Reduction In Glycosaminoglycan Content Research Articles

Elevated fluid and glycosaminoglycan content in the Achilles tendon contribute to higher intratendinous pressures: Implications for Achilles tendinopathy

BackgroundTendinopathy alters the compositional properties of the Achilles tendon by increasing fluid and glycosaminoglycan content. It has been speculated that these changes may affect intratendinous pressure, but the extent of this relationship remains unclear. Therefore, we aimed to investigate the impact of elevated fluid and glycosaminoglycan content on Achilles tendon intratendinous pressure and to determine whether hyaluronidase (HYAL) therapy can intervene in this potential relationship. MethodsTwenty paired fresh-frozen cadaveric Achilles tendons were mounted in a tensile-testing machine and loaded up to 5% strain. Intratendinous resting (at 0% strain) and dynamic pressure (at 5% strain) were assessed using the microcapillary infusion technique. First, intratendinous pressure was measured under native conditions before and after infusion of 2 mL physiological saline. Next, 80 mg of glycosaminoglycans were administered bilaterally to the paired tendons. The right tendons were additionally treated with 1500 units of HYAL. Finally, both groups were retested, and the glycosaminoglycan content was analyzed. ResultsIt was found that both elevated fluid and glycosaminoglycan content resulted in higher intratendinous resting and dynamic pressures (p < 0.001). HYAL treatment induced a 2.3-fold reduction in glycosaminoglycan content (p = 0.002) and restored intratendinous pressures. ConclusionThe results of this study demonstrated that elevated fluid and glycosaminoglycan content in Achilles tendinopathy contribute to increased intratendinous resting and dynamic pressures, which can be explained by the associated increased volume and reduced permeability of the tendon matrix, respectively. HYAL degrades glycosaminoglycans sufficiently to lower intratendinous pressures and may, therefore, serve as a promising treatment.

Effect of Targeted Cytokine Inhibition on Progression of Post-Traumatic Osteoarthritis Following Intra-Articular Fracture.

Intra-articular fractures (IAF) result in significant and prolonged inflammation, increasing the chances of developing post-traumatic osteoarthritis (PTOA). Interleukin-one beta (IL-1β) and Tumor Necrosis Factor-alpha (TNF-α) are key inflammatory factors shown to be involved in osteochondral degradation following IAF. As such, use of targeted biologics such as Infliximab (INX), a TNF-α inhibitor, and Anakinra (ANR), an interleukin-one (IL-1) receptor antagonist (IL1RA), may protect against PTOA by damping the inflammatory response to IAF and reducing osteochondral degradation. To test this hypothesis, IAFs were induced in the hindlimb knee joints of rats treated with INX at 10 mg/kg/day, ANR at 100 g/kg/day, or saline (vehicle control) by subcutaneous infusion for a period of two weeks and healing was evaluated at 8-weeks post injury. Serum and synovial fluid (SF) were analyzed for soluble factors. In-vivo microcomputed tomography (µCT) scans assessed bone mineral density and bone morphometry measurements. Cationic CA4+ agent assessed articular cartilage composition via ex vivo µCT. Scoring according to the Osteoarthritis Research Society International (OARSI) guidelines was performed on stained histologic tibia sections at the 56-day endpoint on a 0-6 scale. Systemically, ANR reduced many pro-inflammatory cytokines and reduced osteochondral degradation markers Cross Linked C-Telopeptide Of Type II (CTXII, p < 0.05) and tartrate-resistant acid phosphatase (TRAP, p < 0.05). ANR treatment resulted in increased chemokines; macrophage-chemotractant protein-1 (MCP-1), MPC-3, macrophage inhibitory protein 2 (MIP2) with a concomitant decrease in proinflammatory interleukin-17A (IL17A) at 14 days post-injury within the SF. Microcomputed tomography (µCT) at 56 days post-injury revealed ANR Treatment decreased epiphyseal degree of anisotropy (DA) (p < 0.05) relative to saline. No differences were found with OARSI scoring but contrast-enhanced µCT revealed a reduction in glycosaminoglycan content with ANR treatment. These findings suggest targeted cytokine inhibition, specifically IL-1 signaling, as a monotherapy has minimal utility for improving IAF healing outcomes but may have utility for promoting a more permissive inflammatory environment that would allow more potent disease modifying osteoarthritis drugs to mitigate the progression of PTOA after IAF.

Does the Intervertebral Disc Adapt to Load?

Abstract: Many in the scientific community have been debating the extent to which the spine can adapt. While it is widely accepted that vertebral bones can adapt and become stronger/denser with appropriately dosed loading, there are still many unanswered questions regarding intervertebral discs (IVDs). Recently, some are claiming that IVDs can adapt to stress under load and become stronger and more resilient over time based on the findings from a recent study “Imaging of exercise-induced spinal remodeling in elite rowers” by Frenken et al 2022 in the Journal of Science and Medicine in Sport. The Frenken paper investigates the Glycosaminoglycan (GAG) content of the IVD amongst elite rowers over the course of a training period. They found a temporary increase in this protein and believed that it indicates lumbar disc “remodeling effects” in response to training. This study does show that repetitive loading of the spine does support nutrient transport across the vertebral endplates and an increase in GAG. However, with research showing that a reduction in GAG content is associated with IVD degeneration (a negative adaptation), many have been led to believe the opposite to be accurate; an increase in GAG content means the disc is positively adapting. However, this assumption may be incorrect as it is based on an association, not causation. The proof of nutrient availability isn’t the same as mechanism utility. If in fact, the disc was truly adapting we would likely NOT see an overwhelming amount of research showing an extremely high prevalence of disc degeneration in rowers. The Frenken paper and many more do support the notion that the GAG content alters in response to certain stimuli; however, we can NOT conclude that a temporary increase in GAG content supports the hypothesis that the IVD adapts positively to load.

B3galt6 Knock-Out Zebrafish Recapitulate β3GalT6-Deficiency Disorders in Human and Reveal a Trisaccharide Proteoglycan Linkage Region.

Proteoglycans are structurally and functionally diverse biomacromolecules found abundantly on cell membranes and in the extracellular matrix. They consist of a core protein linked to glycosaminoglycan chains via a tetrasaccharide linkage region. Here, we show that CRISPR/Cas9-mediated b3galt6 knock-out zebrafish, lacking galactosyltransferase II, which adds the third sugar in the linkage region, largely recapitulate the phenotypic abnormalities seen in human β3GalT6-deficiency disorders. These comprise craniofacial dysmorphism, generalized skeletal dysplasia, skin involvement and indications for muscle hypotonia. In-depth TEM analysis revealed disturbed collagen fibril organization as the most consistent ultrastructural characteristic throughout different affected tissues. Strikingly, despite a strong reduction in glycosaminoglycan content, as demonstrated by anion-exchange HPLC, subsequent LC-MS/MS analysis revealed a small amount of proteoglycans containing a unique linkage region consisting of only three sugars. This implies that formation of glycosaminoglycans with an immature linkage region is possible in a pathogenic context. Our study, therefore unveils a novel rescue mechanism for proteoglycan production in the absence of galactosyltransferase II, hereby opening new avenues for therapeutic intervention.

Production and Characterisation of Acellular Human Liver Matrix: Potential Auxiliary Liver Graft

Shortage of donor organs combined with ever increasing liver disease has directed attention towards growing implantable liver tissue in the laboratory setting. Recent developments in decellularisation and recellularisation techniques have opened exciting facets for liver whole organ reengineering. The aim of this study was to develop a decellularisation protocol for human liver, assess the structural and biochemical integrity and assess the biocompatibility of the acellular matrix. Discarded human livers were split and the left lateral and right posterior lobes with a mean weight of 399 ± 162 g were used (n=8 lobes). Perfusion decellularisation was carried out through the hepatic artery and portal vein. Hypotonic buffer, 0.1 % (w/v) sodium dodecyl sulphate, hypertonic buffer and nuclease solutions were used to remove cells plus nuclear material. Following several iterations of a previously patented Leeds University decellularisation protocol, greater than 91.0 % (w/w) DNA was removed from all areas of liver tissue. Fluoroscopy confirmed integrity of vascular tree. Histological analysis demonstrated lack of cells and maintenance of the portal triad histioarchitecture. Immunohistochemistry demonstrated positive staining for key extracellular proteins such as collagen type I and III, fibronectin and laminin. Scanning electron microscopy showed an intact extracellular matrix. Biochemical assessment confirmed significant reduction in glycosaminoglycan content, but there was increase in collagen content, the latter likely due to extraction by dry weight of other soluble components in the matrix. The acellular tissues and extracts were not cytotoxic to either murine 3T3 or baby hamster kidney cells. Preliminary recellularisation (n=3 lobes) showed the matrix to be biocompatible to primary cryopreserved human hepatocytes seeded at 30,000 cells. The study has developed a suitable protocol for decellularisation of human liver lobes, without adversely affecting the extracellular matrix. The biocompatible acellular scaffold has the potential to be recellularised with hepatocytes, with a view to engineering a transplantable auxiliary liver graft.

Genistein reduces glycosaminoglycan levels in a mouse model of mucopolysaccharidosis type II

Mucopolysaccharidoses (MPS) are lysosomal storage disorders resulting from a deficit of specific lysosomal enzymes catalysing glycosaminoglycan (GAG) degradation. The typical pathology involves most of the organ systems, including the brain, in its severe forms. The soy isoflavone genistein has recently attracted considerable attention as it can reduce GAG synthesis in vitro. Furthermore, genistein is able to cross the blood-brain barrier in the rat. The present study was undertaken to assess the ability of genistein to reduce urinary and tissue GAG levels in vivo. We used mice with genetic deletion of iduronate-2-sulphatase (one of the GAG catabolizing enzymes) which provide a model of MPS type II. Two doses of genistein, 5 or 25 mg.kg(-1).day(-1), were given, in the diet for 10 or 20 weeks. Urinary and tissue GAG content was evaluated by biochemical and histochemical procedures. Urinary GAG levels were reduced after 10 weeks' treatment with genistein at either 5 or 25 mg.kg(-1).day(-1). In tissue samples from liver, spleen, kidney and heart, a reduction in GAG content was observed with both dosages, after 10 weeks' treatment. Decreased GAG deposits in brain were observed after genistein treatment in some animals. There was decreased GAG storage in the MPSII mouse model following genistein administration. Our results would support the use of this plant-derived isoflavone in a combined therapeutic protocol for treatment of MPS.

Donor sex and age influence the chondrogenic potential of human femoral bone marrow stem cells

Damaged articular cartilage does not heal well and can progress to osteoarthritis (OA). Human bone marrow stem cells (BMC) are promising cells for articular cartilage repair, yet age- and sex-related differences in their chondrogenesis have not been clearly identified. The purpose of this study is to test whether the chondrogenic potential of human femoral BMC varies based on the sex and/or age of the donor. BMC were isolated from 21 males (16-82 years old (y.o.)) and 20 females (20-77 y.o.) during orthopaedic procedures. Cumulative population doubling (CPD) was measured and chondrogenesis was evaluated by standard pellet culture assay in the presence or absence of transforming growth factor beta 1 (TGFbeta1). Pellet area was measured, and chondrogenic differentiation was determined by Toluidine blue and Safranin O-Fast green histological grading using the Bern score and by glycosaminoglycan (GAG) content. No difference in CPD was observed due to donor sex or age. The increase in pellet area with addition of TGFbeta1 and the Bern score significantly decreased with increasing donor age in male BMC, but not in female BMC. A significant reduction in GAG content per pellet was also observed with increasing donor age in male BMC. This was not observed in female BMC. This study showed an age-related decline in chondroid differentiation with TGFbeta1 stimulation in male BMC, but not in female BMC. Understanding the mechanisms for these differences will contribute to improved clinical use of autologous BMC for articular cartilage repair, and may lead to the development of customized age- or sex-based treatments to delay or prevent the onset of OA.

A Fibronectin Fragment Alters the Metabolism by Rabbit Intervertebral Disc Cells In Vitro

A biochemical and gene expression study was conducted to determine the effects of the 30-kDa N-terminal fibronectin fragment (Fn-f) on the glycosaminoglycan content of nucleus pulposus (NP) explant cultures, and on the gene expression profile of NP cells in alginate culture. To determine the effects of Fn-f on NP cells in alginate culture and disc explant cultures. The macroscopic and histologic features of disc degeneration have been well described, but the molecular biology of disc degeneration remains poorly understood. Although fibronectin and fibronectin fragments are known to accumulate in degenerative discs, the role of fibronectin fragments on the degenerative process has not been elucidated. This study sought to define the effects of Fn-f on the expression of key matrix and degradative genes and on disc matrix proteins. New Zealand white rabbits discs were harvested. NP cells were either isolated and grown in alginate culture or cultured as explanted tissue. The cultured cells were exposed to 10 nmol/L, 100 nmol/L, and 1 micromol/L concentrations of 30-kDa N-terminal Fn-f or a control substance and then analyzed histologically, biochemically, and with gene expression studies. Alginate-cultured NP cells maintained a histologic appearance and phenotypic expression pattern similar to disc cells in vivo. Exposure of these cells to Fn-f led to the up-regulation of the MMP-9, MMP-13, and Fas genes and the down-regulation of the Type II collagen and aggrecan genes. In explant culture, Fn-f exposure led to a 60% reduction in glycosaminoglycan content compared with controls. Treatment of NP cells in vitro with Fn-f led to changes in matrix proteins and gene expression similar to those seen during disc degeneration in vivo. This supports a possible detrimental role of the N-terminal fibronectin fragment in degenerative disc disease.

Effect of oral glucosamine on cartilage degradation in a rabbit model of osteoarthritis

To determine whether oral glucosamine alleviates cartilage degradation in an animal model of osteoarthritis (OA). The effect of 8 weeks of daily oral glucosamine hydrochloride on degeneration of articular cartilage was evaluated in rabbits in which anterior cruciate ligament transection (ACLT) was performed to induce OA. Animals were treated with glucosamine (n = 16) or a placebo (n = 16) and necropsied at 11 weeks. Seven unoperated rabbits served as controls. The articular cartilage was evaluated macroscopically and histologically and analyzed for total type II collagen and glycosaminoglycan (GAG) content. Histologic analysis revealed that loss of proteoglycan, based on Safranin O-fast green staining, was significantly reduced in the lateral tibial plateau cartilage of ACL-transected limbs in the glucosamine group compared with ACL-transected limbs in the placebo group, with a similar, but not significant, trend for the lateral femoral condylar cartilage. Likewise, macroscopic analysis of cartilage showed that the lateral tibial plateau alone had a significantly lower rate of disease in the glucosamine group, which was consistent with the results of the independent histologic assessment. However, no significant treatment effect was detected when composite histologic scores were analyzed. A significant reduction in GAG content was observed in the femoral condyles of placebo-treated ACL-transected joints, but not in the same region of glucosamine-treated ACL-transected joints, compared with their respective contralateral unoperated joints. Oral administration of glucosamine had a detectable, site-specific, partial disease-modifying effect in this model of OA. From a clinical perspective, the administration of glucosamine did not prevent fibrillation and/or erosions of the articular cartilage in all of the treated animals, and no effects were detected in the medial joint compartments.

Inhibition of interleukin 1 beta induced rat and human cartilage degradation in vitro by the metalloproteinase inhibitor U27391.

Interleukin 1 induced proteoglycan loss from cartilage in vitro was prevented by a biochemical inhibitor of metalloproteinase activity. The inhibitor also partially relieved the inhibition of proteoglycan synthesis caused by interleukin 1. The loss of glycosaminoglycan by rat and human femoral head cartilage in response to human recombinant interleukin 1 beta (rhIL-1 beta) was established, and the modulation of this loss by the metalloproteinase inhibitor U27391 was investigated. Rat femoral head cartilage consistently lost glycosaminoglycan in response to rhIL-1 beta whereas only a proportion (30%) of normal human femoral head cartilage did so. Concentrations of 10-100 mumol/l U27391 inhibited the action of rhIL-1 beta on rat femoral head cartilage, reversing both the loss of glycosaminoglycan and the inhibition of glycosaminoglycan synthesis. U27391 also prevented the reduction in glycosaminoglycan content of those human femoral head cartilage explants responsive to rhIL-1 beta. Metalloproteinase inhibition therefore prevents rhIL-1 beta induced glycosaminoglycan loss by rat and human femoral head cartilage, suggesting that inhibitors of such enzymes may prove to be of therapeutic benefit in erosive diseases in humans.

The effect of chronic paracetamol administration to rats on the glycosaminoglycan content of patellar cartilage

Male Wistar rats were treated with paracetamol (200 mg/kg twice a day) for 2, 3, 4 and 9 weeks. During the first four weeks of paracetamol administration the serum sulfate concentration was significantly decreased. However, during the fourth until the ninth week, the serum sulfate concentration was only diminished to a small and insignificant extent. The paracetamol administration did not lead to serious liver or renal toxicity, as determined by alanine aminotransferase and creatinine levels in the serum of the rats. The paracetamol-induced serum sulfate depletion, observed during the first four weeks of the experiment, led to a significantly lower glycosaminoglycan content of the patellar cartilage of the rats after three and four weeks paracetamol treatment. When after the fourth week the serum sulfate concentration rose to nearly normal levels also the glycosaminoglycan content in the rat patellar cartilage reached control levels. These data indicate that the serum sulfate depletion might be the causative factor for the observed reduction in glycosaminoglycan content of rat patellar cartilage.

Fluoride-induced changes in the tooth glycosaminoglycans: an in vivo study in the rabbit.

Effect of high fluoride ingestion on the tooth matrix glycosaminoglycans was studied in rabbits administered 10 mg NaF/kg body weight orally at 24-h intervals for a period of 9 months. Fluoride-treated tooth showed a significant reduction in glycosaminoglycan content as compared to the normal tooth. Sephadex G-75 chromatography and DEAE-cellulose ion exchange chromatography revealed the presence of small molecular weight glycosaminoglycan molecules and an increase in the charge density heterogeneity in the sulphated glycosaminoglycans of the fluoride-treated rabbit tooth as compared to the controls. These changes may be related to the dedifferentiated tooth matrix and an increase in the dermatan sulphate content in the fluoride-treated tooth matrix reported earlier.

Hypertrophic repair of articular cartilage in experimental osteoarthrosis.

Section of the anterior cruciate ligament has been performed in the knee of 11 mature dogs. The macroscopically normal cartilage from patella and femoral trochlea of animals killed from 2 to 32 weeks after operation was used for histological, histomorphometrical, and biochemical analysis. Previously undescribed degenerative lesions of the superficial matrix were observed, and there was evidence for secondary healing of these lesions. An early and progressive decrease in superficial cell density and a later progressive increase in cartilage thickness without any change in the cell density of the middle and deep cartilage layers was found. A slight increase in water content with no reduction in glycosaminoglycan content was observed. The results suggest that joint laxity results initially in superficial degenerative changes and later in hypertrophic regenerative changes due to cell proliferation and increased matrix synthesis. Hypertrophic remodeling of articular cartilage in response to abnormal mechanical stresses is postulated.