronically consuming APAP.     

A leucine-rich diet and exercise affect the biomechanical characteristics of the digital flexor tendon in rats after nutritional recovery

An increase in the capacity of athletic performance depends on adequate nutrition, which ensures optimal function of the musculoskeletal system, including tendon stability. However, little is known about the status of tendons and extracellular matrix modifications during malnutrition and nutritional recovery when leucine is used in response to exercise conditioning. The purpose of this study was to evaluate the collagen content and biomechanical aspects of the deep digital flexor tendon (DDFT) in malnourished rats submitted to nutritional recovery (control diet or leucine-rich diet) and aerobic physical activity. After 60 days of undernourishment (6% protein diet), the malnourished rats were subsequently nutritionally recovered with a control diet or leucine-rich diet and trained or not (swimming, without overload) for 5 weeks. The biomechanical analysis and quantification of hydroxyproline were assessed in the DDFT in all experimental groups. The leucine-rich diet increased hydroxyproline content in the tension region, independently of the training. In the compression region, hydroxyproline content was higher in the malnourished and leucine-trained groups. Biomechanical analysis showed a lower load in the malnourished and all-trained groups. The lowest stress was observed with control-trained animals. The nutritional-recovered groups showed higher strain values corresponding to control group, while the lowest values were observed in malnourished and trained groups. The results suggest that a leucine-rich diet stimulates collagen synthesis of the DDFT, especially when in combination with physical exercise, and seems to determine the increase of resistance and the biomechanical characteristics of tendons.     

Cross-Linking in Collagen by Nonenzymatic Glycation Increases the Matrix Stiffness in Rabbit Achilles Tendon

Nonenzymatic glycation of connective tissue matrix proteins is a major contributor to the pathology of diabetes and aging. Previously the author and colleagues have shown that nonenzymatic glycation significantly enhances the matrix stability in the Achilles tendon (Reddy et al., 2002, Arch. Biochem. Biophys., 399, 174–180). The present study was designed to gain further insight into glycation-induced collagen cross-linking and its relationship to matrix stiffness in the rabbit Achilles tendon. The glycation process was initiated by incubating the Achilles tendons (n = 6) in phosphate-buffered saline containing ribose, whereas control tendons (n = 6) were incubated in phosphate-buffered saline without ribose. Eight weeks following glycation, the biomechanical attributes as well as the degree of collagen cross-linking were determined to examine the potential associations between matrix stiffness and molecular properties of collagen. Compared to nonglycated tendons, the glycated tendons showed increased maximum load, stress, strain, Young''s modulus of elasticity, and toughness indicating that glycation increases the matrix stiffness in the tendons. Glycation of tendons resulted in a considerable decrease in soluble collagen content and a significant increase in insoluble collagen and pentosidine. Analysis of potential associations between the matrix stiffness and degree of collagen cross-linking showed that both insoluble collagen and pentosidine exhibited a significant positive correlation with the maximum load, stress, and strain, Young''s modulus of elasticity, and toughness (r values ranging from .61 to .94) in the Achilles tendons. However, the soluble collagen content present in neutral salt buffer, acetate buffer, and acetate buffer containing pepsin showed an inverse relation with the various biomechanical attributes tested (r values ranging from .22 to .84) in the Achilles tendons. The results of the study demonstrate that glycation-induced collagen cross-linking is directly associated with the increased matrix stiffness and other mechanical attributes of the tendon.     

Effect of high intensity aerobic exercise and mesterolone on remodeling of Achilles tendon of C57BL/6 transgenic mice

The effect of mesterolone and intensive treadmill training (6 weeks, 5 days/week, means: 15.82 m/min and 45.8 min/day) in Achilles tendon remodeling was evaluated. Sedentary mice treated with mesterolone (Sed-M) or vehicle (Sed-C, placebo/control) and corresponding exercised (Ex-M and Ex-C) were examined. SDS-polyacrylamide gel electrophoresis was used for determining collagen bands and hydroxyproline concentration. Collagen fibril diameter, the area and number of fibrils contained in an area probe, and the ultrastructure of fibroblasts (tenocytes) were determined. The presence of collagen was notable in the tendons of all groups. Collagen α_1/α₂ bands in Sed-M, Ex-C, and Ex-M were higher than in Sed-C, as shown by hydroxyproline content, but collagen β-chain appeared only in Ex-C. Noticeable bands of non-collagenous proteins were found in Sed-M and Ex-M. The number of fibrils in the area probe increased markedly in Sed-M and Ex-C (12-fold), but their diameter and area were unchanged compared with Sed-C. In Ex-M, the fibril number decreased by three–fold to 3.5-fold compared with Sed-M and Ex-C, whereas diameter and area increased. Sed-C tenocytes appeared quiescent, whereas those in the other groups seemed to be engaged in protein synthesis. The density of tenocytes was smaller in Sed-C than in Ex-C, Sed-M, and Ex-M. Thus, mechanical stimuli and mesterolone alter the morphology of tenocytes and the composition of the tendon, probably through fibrillogenesis and/or increased intermolecular cross-links. The ergogenic effect is evidenced by the activation of collagenous and non-collagenous protein synthesis and the increase in the diameter and area of collagen fibrils. This study might be relevant to clinical sports medicine.     

Inflammatory cells do not decrease the ultimate tensile strength of intact tendons in vivo and in vitro: protective role of mechanical loading.

Although inflammatory cells and their products are involved in various pathological processes, a possible role in tendon dysfunction has never been convincingly confirmed and extensively investigated. The goal of this study was to determine whether or not an acute inflammatory process deprived of mechanical trauma can induce nonspecific damages to intact collagen fibers. To induce leukocyte accumulation, carrageenan was injected into rat Achilles tendons. We first tested the effect of leukocyte recruitment on the concentrations or activities of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases. Second, we analyzed at the biochemical, histological, and biomechanical levels the impact of leukocyte invasion on tendons. Finally, collagen bundles isolated from rat-tail tendons were exposed in vitro to mechanical stress and/or inflammatory cells to determine if mechanical loading could protect tendons from the leukocyte proteolytic activity. Carrageenan-induced leukocyte accumulation was associated with an increased matrix metalloproteinase activity and a decreased content of tissue inhibitors of matrix metalloproteinases. However, hydroxyproline content and load to failure did not change significantly in these tendons. Interestingly, mechanical stress, when applied in vitro, protected collagen bundles from inflammatory cell-induced deterioration. Together, our results suggest that acute inflammation does not induce damages to intact and mechanically stressed collagen fibers. This protective effect would not rely on increased tissue inhibitors of matrix metalloproteinases content but would rather be conferred to the intrinsic resistance of mechanically loaded collagen fibers to proteolytic degradation.     

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.