Total Glycosaminoglycan Content Research Articles

The influence of phenformin on the extracellular matrix of the liver of rats under long-term administration of ethanol [

Experimental and clinical studies have revealed the influence of AMP-activated protein kinase (AMPK) signaling on the development of non-alcoholic liver fibrosis. Currently, the results of experimental studies demonstrate that inhibition of AMPK promotes fibrogenesis, while its activation prevents the development of liver fibrosis. The purpose of this work is to establish the effect of activation of AMP-activated protein kinase by the administration of phenformin on the content of glycosaminoglycans, oxyproline and sialic acids in the liver of rats under the conditions of long-term administration of ethanol. The study was conducted on 24 male Wistar rats. The animals were randomly divided into 4 groups of 6 animals each, on which we modeled ethanol-induced liver damage and administered phenformin hydrochloride at a dose of 10 mg/kg. The experiment lasted 63 days. In the liver of rats, the content of total glycosaminoglycans, the concentration of heparin-heparan, keratan-dermatan and chondroitin fractions of glycosaminoglycans, the content of free oxyproline and sialic acids were studied. Long-term alcoholization leads to a violation of the extracellular matrix of the liver of rats, which is evidenced by a decrease in the concentration of proteoglycans and a redistribution of their fractions in the direction of a decrease in anti-inflammatory and regenerative fractions. Chronic intake of alcohol increases the processes of desialylation of glycoconjugates and the intensity of collagenolysis. Activation of AMP-activated protein kinase by administration of phenformin under the conditions of simulating ethanol-induced liver damage leads to an increase in the concentration of glycosaminoglycans due to the growth of heparin-heparan and chondroitin fractions and reduces the intensity of desialylation of glycoconjugates and collagenolysis in the liver of rats.

Sodium loading in ambulatory patients with heart failure with reduced ejection fraction: Mechanistic insights into sodium handling.

Sodium restriction was not associated with improved outcomes in heart failure patients in recent trials. The skin might act as a sodium buffer, potentially explaining tolerance to fluctuations in sodium intake without volume overload, but this is insufficiently understood. Therefore, we studied the handling of an increased sodium load in patients with heart failure with reduced ejection fraction (HFrEF). Twenty-one ambulatory, stable HFrEF patients and 10 healthy controls underwent a 2-week run-in phase, followed by a 4-week period of daily 1.2 g (51 mmol) sodium intake increment. Clinical, echocardiographic, 24-h urine collection, and bioelectrical impedance data were collected every 2 weeks. Blood volume, skin sodium content, and skin glycosaminoglycan content were assessed before and after sodium loading. Sodium loading did not significantly affect weight, blood pressure, congestion score, N-terminal pro-brain natriuretic peptide, echocardiographic indices of congestion, or total body water in HFrEF (all p > 0.09). There was no change in total blood volume (4748 ml vs. 4885 ml; p = 0.327). Natriuresis increased from 150 mmol/24 h to 173 mmol/24 h (p = 0.024), while plasma renin decreased from 286 to 88 μU/L (p = 0.002). There were no significant changes in skin sodium content, total glycosaminoglycan content, or sulfated glycosaminoglycan content (all p > 0.265). Healthy controls had no change in volume status, but a higher increase in natriuresis without any change in renin. Selected HFrEF patients can tolerate sodium loading, with increased renal sodium excretion and decreased neurohormonal activation.

BIOCHEMICAL MARKERS OF BLOOD SERUM AND ARTICULAR CARTILAGE IN DIFFERENT FORMS OF IDIOPATHIC COXARTHROSIS PROGRESSION

Deformation due to arthrosis of the hip joint affects 40 % of the total number of patients with osteoarthritis, which is from 7 to 25 % of the adult population. For the activity of the pathological process evaluation we studied biochemical changes in the metabolism of collagen, namely: glycosaminoglycans (GAG), hydroxyproline (HP) fractions, collagenase, hyaluronidase activity, etc. Objective. To determine the biochemical changes in blood serum and articular cartilage depending on the form of progression of idiopathic coxarthrosis. Methods. 23 patients were examined (8 (34.8 %) men, 15 (65.2 %) women, age 45‒75 years and more) with idiopathic arthrosis of the hip joint IV stage (Kellgren–Lawrence). Progression forms of idiopathic coxarthrosis were distinguished: group I — rapid (the period from the initial to the final stage of the disease was 5 years and less), II — moderate (5‒10 years), III — slow (over 10 years). Collagenase activity, HP fractions, total GAG content were determined in patients’ blood serum, and collagen content was determined in the cartilage tissue of the femoral head. Results. Compared with the age reference norm in the blood serum of patients of group I activity collagenase increased to 114 %, in II and III — 122 and 135 %, respectively; the content of the free fraction of GP — up to 111, 169, 128 %, respectively; GAG concentrations — up to 110, 122, and 135 %, respectively; protein-bound HP was reduced to 60% in group II, up to 84 % — in group III. In cartilage tissue, the GAG content decreased in group III to 63 %, II — 55 %, I — 47 %; collagen — 32, 25, 24 % in accordance. Conclusions. The course of idiopathic coxarthrosis is determined by metabolic changes in the components of the organic basis of connective and cartilaginous tissues in blood serum. The most profound changes were found in the synthetic phase of HP metabolism, especially in the group with a rapid course disease. A decrease in the content of organic components of connective tissue (GAG and collagen) was determined

On the Mechanical Roles of Glycosaminoglycans in the Tensile Properties of Porcine Corneal Stroma.

The cornea is primarily composed of collagen fibrils that are embedded in a ground substance rich in proteoglycans and other glycoproteins. It is known that glycosaminoglycan (GAG) side chains of proteoglycans form anti-parallel duplexes between collagen fibrils. The present work was done in order to investigate the mechanical function of GAGs in defining the tensile properties of porcine corneal stroma. Porcine corneal stromal strips dissected from the nasal-temporal direction were divided into control, buffer-treated, and enzyme-treated groups. The samples in the control group were used immediately after dissection. However, the buffer-treated and enzyme-treated samples were, respectively, incubated for 18 hours at 37°C in a buffer solution made up of 100-mM sodium acetate at pH 6.0 or in an enzyme solution containing keratanase II. The Blyscan assay was used to quantify the total GAG content and assess GAG depletion in the samples treated with the enzyme and buffer solutions. Uniaxial tensile tests were also performed to determine the effect of GAG removal on mechanical properties of the cornea. The GAG content in enzyme-treated samples was significantly lower than that of the normal and buffer-treated specimens (P < 0.05). Moreover, GAG-depleted strips showed significantly softer mechanical responses in comparison with the control and buffer samples (P < 0.05). Removing GAGs from the corneal extracellular matrix led to significant tensile property reduction; supporting the hypothesis that there exists a strong correlation between the GAG content and mechanical properties of the corneal stroma.

Interleukin-1β and methylprednisolone acetate demonstrate differential effects on equine deep digital flexor tendon and navicular bone fibrocartilage cells in vitro

To investigate the effects of interleukin-1β (IL-1β) and methylprednisolone acetate (MPA) on equine intrabursal deep digital flexor tendon (DDFT) and navicular bone fibrocartilage (NBF) cells in vitro. Third passage DDFT and NBF cells from 5 healthy donor horses ages 11-17 years euthanized for reasons unrelated to musculoskeletal conditions. Aggregate cultures were incubated with culture medium alone (control), 10 ng/mL IL-1β, 10 ng/mL IL-1β + 0.05 mg/mL MPA, or 10 ng/mL IL-1β + 0.5 mg/mL MPA for 24 hours. Extracellular matrix (ECM) gene expressions were assessed via real-time polymerase chain reaction (rtPCR). Culture media matrix metalloproteinase (MMP) -3 and -13 concentrations were quantified via ELISA. Total glycosaminoglycan (GAG) content in the cell pellets and culture media was also assessed. IL-1β and IL-1β combined with MPA significantly downregulated ECM gene expression to a greater extent in NBF cells compared with DDFT cells. IL-1β and IL-1β combined with MPA significantly upregulated MMP-3 culture media concentrations in DDFT cells only, and MMP-13 culture media concentrations to a greater extent in NBF cells compared with DDFT cells. NBF cells were more susceptible to IL-1β and MPA-mediated ECM gene expression downregulation in vitro. These results serve as a first step for future work to determine intrabursal corticosteroid regimens that limits or resolve the inflammation as well as take into consideration NBF cell biosynthesis in horses with navicular disease, for which currently no information exists.

Biochemical markers of connective tissue in the pathogenesis of gastroenteritis in dogs and cats

The aim: to analyze and establish the pathogenetic role of biochemical markers of the state of connective tissue in diseases of the stomach and intestines in dogs and cats.&#x0D; Materials and methods. The research was carried out by the method of analysis of sources of scientific literature (PubMed, Elsevier, electronic resources of the V. I. Vernadskyi National Library), because of which a scheme of the pathogenesis of gastroenteritis in dogs and cats with the participation of connective tissue biopolymers was created.&#x0D; Results. In dogs and cats, the issue of the use of biochemical markers for the diagnosis of diseases of the stomach and intestines has not yet been fully clarified. It is known, that in dogs and cats, lymphocytic-plasmacytic enteritis is histologically determined by fibrosis of the intestinal wall, but biochemical tests for the diagnosis of this condition are not given. Among the biochemical markers of inflammatory bowel disease, tumor necrosis factor, C-reactive protein and microalbumin were identified. Although C-reactive protein was elevated in a greater number of diseased animals, this increase was insignificant. Other tests also did not show high diagnostic informativeness. Several stages can be identified in the pathogenesis of alimentary gastroenteritis in dogs and cats. First, the irritating components of food act on the mucous membrane of the stomach and disrupt its secretory and motor functions, which causes gastritis. Thus, the use of indicators of the state of connective tissue in the diagnosis of intestinal diseases in dogs can be used to assess the degree of the inflammatory process.&#x0D; Conclusions. According to the results of the analysis, it was established, that the development of the inflammatory process in the stomach and intestines causes an increase in the content of glycoproteins in the blood serum of cats and dogs, and a decrease in synthetic processes in the liver is accompanied by a decrease in the concentration of glycosaminoglycans (GAG) in the blood serum of sick animals. It should be noted, that this decrease has peculiarities: in dogs, the content of total chondroitin sulfates remained at the level of clinically healthy animals, while the concentration of total GAG decreased. In cats, on the contrary, the content of total chondroitin sulfates decreased, and the fractional composition of GAG remained unchanged. The level of excretion of oxyproline and uronic acids in the urine of animals with gastroenteritis did not change, which indicates the absence of catabolism of collagen and proteoglycans with gastroenteritis

A three-dimensional (3D), serum-free, Collagen Type I system for chondrogenesis of canine bone marrow-derived multipotent stromal cells (cMSCs).

The dog is an underrepresented large animal translational model for orthopedic cell-based tissue engineering. While chondrogenic differentiation of canine multipotent stromal cells (cMSCs) has been reported using the classic micromass technique, cMSCs respond inconsistently to this method. The objectives of this study were to develop a three-dimensional (3D), serum-free, Collagen Type I system to facilitate cMSC chondrogenesis and, once established, to determine the effect of chondrogenic growth factors on cMSC chondrogenesis. Canine MSCs were polymerized in 100 μL Collagen Type I gels (5 mg/mL) at 1 x 106 cells/construct. Constructs were assessed using morphometry, live/dead staining, and histology in 10 various chondrogenic media. Four media were selected for additional in-depth analyses via lactate dehydrogenase release, total glycosaminoglycan content, qPCR (COL1A1, COL2A, SOX9, ACAN, BGLAP and SP7), immunofluorescence, and TUNEL staining. In the presence of dexamethasone and transforming growth factor-β3 (TGF-β3), both bone morphogenic protein-2 (BMP-2) and basic fibroblast growth factor (bFGF) generated larger chondrogenic constructs, although BMP-2 was required to achieve histologic characteristics of chondrocytes. Chondrogenic medium containing dexamethasone, TGF-β3, BMP-2 and bFGF led to a significant decrease in lactate dehydrogenase release at day 3 and glycosaminoglycan content was significantly increased in these constructs at day 3, 10, and 21. Both osteogenic and chondrogenic transcripts were induced in response to dexamethasone, TGF-β3, BMP-2 and bFGF. Collagen Type II and X were detected in all groups via immunofluorescence. Finally, TUNEL staining was positive in constructs lacking BMP-2 or bFGF. In conclusion, the 3D, serum-free, Collagen Type-I assay described herein proved useful in assessing cMSC differentiation and will serve as a productive system to characterize cMSCs or to fabricate tissue engineering constructs for clinical use.

Bio‐equivalence of Potency Adjusted Approved Heparin Solutions Compared to a Newly Developed Heparin Solution

IntroductionPorcine mucosal heparin solutions with a defined potency adjusted concentrations are widely used for the anticoagulation in medical, surgical and interventional indications. Recently, Sintetica has developed a 20.000 IU (48 ml, 416 IU/ml) solution. The active pharmaceutical ingredient used is a porcine mucosal origin with a specific activity of 194 U/mg. The purpose of this investigation is to compare the Sintetica heparin solution with other commercially available potency adjusted heparin solutions such as Fresenius, Roche, Hospira and Medefil heparins to demonstrate their bio‐equivalence in various biological assays.MethodCommercially available heparin solutions were obtained from Sintetica, Roche, Fresenius Kabi, Hospira and Medefil. USP reference standard was obtained from US Pharmacopeia. USP compliant anti‐Xa and anti‐IIa method based on chromogenic substrate assays were obtained from Aniara Diagnostics (Westchester, OH). Thrombin generation studies were carried out using a fluorokinetic method (Diagnostico France). The quantification of glycosaminoglycan (GAG) content was made by using a fluorescence probe (Red Probe, Munster, Germany). The clot based assays and amidolytic assays were carried out on ACL Elite analyzer. HIT mediated platelet aggregation studies were carried out using HIT antibodies from clinically diagnosed patient serum. All solutions were initially diluted to a working concentration of 100 USP U/ml in sterile saline. Working concentrations were prepared at 10 and 1 U/ml. For the potency measurements calibration curves were prepared with the USP standard for cross‐referencing. NHP was supplemented with each of these heparins in 0 to 1 U/ml concentrations. All other tests were carried out according to standard operating procedures.ResultsIn the USP compliant anti‐Xa and anti‐IIa assays all standardized solutions provided comparable results. Similarly in the clot‐based assays such as the aPTT, thrombin time, prothrombinase‐induced clotting time, all solutions produced comparable anticoagulant effects. Similarly in the thrombin generation assays all products produced comparable inhibitory responses of thrombin generation in terms of peak thrombin, area‐under‐the‐curve and the lag time. The HIT antibody mediated platelet aggregation was similar with all solutions. Total GAG content as measured by Red Probe was also comparable with calculated amounts of 0.520 mg/100U for Sintetica, 0.540 mg/100U for Fresenius, 0.510 mg/100U for Hospira, 0.540 mg/100U for Medefil and 0.530 mg/100Us for Roche’s product.ConclusionThese result demonstrate that commercially available potency adjusted heparin solutions and the developed Sintetica heparin solution exhibit comparable anticoagulant and anti‐protease effects. Based on these results the Sintetica product is similar to the commercially available clinically approved potency adjusted heparin solutions. Furthermore these results suggest that these solutions are interchangeable for clinical use.

The degenerative impact of hyperglycemia on the structure and mechanics of developing murine intervertebral discs.

IntroductionDiabetes has long been implicated as a major risk factor for intervertebral disc (IVD) degeneration, interfering with molecular signaling and matrix biochemistry, which ultimately aggravates the progression of the disease. Glucose content has been previously shown to influence structural and compositional changes in engineered discs in vitro, impeding fiber formation and mechanical stability.MethodsIn this study, we investigated the impact of diabetic hyperglycemia on young IVDs by assessing biochemical composition, collagen fiber architecture, and mechanical behavior of discs harvested from 3‐ to 4‐month‐old db/db mouse caudal spines.ResultsWe found that discs taken from diabetic mice with elevated blood glucose levels demonstrated an increase in total glycosaminoglycan and collagen content, but comparable advanced glycation end products (AGE) levels to wild‐type discs. Diabetic discs also contained ill‐defined boundaries between the nucleus pulposus and annulus fibrosus, with the latter showing a disorganized and unaligned collagen fiber network at this same boundary.ConclusionsThese compositional and structural changes had a detrimental effect on function, as the diabetic discs were twice as stiff as their wild‐type counterparts and demonstrated a significant resistance to deformation. These results indicate that diabetes may predispose the young disc to DDD later in life by altering patterns of extracellular matrix deposition, fiber formation, and motion segment mechanics independently of AGE accumulation.

Contrast-Enhanced Micro-Computed Tomography for 3D Visualization and Quantification of Glycosaminoglycans in Different Cartilage Types.

To compare CA4+-enhanced micro-computed tomography (microCT) of bovine articular, meniscal, nasal, and auricular cartilage, each of which possesses a different extracellular matrix (ECM) composition and structure. The diffusion kinetics of CA4+ in different native cartilage types were assessed over 20 hours. The feasibility of CA4+-enhanced microCT to visualize and quantify glycosaminoglycans (GAGs) in these different tissues was tested using safranin-O staining and 1,9-dimethylmethylene blue assay. The diffusion kinetics of CA4+ in auricular cartilage are significantly slower compared with all other cartilage types. Total GAG content per volume correlates to microCT attenuation with an R2 value of 0.79 for all cartilage types. Three-dimensional contrast-enhanced microCT images of spatial GAG distribution reflect safranin-O staining and highlight the differences in ECM structure, with heterogeneous regions with higher GAG concentrations highlighted by the contrast agent. CA4+-enhanced microCT enables assessment of 3-dimensiona distribution and GAG content in different types of cartilage and has promise as an ex vivo diagnostic technique to monitor matrix development in different tissues over time as well as tissue-engineered constructs.

The study of fibroblast growth factor-2, sulfated glycosaminoglycans and oxyproline with mitral valve prolapse in combination with type 1 diabetes mellitus

The aim. The aim of the study was to conduct a comparative analysis of fibroblast growth factor-2, the total content and fractions of sulfated glycosoaminoglycans as well as free and peptide-bounding oxyproline as the markers of connective tissue metabolism in young patients with mitral valve prolapse, type 1 diabetes mellitus and the combination of both indications.&#x0D; Materials and methods. 93 patients between 19 and 33 years old with either mitral valve prolapse, or type 1 diabetes or the combination of both indications were examined. Group 1 was represented by 36 patients with the monomorbid type 1 diabetes mellitus. Group 2 consisted of 33 patients with type 1 diabetes mellitus and mitral valve prolapse. The comparison group included 24 people with diagnosed mitral valve prolapse. The concentration of fibroblast growth factor-2 in blood plasma was determined by the enzyme immunoassay using a Quantikine reagent kit (Human FGF basic Immunoassay), manufactured by R&amp;D Systems, Inc. (USA) and expressed in pg/ml. The total content and fractions of glycosaminoglycans in blood serum were determined by the method of N. G. Stern et al. and expressed in units of optical density. The level of free and peptide-bounding oxyproline in blood serum were determined by the method of P. N. Sharaev and expressed in µmol/L.&#x0D; Results. Fibroblast growth factor-2 was higher in group 2, where it was 23.7±0.25 pg/ml compared to the control group – 14.20±0.22 pg/ml (p &lt;0.01). There was also a significant difference in the levels of fibroblast growth factor-2 between groups 1 where it equaled 15.33±0.24 pg/ml and 2 – 23.71±0.25 pg/ml (p&lt;0.01). The total content of glycosaminoglycans in comparison with the control group, where it was equal to 9.7±0.62 odu, was higher in all groups of patients: in group 1 it was 12.07±1.04 odu, in the comparison group it was 11.75±0.83 odu, in the group with the combined pathology it was 13.32±1.59 odu (p&lt;0.05). The values of glycosaminoglycans II fraction, were higher in group 2 – 4.96±0.59 odu. The level of peptide-bound oxyproline, just as the level of free oxyproline, significantly increased in patients with comorbid pathology in group 2, compared to the control group: 16.06±1.54 µmol/l versus 8.7±0.81 µmol/l (p&lt;0.01) respectively.&#x0D; Conclusions. The values of fibroblast growth factor-2, glycosaminoglycans II fraction, free and peptide-bound oxyproline were significantly higher in patients with diabetes mellitus and mitral valve prolapse in comparison with the control group. Fibroblast growth factor-2 was higher in patients with comorbid pathology compared to the monomorbid diabetes patients.

Determination of glycosaminoglycans in biological matrices using a simple and sensitive reversed-phase HPLC method with fluorescent detection

This paper suggests a sensitive reversed-phase gradient HPLC method combined with fluorescence detection that has been developed, optimized and tested via the quantitative analysis of authentic biological material in an effort to determine and subsequently compare the total content of glycosaminoglycans (GAGs) in various collagen-based biomaterials intended for medical application.The proposed analytical method enabled the identification and separation of the GAGs present from the other components in the samples using commonly-available laboratory equipment; moreover, the very low detection limit of the method permits the determination of GAGs even for very small samples.This study describes the development of the method, including the isolation and processing of the collagen samples prior to HPLC analysis and the optimal parameters applied during the chromatographic analysis. The application of the method in laboratory practice is documented by means of several examples of the determination of GAGs employing both commercial standards and real collagen samples isolated from various animal tissues.

Long-Term Exposure to Temozolomide Affects Locomotor Activity and Cartilage Structure of Elderly Experimental Rats.

Chemotherapy with temozolomide (TMZ) is an essential part of anticancer therapy of various malignant tumours; however, its long-term effects on patients’ health and life quality need to be further investigated. Here, we studied the effects of TMZ and/or companion drug dexamethasone (DXM) on the locomotor activity and cartilage structure of elderly Wistar rats (n = 40). Long-term TMZ treatment selectively inhibited the horizontal, but not vertical locomotor activity of the rats (6.7-fold, p < 0.01) and resulted in delamination of the superficial epiphyseal cartilage of the femoral epiphysis of knee joints, a 2-fold decrease in mean thickness of epiphyseal cartilage (p < 0.001), and changes in the proliferative and maturation cartilage zones ratio. The simultaneous use of DXM attenuated TMZ-induced changes in cartilage thickness and integrity and compensated the decrease in horizontal locomotor activity of experimental animals. Nevertheless, combined TMZ/DXM treatment still significantly affected the structure of proximal tibial, but not distal femoral epiphysis of knee joints of the rats. These changes were accompanied by the increased content of total glycosaminoglycans (GAGs) and their partial re-localisation from chondrocytes into tissue matrix, as well as the decrease in sulfated GAGs content in both compartments. Taken together, the results demonstrate that long-term treatment with TMZ results in a significant decrease in locomotor activity of elderly Wistar rats and the reorganisation of their knee joint cartilage structure, while DXM treatment attenuates those effects. So, use of DXM or chondroprotective drugs might be beneficial to maintain quality of life for TMZ-treated cancer patients.

Effects of soy isoflavones extract on the lipid profile and uterus in ovariectomized rats

Aim Although soy isoflavones (ISO) have been shown to relief postmenopausal symptoms, it remains inconclusive whether ISO can improve lipid-profile without uterotrophic effects under estrogen-deficiency. Thus, we investigated the effects of ISO on lipid-profile and uterus of ovariectomized (Ovx) rats. Materials and methods Twenty-five adult rats were Ovx or Sham-operated (Sham) and assigned into five groups: Sham and Ovx groups, administered with vehicle solutions; Ovx-E, treated with 10 µg/kg of 17β-Estradiol; Ovx-ISO, treated with 200 mg/kg of ISO; Ovx-E + ISO, treated with estradiol + ISO combined. After fifty days of treatments, rats were euthanized and uterine horns were processed for histomorphometry or to collagen fibers and glycosaminoglycans evaluations. Blood samples were collected to evaluate levels of triglycerides, total cholesterol (TC) and its fractions (HDL/VLDL). Data were subjected to statistical analysis (p < .05). Results Uterus weight was lower in Ovx group than the Sham and Ovx-E groups, whereas it was similar between Ovx and Ovx-ISO groups. Histomorphometry showed atrophic uterus in Ovx and Ovx-ISO groups, whereas uterotrophic effects were noticed in Ovx-E and Ovx-E + ISO groups. Collagen fibers-birefringence was higher in Sham, Ovx, and Ovx-ISO groups than in Ovx-E and Ovx-E + ISO groups. Sulfated glycosaminoglycans content was similar among Sham, Ovx, and Ovx-ISO groups, while it was higher in estrogen-treated groups; total glycosaminoglycans content was similar among groups. TC and HDL was higher in Ovx-ISO group, whereas VLDL and triglycerides levels was higher in Ovx-E + ISO group and similar among other groups. Conclusion Soy isoflavones at 200 mg/kg have slight beneficial effects on lipid-profile without uterotrophic effects in Ovx rats.

Low energy extracorporeal shock wave therapy combined with low tension traction can better reshape the microenvironment in degenerated intervertebral disc regeneration and repair

BACKGROUND Low-tension traction is more effective than high-tension traction in restoring the height and rehydration of a degenerated disc and to some extent the bony endplate. This might better reshape the microenvironment for disc regeneration and repair. However, the repair of the combination of endplate sclerosis, osteophyte formation, and even collapse leading to partial or nearly complete occlusion of the nutrient channel is greatly limited. PURPOSE To evaluate the effectiveness of low-intensity extracorporeal shock wave therapy (ESWT) combined with low tension traction for regeneration and repair of moderately and severely degenerated discs; to explore the possible mechanism of action. STUDY DESIGN Animal study of a rat model of degenerated discs. METHODS A total of 35 6-month old male Sprague-Dawley rats were randomly assigned to one of five groups (n = 7, each group). In Group A (model group), caudal vertebrae were immobilized using a custom-made external device to fix four caudal vertebrae (Co7 - Co10) while Co8 - Co9 underwent 4 weeks of compression to induce moderate disc degeneration. In Group B (experimental control group), as in Group A, disc degeneration was successfully induced after which the fixed device was removed for 8 weeks of self-recovery. The remaining three groups of rats represented the intervention Groups (C - E): after successful generation of disc degeneration in Group C (com - 4w/tra - 4w) and Group D (com - 4w/ESWT), as described for group A, low-tension traction (in-situ traction) or low-energy ESWT was administered for 4 weeks (ESWT parameters: intensity: 0.15Mpa; frequency: 1Hz; impact: 1000 each time; once/week, 4 times in total); Group E (com - 4w/tra - 4w/ESWT): disc degeneration as described for group A, low-tension traction combined with low-energy ESWT was conducted (ESWT parameters as Group D). After experimentation, caudal vertebrae were harvested and disc height, T2 signal intensity, disc morphology, total glycosaminoglycan content, gene expression, structure of the Co8 - Co9 bony endplates and elastic moduli of the discs were measured. RESULTS After continuous low-tension traction, low energy ESWT intervention or combined intervention, the degenerated discs effectively recovered their height and became rehydrated. However, the response in Group D was weaker than in the other intervention groups in terms of restoration of intervertebral disc height, while Group E was superior in disc rehydration. Tissue regeneration was evident in Groups C - E using different interventions. No apparent tissue regeneration was observed in the experimental control group (Group B). The histological scores of the three intervention groups (Groups C - E) were lower than those of Groups A or B (p 0.05). Compared with the intervention groups (Groups C - E), total GAG content of the nucleus pulposus (NP) in Group B did not increase significantly (p > 0.05). There was also no significant difference in the total GAG content between Groups A and B (p > 0.05). Of the three intervention groups, the recovery of NP GAG content was greatest in Group E. The expression of collagen I and II, and aggrecan in the annulus fibrosus (AF) was up-regulated (p CONCLUSIONS Low energy ESWT combined with low tension traction provided a more stable intervertebral environment for the regeneration and repair of moderate and severe degenerative discs. Low energy ESWT promoted the regeneration of disc matrix by reducing MMP-3, MMP-13, and ADAMTS-4 resulting in inhibition of collagen degradation. While axial traction promoted the recovery of height and rehydration of the IVD, combined with low energy ESWT, the micro-nano structure of the bony endplate underwent positive reconstruction, tension in the annulus of the AF and nuclear stress of the NP declined, and the biomechanical microenvironment required for IVD regeneration and repair was reshaped.

Structural analysis of urinary glycosaminoglycans from healthy human subjects.

Urinary glycosaminoglycans (GAGs) can reflect the health condition of a human being, and the GAGs composition can be directly related to various diseases. In order to effectively utilize such information, a detailed understanding of urinary GAGs in healthy individuals can provide insight into the levels and structures of human urinary GAGs. In this study, urinary GAGs were collected and purified from healthy males and females of adults and young adults. The total creatinine-normalized urinary GAG content, molecular weight distribution and disaccharide compositions were determined. Using capillary zone electrophoresis (CZE)-mass spectrometry (MS) and CZE-MS/MS relying on negative electron transfer dissociation, the major components of healthy human urinary GAGs were determined. The structures of 10 GAG oligosaccharides representing the majority of human urinary GAGs were determined.

Association between Extracellular Matrix Accumulation and Oxidative Stress-Induced Apoptosis in Chondrocytes Cultured on 3D-Porous Scaffolds in Static versus Dynamic Cultures

The aim of this study was to investigate the relation between the amount of glycosaminoglycans (GAGs) secreted and the level of apoptosis induced by hydrogen peroxide (H2O2) in porcine chondrocytes cultured on two different biodegradable PCL/PHBV scaffolds: one had primary pores solely, while the other possessed both primary and secondary pores, namely PCL/PHBV/0%NaCl and PCL/PHBV/50%NaCl scaffolds, respectively. The scaffolds with an average primary pore size of 200 μm were primarily fabricated by a fused deposition modeling (FDM) machine, and secondary pores on the material surface were then generated after NaCl leaching in a sodium hydroxide solution. The investigation was performed comparatively under static and dynamic environments. In static culture study, more pronounced GAG accumulation was found on the PCL/PHBV/50%NaCl scaffold, suggesting that the secondary pores could promote the chondrogenic function of chondrocytes, and the primary pores could further accommodate the cells to grow and function more productively. Meanwhile, the significant reductions in the levels of apoptosis of chondrocytes, studied in a H2O2-dose dependent manner (0-2 mM), were observed with a use of a flow cytometer when the chondrocytes were cultured on the PCL/PHBV/50%NaCl scaffold in which more GAG content was found. In dynamic culture study, although the total GAG content detected on the PCL/PHBV/50%NaCl scaffold was still significantly higher than that measured on the PCL/PHBV/0%NaCl scaffold, these GAG accumulations were tremendously greater than those found in the static culture. As a consequence, far less oxidative stress-induced apoptotic death was observed in the cells cultured under the dynamic culture. Moreover, cells seemed to be well protected from apoptosis, in the presence of considerable amounts of GAGs; similar degrees of apoptosis (~25%) were observed on the cells cultured on each scaffold at all H2O2 concentrations studied. The obtained results had demonstrated that the degree of chondrocyte apoptosis was somehow related to the level of GAG accumulation.

Assessment of changes in the micro-nano environment of intervertebral disc degeneration based on Pfirrmann grade

BACKGROUND CONTEXTPfirrmann grading can be used to assess intervertebral disc degeneration (IVDD). There is growing evidence that IVDD is not simply a structural disorder but also involves changes to the substructural characteristics of the disc. Whether Pfirrmann grade can accurately represent these micro-nano environmental changes remains unclear. PURPOSEWe aimed to assess the micro-nano structural characteristics of the degenerative disc to provide more specific biomechanical information than the Pfirrmann score. STUDY DESIGNA micro- and nano-level structural analysis of degenerative discs of rat tails. METHODSIn this study, 12-week-old adult male Sprague-Dawley rats were divided randomly into five groups: control (no intervention to the intervertebral disc of the tail) and four intervention groups that all had caudal vertebrae immobilized using a custom-made external device to fix four caudal vertebrae (Co7–Co10) but with variable subsequent compression of Co8 and Co9 for 2, 4, 6, or 8 weeks. Magnetic resonance imaging detection of rat coccygeal vertebrae was conducted at each time node of the experiment, and the T2 signal intensity and disc space were evaluated. Animals were euthanized and the caudal vertebrae were harvested for further analysis. Histopathology, glycosaminoglycan (GAG) content, histologic score, end plate structure, and elastic modulus of the intervertebral discs were evaluated. RESULTSIVDD was observed at an earlier Pfirrmann grade (Pfirrmann II) under the microscope. With an increase in Pfirrmann grade to III–V, the pore structure of the bony end plate changed significantly and the number of pores decreased gradually. Furthermore, the total GAG content of the nucleus pulposus decreased from an average of 640.33 μg GAG/ng DNA in Pfirrmann grade I to 271.33 μg GAG/ng DNA in Pfirrmann grade V (p < .0001). At the early stage of clinical degeneration of intervertebral discs (Pfirrmann grades II and III), there were significant changes in mechanical properties of the outer annulus fibrosus compared with the inner layer (p < .05). Further, the fibril diameters exhibited significant changes compared with the control group (p < .05). CONCLUSIONSOur study found that the Pfirrmann grading system combined with intervertebral disc micro-nano structural changes more comprehensively reflected the extent of disc degeneration. These data may help improve our understanding of the pathogenesis and process of clinical disc degeneration.

Plasma Glycosaminoglycans, Cortisol, Iron, and Hemoglobin in Term and Nursing Mares

The purpose of this study was to evaluate the contribution of the total content of glycosaminoglycans (GAGs), cortisol, iron, and hemoglobin (Hb) in term and nursing mares. Twenty-four healthy mares, aged 7 ± 1.5 years, were examined. Eighteen pregnant mares were considered the observational group, and six nonpregnant mares were considered the control group, by random selection. Blood samples were taken at the 10 and 11 months of gestation and at 15 and 30 postpartum days for the observational group and in baseline condition for the control group. Two-way analysis of variance showed a significant interaction between peripartum, postpartum, and time points for GAGs (F = 34.75; P < .0001) and Hb (F = 8.77; P = .007) changes. A significant effect of peripartum (F = 64.96; P < .0001) on the Hb changes was observed, with higher concentrations at 15 days of postpartum (P < .01) than 11 months of pregnancy. Compared with control group, observational group showed lower Hb values along peripartum (P < .01) and at 30 days of postpartum (P < .01). A significant and positive correlation between GAGs and cortisol concentrations (r = 0.99; P = .008) was also shown. The present study contributes to a deeper knowledge on the dynamic endocrine–metabolic profile of mare throughout the transition from one to another physiological stage.

Controlled immobilization-traction based on intervertebral stability is conducive to the regeneration or repair of the degenerative disc: an in vivo study on the rat coccygeal model

Previous studies have shown the potential for intervertebral disc tissue regeneration is very limited. While in vivo and in vitro studies have shown that traction can restore disc height and internal pressure, in many clinical studies it was shown that axial mechanical traction for the treatment of low back pain is ineffective. The aim of this study was to identify how the disc could be distracted, how to define the state of traction, and to further examine the feasibility of regenerating or restoring the degenerative disc by means of traction. A macro- and microlevel structural analysis of degenerative discs of rat tail before and after controlled immobilization-traction. In this study, 49 6-month-old male Sprague-Dawley rats were randomly assigned to one of seven groups. Group A was the sham control group in which caudal vertebrae were instrumented with K-wires only. In Group B (model group), caudal vertebrae were immobilized using a custom-made external device to fix four caudal vertebrae (Co7-Co10) and Co8-Co9 underwent 4 weeks of compression to induce moderate disc degeneration. In Group C, vertebrae Co8-Co9 underwent 4 weeks of compression to induce moderate disc degeneration, followed by removal of the external apparatus. Rats in the other four groups (Groups D-G), Co8-Co9 underwent 4 weeks of compression to induce moderate disc degeneration followed by 2 weeks, 4 weeks, 6 weeks, and 8 weeks of distraction, respectively. Caudal vertebrae were harvested and disc height, T2 signal intensity of the discs, disc morphology, total glycosaminoglycan content of the nucleus pulposus and the structure of the Co8-Co9 end plate were evaluated. After 4 weeks of compression, the intervertebral height and T2 signal intensity of Co8-Co9 vertebrae of rats in Groups B to G were significantly reduced compared with Group A (sham group, all p<.0001). Histological scores of rats in Group B averaged 10.14 and the total glycosaminoglycan (GAG) of nucleus pulposus averaged 238.21μg GAG/ng DNA. The bony end plate structure showed significant changes in comparison with the control Group. After 2 weeks to 8 weeks of traction, the disc space and T2 signal intensity of Co8-Co9 vertebrae in Group E were significantly recovered compared to that of rats in Group B (p<.0001), and the intervertebral height of the Co8-Co9 in Group D, Group F, and Group G when compared with Group B (p<.0001). Meanwhile, the T2 signal intensity of Co8-Co9 in Group D, F, and G when compared with Group B (p<.001). Histological scores dropped from an average of 10.14 in Group B to 5.57 in Group E, and 5.86 in Group F (all p<.0001). Furthermore, the total GAG content of the nucleus pulposus increased from an average of 238.21μg GAG/ng DNA in Group B to 601.02μg GAG/ng DNA in Group E (p<.0001). The number of pores of end plates in rats in Groups D and E both were significantly increased when compared to that of rats in Group B (Groups D vs Groups B, p<.05; Groups E vs Groups B, p<.0001). A mechanical degenerative model was successfully established by using a custom-made device. We demonstrated that disc degeneration is a cascade of biochemical, mechanical, and structural changes mediated by cells in an abnormal mechanical environment. Not all levels of disc degeneration can be regenerated or repaired. Regeneration or recovery of disc degeneration requires specific conditions. Based on the immobilization-traction mode, the cascade cycle of disc degeneration is interrupted. Traction of 2 to 6 weeks is a sensitive period for regeneration of the degenerative disc. Moreover, the duration and extent of the traction loading must be moderately controllable, and beyond the limits that can lead to significant degeneration. These data may help improve our understanding of the pathogenesis of clinical disc degeneration and how to optimize the use of traction devices for possible regeneration.