Concentration Of Sulphated Glycosaminoglycans Research Articles

Bone marrow mesenchymal stromal cell-derived extracellular matrix displays altered glycosaminoglycan structure and impaired functionality in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of hematologic malignancies characterized by clonal hematopoiesis, one or more cytopenias such as anemia, neutropenia, or thrombocytopenia, abnormal cellular maturation, and a high risk of progression to acute myeloid leukemia. The bone marrow microenvironment (BMME) in general and mesenchymal stromal cells (MSCs) in particular contribute to both the initiation and progression of MDS. However, little is known about the role of MSC-derived extracellular matrix (ECM) in this context. Therefore, we performed a comparative analysis of in vitro deposited MSC-derived ECM of different MDS subtypes and healthy controls. Atomic force microscopy analyses demonstrated that MDS ECM was significantly thicker and more compliant than those from healthy MSCs. Scanning electron microscopy showed a dense meshwork of fibrillar bundles connected by numerous smaller structures that span the distance between fibers in MDS ECM. Glycosaminoglycan (GAG) structures were detectable at high abundance in MDS ECM as white, sponge-like arrays on top of the fibrillar network. Quantification by Blyscan assay confirmed these observations, with higher concentrations of sulfated GAGs in MDS ECM. Fluorescent lectin staining with wheat germ agglutinin and peanut agglutinin demonstrated increased deposition of N-acetyl-glucosamine GAGs (hyaluronan (HA) and heparan sulfate) in low risk (LR) MDS ECM. Differential expression of N-acetyl-galactosamine GAGs (chondroitin sulfate, dermatan sulfate) was observed between LR- and high risk (HR)-MDS. Moreover, increased amounts of HA in the matrix of MSCs from LR-MDS patients were found to correlate with enhanced HA synthase 1 mRNA expression in these cells. Stimulation of mononuclear cells from healthy donors with low molecular weight HA resulted in an increased expression of various pro-inflammatory cytokines suggesting a contribution of the ECM to the inflammatory BMME typical of LR-MDS. CD34+ hematopoietic stem and progenitor cells (HSPCs) displayed an impaired differentiation potential after cultivation on MDS ECM and modified morphology accompanied by decreased integrin expression which mediate cell-matrix interaction. In summary, we provide evidence for structural alterations of the MSC-derived ECM in both LR- and HR-MDS. GAGs may play an important role in this remodeling processes during the malignant transformation which leads to the observed disturbance in the support of normal hematopoiesis.

The effect of the sulfated glycosaminoglycans complex on the human corneal epithelial cells proliferation: an <i>in vitro</i> experiment

Aim to study the proliferative effect of various concentrations of sulfated glycosaminoglycans (sGAG) on the culture of human corneal epithelial cells.
 Material and methods. The study focused on the properties of a mixture of sGAG consisting of chondroitin-4-sulfate, chondroitin-6-sulfates and keratan sulfates obtained by isolation from the transparent unchanged stroma of the cornea of farm animals. The material for the in vitro study was the cells of the anterior epithelium of the human cornea.
 In the experimental group No.1, a mixture of sGAG was added to the culture medium at a concentration of 0.1%, in the experimental group No.2 at a concentration of 0.5%, in the experimental group No.3 at a concentration of 1.0%. In the control group No.1, the medium was replaced without the addition of solutions, cell seeding was carried out similarly to seeding with experimental samples. The negative control group included the holes of the tablet without the addition of medium and solution.
 Results. The highest levels of the cellular index (CI) were obtained in the experimental group No.3 (1.00% of sGAG) and amounted to 2.86 0.11 (the total increase in indicators was 60.67%). In the experimental group No.2 (0.50% sGAG), the cellular index levels were 2.650.24 (the total increase in indicators was 52.29%), which was significantly different from the experimental group No.1 (0.10% sGAG) and the control group No.1, where these indicators were 2.54 0.21 (35.81%) and 2.370.02 (25.39%) respectively.
 Conclusion. The final CI indicators show that solutions of the sGAG mixture at concentrations of 0.5% and 1.0% have a more pronounced proliferative effect on human corneal epithelial cells in vitro from the range of concentrations under study. The results obtained open up the prospects for the use of sGAG in the development of new approaches to the treatment of patients undergoing a chronic drug therapy or patients with concomitant corneal diseases.

Investigation of synovial fluid lubricants and inflammatory cytokines in the horse: a comparison of recombinant equine interleukin 1 beta-induced synovitis and joint lavage models

BackgroundLameness is a debilitating condition in equine athletes that leads to more performance limitation and loss of use than any other medical condition. There are a limited number of non-terminal experimental models that can be used to study early inflammatory and synovial fluid biophysical changes that occur in the equine joint. Here, we compare the well-established carpal IL-1β-induced synovitis model to a tarsal intra-articular lavage model, focusing on serial changes in synovial fluid inflammatory cytokines/chemokines and the synovial fluid lubricating molecules lubricin/proteoglycan 4 and hyaluronic acid. The objectives of this study were to evaluate clinical signs; synovial membrane and synovial fluid inflammation; and synovial fluid lubricants and biophysical properties in response to carpal IL-1β synovitis and tarsal intra-articular lavage.ResultsHyaluronic acid (HA) concentrations, especially high molecular weight HA, and synovial fluid viscosity decreased after both synovitis and lavage interventions. Synovial fluid lubricin concentrations increased 17–20-fold for both synovitis and lavage models, with similar changes in both affected and contralateral joints, suggesting that repeated arthrocentesis alone resulted in elevated synovial fluid lubricin concentrations. Synovitis resulted in a more severe inflammatory response based on clinical signs (temperature, heart rate, respiratory rate, lameness and joint effusion) and clinicopathological and biochemical parameters (white blood cell count, total protein, prostaglandin E2, sulfated glycosaminoglycans, tumor necrosis factor-α and CC chemokine ligands − 2, − 3, − 5 and − 11) as compared to lavage.ConclusionsSynovial fluid lubricin increased in response to IL-1β synovitis and joint lavage but also as a result of repeated arthrocentesis. Frequent repeated arthrocentesis is associated with inflammatory changes, including increased sulfated glycosaminoglycan concentrations and decreased hyaluronic acid concentrations. Synovitis results in more significant inflammatory changes than joint lavage. Our data suggests that synovial fluid lubricin, TNF-α, CCL2, CCL3, CCL5, CCL11 and sGAG may be useful biomarkers for synovitis and post-lavage joint inflammation. Caution should be exercised when performing repeated arthrocentesis clinically or in experimental studies due to the inflammatory response and loss of HA and synovial fluid viscosity.

Do Counter‐Current Exchange and Glycosaminoglycan Buffering Contribute in Salt Storage in Skin of Rats?

We have previously shown that there is an osmolality and urea gradient from epidermis to dermis and subcutis. This gradient may be a result of the proposed counter-current mechanism for electrolyte homeostasis in skin. Our aims were to test whether there are gradients in salt concentration in skin generated by counter-current exchange and whether glycosaminoglycans (GAGs) can store sodium making it ‘inactive’ in relation to fluid exchange and thereby act as buffers during high salt intake. Sprague-Dawley rats were given low salt diet (LSD, <0.1% NaCl in chow and tap water), high salt diet (HSD, 8% NaCl in chow and 1% saline to drink), and DOCA-salt diet (50mg/week deoxycorticosterone acetate, 150mg/21 days and 1% saline to drink). Rats were randomly assigned to either LSD (n=15), HSD (n=7) or DOCA salt (n=8) diet for measurements of extracellular volume (ECV) in tangential sequential skin sections. Two weeks into the diet regimen the rats were anesthetized, neprectomized and given the extracellular tracer 51Cr-EDTA i.v. that was allowed 120 min circulation time. Directly thereafter, shaved back skin was further processed by cutting sequential sections of 40 µm (epidermis) and 100 µm (dermis and subcutis) thickness in a cryostat taking care to avoid evaporation. The skin sections were weighed and counted in a gamma counter and the extracellular volume found as the plasma equivalent space of 51Cr-EDTA. After counting, the samples were dried until constant weight, then eluted in ultrapure water for seven days. The eluate was analyzed in an ion chromatograph to determine sodium and potassium content in each layer. Sulfated GAGs were determined in whole skin, dermis and subcutis using Blyscan sGAG colorimetric assay to determine sGAG content. When relating sodium content to ECV we found that there was a gradient from epidermis to dermis, and that this gradient was more pronounced in the DOCA rats. The LSD group had a mean sodium of 332.7±194.3 mM in epidermis and 118.3±20.15 mM in the following layer at 140±mM, with corresponding concentrations of 219.2±52 mM and 121.1±41.4 mM in HSD, and 757.8±426.1 mM and 123.7±11.45 mM in DOCA. There was no difference in sulfated GAGs in skin between LSD, HSD and the DOCA salt group in any of the skin regions. Our results suggest that there may be a gradient of salt in the skin with a higher concentration in ECV in epidermis supporting the notion of a counter-current mechanism. However, our data do not support that an increased concentration of sulfated GAGs contribute to salt buffering during high salt conditions.

Altered Structure and Function of Mesenchymal Stromal Cell-Derived Extracellular Matrix in MDS Can be Restored By Luspatercept

Introduction The involvement of the bone marrow microenvironment (BMME) into disease progression and therapeutic response of myelodysplastic syndromes (MDS) is indisputable. Hereby, mesenchymal stromal cells (MSCs) play an important role for both the support of the leukemic clone and the remaining healthy hematopoietic stem and progenitor cells (HSPCs). The extracellular matrix (ECM) secreted by MSCs regulates stem cell fate through the modulation of cytokine and growth factor delivery and may also be targeted by clinically available drugs such as luspatercept, a novel recombinant fusion protein containing modified extracellular domain of activin receptor IIB. Luspatercept is a first-in-class erythroid maturation agent with promising results in lower-risk MDS patients with red blood cell transfusion dependency. Aim To shed light on the largely unknown composition and function of the MSC-derived ECM, we have characterized ECM from MDS patients vs. healthy controls and elucidate how luspatercept may modulate their functional characteristics. Methods Bone marrow-derived MSCs from patients with lower-risk MDS and age-matched healthy donors (HD) were treated with RAP-536, a murine homologue of luspatercept harboring the same activin receptor IIB domain. MSCs of three patients were treated with RAP-536 and RNA sequencing was carried out. Gene expression and pathway analyses were performed using the Reactome tool (https://reactome.org). Candidate genes were validated by quantitative real-time PCR (qPCR). For the generation of ECM, MSCs were seeded on poly-octadecene-alt-maleic anhydride and human fibronectin coated glass slides in the presence or absence of RAP-536. To yield cell-free ECM structures, cultures were decellularized at day 10 and analyzed by scanning electron microscopy (SEM), sulfated glycosaminoglycan (GAG), fibronectin and collagen staining as well as GAG quantification (Blyscan assay). Moreover, purified HD CD34+ HSPCs were cultured on ECM scaffolds for 6 and 9 days, respectively. Subsequently, expansion of adherent and supernatant cells was determined and the phenotype was analyzed by flow cytometry. Results RNA sequencing of MDS MSCs after six days of RAP-536 treatment revealed a total of 58 significantly regulated genes, thereof 24 up- and 34 down-regulated genes. Gene enrichment and pathway analyses revealed a striking involvement in ECM organization, collagen biosynthesis and formation. Moreover, integrin cell surface interaction genes showed significantly differential expression. Focusing on collagens as important ECM components, we identified Col7A1 and Col4A2 to be down-regulated. Indeed, both collagen mRNAs were significantly decreased by 46% and 25%, respectively, in MSCs after RAP-536 treatment compared to untreated controls. SEM characterization and immunofluorescence staining of the ECM showed a more compact fiber network produced by MDS MSCs. Moreover, MDS ECM contained higher levels of collagen and GAGs. Blyscan assay confirmed the latter observation, showing significantly higher sulfated GAG concentrations in MDS ECM. Interestingly, trapping of TGFβ superfamily ligands, such as GDF-11, by RAP-536 clearly reduced Col4 staining intensity in MDS MSC ECM. Structural and compositional ECM differences had functional impact on the expansion of HSPCs cultured on the matrices. Significant higher total cell numbers were detected on healthy ECM (18.3-fold vs. 12.1-fold expansion, *p&lt; 0.05) but not on MDS ECM (12.9-fold) after 9 days of culture. The number of adherent cells increased 8.5-fold on healthy and 4.3-fold on MDS ECM and could be further increased after RAP-536 treatment of MSCs. Using flow cytometry, we found a 3.1-fold increased proportion of CD90+ HSPCs in the adherent fraction on healthy but only 1.8-fold on MDS MSC ECM. Integrin αIIb (CD41), αV (CD51) and β3 (CD61) were found to be significantly higher expressed in the adherent HSPC fraction. RAP-536 treatment resulted in up to 20% higher expression of both CD90 and integrin subunits. Summary We demonstrate an association between induced collagen abundance and reduced hematopoietic support in ECM derived from MDS MSCs and conclude that compact MDS ECM structure induced by TGFβ superfamily members may alter the cytokine environment for HSPCs. Consequently, TGFβ ligand trapping by RAP-536/luspatercept leads to ECM re-organization and thus an improved hematopoietic support. Disclosures Stoelzel: Shire: Consultancy, Other: Travel funding; Neovii: Other: Travel funding; JAZZ Pharmaceuticals: Consultancy. Platzbecker:Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Concentration of sulfated glycosaminoglycans in the mammary tissue of female rats with the aging and about hormonal influence

It was to evaluate the concentration of sulfate glycosaminoglycans (GAG) in mammary tissue of the young and adult female rats and ovariectomized females rats after hormonal stimulation. For this purpose, 60 female rats were divided into six groups with 10 animals/each: nonovariectomized groups: G1 (5 months), and G2 (15 months) and ovariectomized groups: OG (vehicle); EG: (estradiol, 7 days of treatment), PG (progesterone acetate, 23 days of treatment) and EPG: (estradiol (7 days of treatment) and next progesterone acetate (23 days of treatment). Twenty-four hours after the last treatment, all animals were euthanized, the mammary tissue removed, processed for biochemical evaluation and quantification of the GAG. The comparison between groups showed that the concentration dermatan sulfate (DS) G1 was lower compared to G2, OG, EG (p < .05) and G2 was lower compared to OG (p < .05), and OG was higher compared to EG, GP, EPG (p < .05); and heparan sulfate (HS) G1 was higher compared to G2 (p < .05), and G2 was higher compared to OG, EP, PG and EPG (p < .05). These changes in the extracellular matrix might explain, at least in part, hormonal influence about sulfated glycosaminoglycans in response to physiological state/age, and in response to hormonal treatment in the mammary tissues.

The role of miR-320a and IL-1β in human chondrocyte degradation.

ObjectivesThis study aimed to explore the role of miR-320a in the pathogenesis of osteoarthritis (OA).MethodsHuman cartilage cells (C28/I2) were transfected with miR-320a or antisense oligonucleotides (ASO)-miR-320a, and treated with IL-1β. Subsequently the expression of collagen type II alpha 1 (Col2α1) and aggrecan (ACAN), and the concentrations of sulfated glycosaminoglycans (sGAG) and matrix metallopeptidase 13 (MMP-13), were assessed. Luciferase reporter assay, qRT-PCR, and Western blot were performed to explore whether pre-B-cell leukemia Homeobox 3 (PBX3) was a target of miR-320a. Furthermore, cells were co-transfected with miR-320a and PBX3 expressing vector, or cells were transfected with miR-320a and treated with a nuclear factor kappa B (NF-κB) antagonist MG132. The changes in Col2α1 and ACAN expression, and in sGAG and MMP-13 concentrations, were measured again. Statistical comparisons were made between two groups by using the two-tailed paired t-test.ResultsExpression of miR-320a was elevated in OA cartilage tissues and chondrocytes, and in IL-1β-stimulated C28/I2 cells (p < 0.05 or p < 0.01). MiR-320a overexpression enhanced IL-1β-induced down-regulation of Col2α1 and ACAN and sGAG, and increased the IL-1β-induced overexpression of MMP-13 (p < 0.01). PBX3 was a direct target of miR-320a. PBX3 and MG132 co-transfection attenuated the effects of miR-320a on the expression of Col2α1, ACAN, sGAG and MMP-13(p < 0.01).ConclusionOverexpression of miR-320a might enhance IL-1β-induced cartilage degradation factors. These effects might be via targeting PBX3 and regulating NF-κB.Cite this article: Y. Jin, X. Chen, Z. Y. Gao, K. Liu, Y. Hou, J. Zheng. The role of miR-320a and IL-1β in human chondrocyte degradation. Bone Joint Res 2017;6:–203. DOI: 10.1302/2046-3758.64.BJR-2016-0224.R1.

Sulfated glycosaminoglycans in human vocal fold lamina propria

IntroductionThe distribution, concentration and function of glycosaminoglycans in the various vocal fold tissues are still unclear. ObjectiveTo evaluate the distribution and concentration of sulfated glycosaminoglycans in different layers of the human vocal fold according to gender and age. MethodsWe used 11 vocal folds obtained from cadavers (7 men and 4 women) with no laryngeal lesion, less than 12h after death, and aged between 35 and 98 years. The folds underwent glycosaminoglycans extraction from the cover and ligament, and post-electrophoresis analysis. Data were compared according to the layer, age and gender. ResultsThe concentration of dermatan sulfate was significantly higher in all layers. No differences were observed in the total concentrations of glycosaminoglycans in layers studied according to gender. It is significantly lower in the cover of individuals aged below 60 years. ConclusionDermatan sulfate, chondroitin sulfate, and heparan sulfate were observed in the human vocal folds cover and ligament of both genders, with the concentration of dermatan sulfate being significantly higher in all layers. Glycosaminoglycans concentration on the cover is significantly lower in individuals below 60 years compared with elderly.

Low-level laser therapy stimulates tissue repair and reduces the extracellular matrix degradation in rats with induced arthritis in the temporomandibular joint.

The objective of this study was to characterize morphological and biochemistry action of low-level laser therapy (LLLT) on induced arthritis in the temporomandibular joint (TMJ) of rats. Twenty-four male Wistar rats were randomly divided into groups with 12 animals each: (AG) group with arthritis induced in the left TMJ and (LG) group with arthritis induced in the left TMJ and treated with LLLT (830nm, 30mW, 3 J/cm(2)). Right TMJs in the AG group were used as noninjected control group (CG). Arthritis was induced by intra-articular injection of 50μl Complete Freund's Adjuvant (CFA) and LLLT began 1week after arthritis induction. Histopathological analysis was performed using sections stained with hematoxylin-eosin, Toluidine Blue, and picrosirius. Biochemical analysis was determined by the total concentration of sulfated glycosaminoglycans (GAGs) and evaluation of matrix metalloproteinases (MMP-2 and MMP-9). Statistical analysis was performed using paired and unpaired t tests, with p < 0.05. Compared to AG, LG had minor histopathological changes in the TMJ, smaller thickness of the articular disc in the anterior (p < 0.0001), middle (p < 0.0001) and posterior regions (p < 0.0001), high birefringence of collagen fibers in the anterior (p < 0.0001), middle (p < 0.0001) and posterior regions (p < 0.0001) on the articular disc, and statistically lower activity of MMP-2 latent (p < 0.0001), MMP-2 active (P = 0.02), MMP-9 latent (p < 0.0001), and MMP-9 active (p < 0.0001). These results suggest that LLLT can increase the remodeling and enhancing tissue repair in TMJ with induced arthritis.

Hyaluronan Does Not Regulate Human Epidermal Keratinocyte Proliferation and Differentiation

Hyaluronan (HA) is synthesized by three HA synthases (HAS1, HAS2, and HAS3) and secreted in the extracellular matrix. In human skin, large amounts of HA are found in the dermis. HA is also synthesized by keratinocytes in the epidermis, although its epidermal functions are not clearly identified yet. To investigate HA functions, we studied the effects of HA depletion on human keratinocyte physiology within in vitro reconstructed human epidermis. Inhibition of HA synthesis with 4-methylumbelliferone (4MU) did not modify the expression profile of the epidermal differentiation markers involucrin, keratin 10, and filaggrin during tissue reconstruction. In contrast, when keratinocytes were incubated with 4MU, cell proliferation was decreased. In an attempt to rescue the proliferation function, HA samples of various mean molecular masses were added to keratinocyte cultures treated with 4MU. These samples were unable to rescue the initial proliferation rate. Furthermore, treatments with HA-specific hyaluronidase, although removing almost all HA from keratinocyte cultures, did not alter the differentiation or proliferation processes. The differences between 4MU and hyaluronidase effects did not result from differences in intracellular HA, sulfated glycosaminoglycan concentration, apoptosis, or levels of HA receptors, all of which remained unchanged. Similarly, knockdown of UDP-glucose 6-dehydrogenase (UGDH) using lentiviral shRNA effectively decreased HA production but did not affect proliferation rate. Overall, these data suggest that HA levels in the human epidermis are not directly correlated with keratinocyte proliferation and differentiation and that incubation of cells with 4MU cannot equate with HA removal.

Effects of high molecular weight hyaluronic acid on induced arthritis of the temporomandibular joint in rats

High molecular weight hyaluronic acid (HMWHA) has been used to treat temporomandibular joint (TMJ) disorders, but controversial results have been described. This study aimed to characterize the morphological and biochemical actions of HMWHA on induced arthritis of the TMJ. Twenty-four male Wistar rats were used, and arthritis of the TMJ was induced through an intra-articular injection of Complete Freund's Adjuvant (CFA) (50μl). One week after arthritis induction, the animals were treated with HMWHA (once per week for three weeks). Histological analyses were performed using sections stained with hematoxylin-eosin, toluidine blue and Picrosirius. Were also performed histomorphometric analysis and birefringence of collagenous fibers (polarization microscopy). Biochemical analyses of TMJ tissues were carried out through measurements of sulfated glycosaminoglycans and zymography for evaluation of metalloproteinase-2 and -9 (MMP-2 and -9). Data were analyzed using paired t-test and unpaired t-test, with a 5% significance level. HMWHA reduced histologic changes and thickness of the articular disc, led to a greater arrangement of collagenous fibers, lower concentration of sulfated glycosaminoglycans and lower activity in all isoforms of MMP-2 and -9 in TMJs with induced arthritis. These findings suggest that HMWHA may exert a protective effect on the TMJ.

Soft Tissue Knee Injury With Concomitant Osteochondral Fracture Is Associated With Higher Degree of Acute Joint Inflammation

Background: Osteochondral fractures are often seen on magnetic resonance imaging (MRI) of acutely injured knees, but their existence has gained little interest because of a lack of knowledge of their relation to treatment options and outcome. It is not clear whether acute phase synovial fluid (SF) concentrations of cartilage and bone markers and proinflammatory cytokines are different between traumatically injured knees with or without osteochondral fracture. Hypothesis: Acutely injured knees with an osteochondral fracture, particularly fractures with disrupted cortical bone, have higher concentrations of bone markers and cytokines than do knees without an osteochondral fracture. Study Design: Cross-sectional study; Level of evidence, 3. Methods: Synovial fluid (hemarthrosis) was aspirated (median 1 day after injury) and 1.5-T MRI was performed (median 8 days after injury) in the acutely injured knee of 98 individuals (26% women; mean age, 23 years). As visualized on MRI, 39% knees had an osteochondral fracture with disrupted cortical bone, 30% had an osteochondral fracture with intact cortical bone, and 32% did not have an osteochondral fracture. Concentrations of sulfated glycosaminoglycan, ARGS aggrecan, cartilage oligomeric matrix protein, osteocalcin, secreted protein acidic and rich in cysteine (SPARC), osteopontin and proinflammatory cytokines (interleukin [IL]–1β, IL-6, IL-8, and tumor necrosis factor [TNF]–α) were analyzed. Results: After adjusting for days between injury and SF aspiration, age at injury, and sex, knees with any osteochondral fracture (with or without disrupted cortical bone) had significantly higher SF concentrations of TNF-α (median [interquartile range (IQR)] = 9 [7-12] pg/mL vs 7 [5-14] pg/mL; P = .013), whereas knees with an osteochondral fracture with disrupted cortical bone had significantly higher SF concentrations (medians [IQRs]) of SPARC (492 [328-754] ng/mL vs 407 [140-685] ng/mL; P = .030), IL-8 (278 [148-628] pg/mL vs 138 [67-413] pg/mL; P = .028), and TNF-α (11 [7-15] pg/mL vs 7 [5-14] pg/mL; P = .004) compared with knees without an osteochondral fracture. Conclusion: In acutely injured knees with hemarthrosis, a concomitant osteochondral fracture with disrupted cortical bone is associated with a higher degree of joint inflammation.

Effects of Autologous Conditioned Plasma® (ACP) on the healing of surgically induced core lesions in equine superficial digital flexor tendon

Tendon pathologies are among the most common musculoskeletal disorders in horses. After damage the tendon repairs by forming disorganized scar tissue that is of inferior functional quality than normal tendon, leading to high re-injury rates. Many of the currently available treatment modalities cannot significantly reduce this high recurrence rate. Autologous Conditioned Plasma (ACP, Arthrex Inc., USA) has been described in the literature as a leukocyte-reduced platelet concentrate. This blood product has been used in equine and human medicine for the treatment of tendon and ligament injuries. However, the effect of this therapeutical approach on tendon healing is unknown. Core lesions were surgically induced in the Superficial Digital Flexor Tendons (SDFT) of both fore- and hind limbs in eight healthy horses. At days 7 and 15 after lesion induction one randomly assigned fore- and hindlimb was treated with ACP and the contralateral one with saline. This study used data from the forelimbs SDFTs only. Gray-scale and color Doppler ultrasonographic parameters monitored throughout the study did not differ significantly at any time point. 22 weeks after the last treatment, the ACP treated tendons presented a significantly lower concentration of sulphated glycosaminoglycans (GAGs) (p≤0.05) when compared to saline. Other compositional, biomechanical and histological parameters presented no significant differences. Our study indicates that 2 intra-tendinous ACP treatments (without anticoagulant) during the proliferative phase of healing in surgically induced tendon core lesions have a limited effect on tendon healing when comparing ultrasonographic, biochemical, biomechanical and histological parameters with the control treatment. Long-term placebo controlled clinical trials with more horses are warranted to determine if this effect is clinically significant.

The expression of glycosaminoglycans and proteoglycans in the uterine cervix of albino rats after local hyaluronidase infusion

Objective: To assess the local effect of hyaluronidase injection on the expression of glycosaminoglycans (GAGs) and proteoglycans (PGs) in the extracellular matrix of the uterine cervix from pregnant albino rats.Methods: Ten pregnant rats were divided into two groups on day 18 of pregnancy. The experimental group (Gexp) of rats received an intracervical infusion of 0.02 mL of hyaluronidase diluted to 1 mL with distilled water, whereas the control group (Gc) received 1 mL of distilled water. On day 20 of pregnancy, the pregnant rats were sacrificed and the uterine cervixes from all rats were then dissected. The qualitative expression of hyaluronic acid (HA) was assessed by immunohistochemistry and quantified by sandwich ELISA. To compare the quantitative GAG values between groups, a Student’s t-test for independent samples was performed. PGs were also assessed by immunohistochemical analysis.Results: The electrophoretic profile of newly synthesized radioactively labeled GAGs degraded by specific enzymes showed that there were two predominant GAGs in both Gc and Gexp, i.e. heparan sulfate (HS) and a mixture of hondroitin sulfate (CS) and dermatan sulfate (DS). The concentrations of GAGs showed a significant reduction of CS/DS (p < 0.004) and HS (p < 0.005) relative to Gc. HA staining was less intense in the lamina propria and area surrounding the blood vessels in Gexp compared to Gc. The HA contents were also significantly reduced (p < 0.012).Conclusions: Intracervical hyaluronidase infusion promoted a significant reduction in the concentration of sulfated GAGs as assessed by both qualitative (histochemical) and quantitative (fluorometric) measurements of HA.

In vitro effects of meloxicam on metabolism in articular chondrocytes from dogs with naturally occurring osteoarthritis

To assess effects of in vitro meloxicam exposure on metabolism in articular chondrocytes from dogs with naturally occurring osteoarthritis. Femoral head cartilage from 16 dogs undergoing total hip replacement. Articular cartilage samples were obtained. Tissue sulfated glycosaminoglycan (SGAG), collagen, and DNA concentrations were measured. Collagen, SGAG, chondroitin sulfate 846, NO, prostaglandin E2 (PGE2), and matrix metalloproteinase (MMP)-2, MMP-3, MMP-9, and MMP-13 concentrations in culture medium were analyzed. Aggrecan, collagen II, MMP-2, MMP-3, MMP-9, MMP-13, ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS)-4, ADAMTS-5, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, TIMP-3, interleukin-1β, tumor necrosis factor-α, cyclooxygenase-1, cyclooxygenase-2, and inducible nitric oxide synthase gene expression were evaluated. Comparisons between tissues cultured without (control) and with meloxicam at concentrations of 0.3, 3.0, and 30.0 μg/mL for up to 30 days were performed by means of repeated-measures analysis. Meloxicam had no effect on chondrocyte SGAG, collagen, or DNA concentrations. Expression of ADAMTS-5 was significantly decreased in all groups on all days, compared with the day 0 value. On day 3, culture medium PGE2 concentrations were significantly lower in all meloxicam-treated groups, compared with values for controls, and values remained low. Culture medium MMP-3 concentrations were significantly lower on day 30 than on day 3 in all meloxicam-treated groups. Results suggested that in vitro meloxicam treatment of osteoarthritic canine cartilage for up to 30 days did not induce matrix degradation or stimulate MMP production. Meloxicam lowered PGE2 release from this tissue, and effects on tissue chondrocyte content and matrix composition were neutral.

Aggrecanase cleavage in juvenile idiopathic arthritis patients is minimally detected in the aggrecan interglobular domain but robust at the aggrecan C‐terminus

To investigate aggrecan degradation in juvenile idiopathic arthritis (JIA). The pattern and abundance of aggrecan fragments in synovial fluid (SF) aspirates from JIA patients were analyzed and compared with aggrecan fragments in SF from patients with other arthritides, children with knee injury, and a knee-healthy reference group. Concentrations of sulfated glycosaminoglycan (sGAG) in SF were measured by Alcian blue precipitation assay. Aggrecan fragments were purified by dissociative CsCl density-gradient centrifugation, deglycosylated, and analyzed by Western blot using antibodies specific for either aggrecanase-derived ARGS, SELE, and KEEE neoepitopes or the aggrecan G3 domain. The concentration of sGAG in SF from patients with JIA was significantly lower compared with that in SF from patients with osteoarthritis (OA) (P < 0.001), patients with juvenile knee injury (P = 0.006), and knee-healthy controls (P = 0.022). Western blot analysis revealed KEEE, SELE, and G3 fragments generated by aggrecanase cleavage in the chondroitin sulfate-rich region of aggrecan in patients with JIA. The pattern of aggrecan fragments in JIA patients was not identical to that in pooled OA SF, although there were notable similarities. Surprisingly, aggrecanase-derived ARGS fragments were barely detectable in JIA SF, in marked contrast to levels in OA SF. Aggrecanases appear to cleave minimally in the interglobular domain of aggrecan in JIA patients despite robust levels of cleavage in the chondroitin sulfate-rich region. These results suggest that in JIA, unlike other arthritides, aggrecanase cleavage in the aggrecan interglobular domain might not be a major pathogenic event.

Assessment of contrast‐enhanced computed tomography for imaging of cartilage during fracture healing

Assessment of the early stages of fracture healing via X-rays and computed tomography is limited by the low radio-opacity of cartilage. We validated a method of contrast-enhanced computed tomography (CECT) for non-destructive identification of cartilage within a healing fracture callus. Closed, stabilized fractures in femora of C57BL/6 mice were harvested on post-operative day 9.5 and imaged ex vivo with micro-computed tomography (µCT) before and after incubation in a cationic contrast agent that preferentially accumulates in cartilage due to the high concentration of sulfated glycosaminoglycans in the tissue. Co-registration of the pre- and post-incubation images, followed by image subtraction, enabled two- and three-dimensional delineation of mineralized tissue, soft callus, and cartilage. The areas of cartilage and callus identified with CECT were compared to those identified with the gold-standard method of histomorphometry. No difference was found between the areas of cartilage measured by the two methods (p = 0.999). Callus area measured by CECT was smaller than, but strongly predictive of (R(2) = 0.80, p < 0.001), the corresponding histomorphometric measurements. CECT also enabled qualitative identification of mineralized cartilage. These findings indicate that the CECT method provides accurate, quantitative, and non-destructive visualization of the shape and composition of the fracture callus, even during the early stages of repair when little mineralized tissue is present. The non-destructive nature of this method would allow subsequent analyses, such as mechanical testing, to be performed on the callus, thus enabling higher-throughput, comprehensive investigations of bone healing.

Evaluation of experimental impact injury for inducing post-traumatic osteoarthritis in the metacarpophalangeal joints of horses

To determine whether a single contusive impact injury to the palmar aspect of the metacarpus would progress to post-traumatic osteoarthritis or palmar osteochondral disease in horses. 12 horses. In each horse, an impact injury was created on the palmar aspect of the medial metacarpal condyle of 1 randomly chosen limb with an impactor device under arthroscopic and fluoroscopic guidance. The opposite limb was sham operated as a control. A low to moderate amount of forced exercise was instituted, and horses were evaluated clinically via lameness examinations weekly for 5 months, then biweekly until endpoint, with synovial fluid analysis performed at 0, 1, 2, 3, 4, 6, 8, and 10 months and radiography at baseline and endpoint. Macroscopic examination, micro-CT, and sample collection for cartilage viability and sulfated glycosaminoglycan content, histologic evaluation, immunohistochemical analysis, and fluorochrome analysis were performed following euthanasia at 1 (3 horses), 4 (4), and 8 to 10 (5) months after surgery. There was variability in impact lesion location, depth, and area on macroscopic inspection, but on histologic evaluation, cartilage defects were less variable. Mean sulfated glycosaminoglycan concentration from cartilage at the impact site was significantly lower than that at a similar site in control limbs. Higher concentrations of cartilage oligomeric matrix protein were observed in synovial fluid from impact-injured joints. The impact injury method caused mild focal osteoarthritic lesions in the metacarpophalangeal joint, but did not progress to palmar osteochondral disease at this site. Repeated injury is probably required for the development of palmar osteochondral disease.

Glycosaminoglycan profiles in the uterus of adult ovariectomized rats treated with estrogen and progestagen

To evaluate the effects of conjugated equine estrogens (CE) alone or in combination with medroxyprogesterone acetate (MPA) on glycosaminoglycans (GAGs) in the cervix and horns of the rat uterus. Thirty days after ovariectomy, adult rats were randomly divided into four groups: GI, control (treated with drug vehicle); GII, CE (50 μg/kg per day); GIII, MPA (0.2mg/kg per day), and GIV, CE+MPA (doses as in GII and GIII). Drugs and vehicle were given by gavage during 28 days. Afterwards the animals were anesthetized, the cervix and uterine horns were dissected out and the middle portion fixed in 10% formaldehyde solution; other portions were fixed in acetone for histological examination and glycosaminoglycan quantification, respectively. Agarose gel electrophoresis was used for sulfated GAG analyses, and hyaluronic acid was assayed with an ELISA-like method. Statistical analysis was done by the Student's t test and the Tukey-Kramer test (P<0.05). The cervix and uterine horn structures presented signs of atrophy in the control group (GI). The other groups, mainly groups III and IV, had histological aspects of proliferation. In all groups the concentration of sulfated GAGs (especially dermatan sulfate) was higher than that of non-sulfated GAGs, both in cervix and in uterine horns. Estrogens increased sulfated GAG concentration at the cervix and the horn, whereas in uterine horns the amounts of sulfated GAGs were decreased after estrogens plus MPA treatment. The concentration of hyaluronic acid in uterine horns was higher than in cervices. The profiling and amounts of glycosaminoglycans in the two portions of the rat uterus are uneven. Dermatan sulfate occurs in higher concentrations in both cervix and uterine horns. Sulfated GAGs in rat cervix were increased by estrogens plus MPA, but were decreased by MPA alone in uterine horns.

Cartilage and bone markers and inflammatory cytokines are increased in synovial fluid in the acute phase of knee injury (hemarthrosis) – a cross-sectional analysis

The aim of this cross-sectional study was to investigate concentrations of cartilage and bone markers, and pro-inflammatory cytokines in synovial fluid (SF) collected at different time-points from acutely injured knees with hemarthrosis and to compare these with SF concentrations of knees of age and gender-matched healthy reference subjects. SF was aspirated from the acutely injured knee of 111 individuals (mean age 27 years, span 13-64 years, 22% women). Concentrations of sulfated glycosaminoglycan (sGAG) were measured by Alcian blue precipitation whereas cartilage ARGS, bone biomarkers [osteocalcin (OCL), secreted protein acidic and rich in cysteine (SPARC) and osteopontin (OPN)] and pro-inflammatory cytokines [interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor (TNF)-α] were analyzed using electrochemiluminescence. Samples were also analyzed with regard to time between injury and aspiration [same day (n=29), 1 day (n=31), 2-3 days (n=19), 4-7 days (n=20) and 8-23 days (n=12)]. SF concentrations of ARGS (P<0.001), SPARC (P<0.001), OPN (P<0.001), and all cytokines (P<0.001), but not sGAG (P=0.06) or OCL (P=0.992), were significantly higher in injured knees compared to knees of reference subjects. The cartilage markers sGAG and ARGS were significantly higher in knees aspirated later than 1 day after injury, whereas concentrations of SPARC and OPN and all cytokines were higher in knees aspirated the same day as the injury and at all time-points thereafter. Our results suggest that an acute knee injury is associated with an instant local biochemical response to the trauma, which may affect cartilage and bone as well as the inflammatory activity.