Glycosaminoglycan Levels Research Articles

Assessment of glycosaminoglycan content in bone using Raman spectroscopy.

Glycosaminoglycans (GAGs) are responsible for preserving bone tissue toughness as well as regulating collagen formation and mineralization in the extracellular matrix. However, current methods for characterization of GAGs in bone are destructive, thus unable to capture in situ changes or differences in GAGs between experimental groups. As an alternative, Raman spectroscopy is a non-destructive method and can detect concurrent changes in GAGs and other bone constituents. In this study, we hypothesized that the two most prominent Raman peaks of sulfated GAGs (at ~1066cm-1 and at ~1378cm-1) could be used to detect differences in GAGs content of bone. To test this hypothesis, three experimental models were utilized: an in vitro model (enzymatic removal of GAGs from human cadaver bone), an ex vivo mouse model (biglycan KO vs. WT), and an ex vivo aging model (comparing cadaveric bone samples from young and old donors). All Raman measurements were compared to Alcian blue measurements to confirm the validity of Raman spectroscopy in detecting GAGs changes in bone. Irrespective of different models, it was found that the ~1378cm-1 peak in Raman spectra of bone was uniquely sensitive to changes of GAGs content in bone when normalized with respect to the phosphate phase (~960cm-1); i.e., 1378cm-1/960cm-1 (peak intensity ratio) or 1370-1385cm-1/930-980cm-1 (integrated peak area ratio). In contrast, the 1070cm-1 peak, which includes another major peak of GAGs (1066cm-1), seemed to be compromised to detect changes of GAGs in bone due to concurrent changes of carbonate (CO3) in the similar peak range. This study validates the ability of Raman spectroscopy to detect in situ treatment-, genotype-, and age-related changes in GAG levels of bone matrix.

Mice lacking nucleotide sugar transporter SLC35A3 exhibit lethal chondrodysplasia with vertebral anomalies and impaired glycosaminoglycan biosynthesis.

SLC35A3 is considered an uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) transporter in mammals and regulates the branching of N-glycans. A missense mutation in SLC35A3 causes complex vertebral malformation (CVM) in cattle. However, the biological functions of SLC35A3 have not been fully clarified. To address these issues, we have established Slc35a3-/-mice using CRISPR/Cas9 genome editing system. The generated mutant mice were perinatal lethal and exhibited chondrodysplasia recapitulating CVM-like vertebral anomalies. During embryogenesis, Slc35a3 mRNA was expressed in the presomitic mesoderm of wild-type mice, suggesting that SLC35A3 transports UDP-GlcNAc used for the sugar modification that is essential for somite formation. In the growth plate cartilage of Slc35a3-/-embryos, extracellular space was drastically reduced, and many flat proliferative chondrocytes were reshaped. Proliferation, apoptosis and differentiation were not affected in the chondrocytes of Slc35a3-/-mice, suggesting that the chondrodysplasia phenotypes were mainly caused by the abnormal extracellular matrix quality. Because these histological abnormalities were similar to those observed in several mutant mice accompanying the impaired glycosaminoglycan (GAG) biosynthesis, GAG levels were measured in the spine and limbs of Slc35a3-/-mice using disaccharide composition analysis. Compared with control mice, the amounts of heparan sulfate, keratan sulfate, and chondroitin sulfate/dermatan sulfate, were significantly decreased in Slc35a3-/-mice. These findings suggest that SLC35A3 regulates GAG biosynthesis and the chondrodysplasia phenotypes were partially caused by the decreased GAG synthesis. Hence, Slc35a3-/- mice would be a useful model for investigating the in vivo roles of SLC35A3 and the pathological mechanisms of SLC35A3-associated diseases.

Characterization of a HIR-Fab-IDS, Novel Iduronate 2-Sulfatase Fusion Protein for the Treatment of Neuropathic Mucopolysaccharidosis Type II (Hunter Syndrome)

Mucopolysaccharidosis type II is a severe lysosomal storage disease caused by deficient activity of the enzyme iduronate-2-sulfatase. The only medicinal product approved by the US Food and Drug Administration for enzyme replacement therapy, recombinant iduronate-2-sulfatase (idursulfase, Elaprase®), is a large molecule that is not able to cross the blood-brain barrier and neutralize progressive damage of the central nervous system caused by the accumulation of glycosaminoglycans. Novel chimeric protein HIR-Fab-IDS is an anti-human insulin receptor Fab fragment fused to recombinant modified iduronate-2-sulfatase. This modification provides a highly selective interaction with the human insulin receptor, which leads to the HIR-Fab-IDS crossing the blood-brain barrier owing to internalization of the hybrid molecule by transcytosis into endothelial cells adjacent to the nervous system by the principle of a 'molecular Trojan horse'. In this work, the physicochemical and biological characterization of a blood-brain barrier-penetrating fusion protein, HIR-Fab-IDS, is carried out. HIR-Fab-IDS consists of an anti-human insulin receptor Fab fragment fused to recombinant iduronate-2-sulfatase. Comprehensive analytical characterization utilizing modern techniques (including surface plasmon resonance and mass spectrometry) was performed using preclinical and clinical batches of HIR-Fab-IDS. Critical quality parameters that determine the therapeutic effect of iduronate-2-sulfatase, as well as IDS enzymatic activity and in vitro cell uptake activity were evaluated in comparison with the marketed IDS product Elaprase® (IDS RP). In vivo efficiency of HIR-Fab-IDS in reversing mucopolysaccharidosis type II pathology in IDS-deficient mice was also investigated. The affinity of the chimeric molecule for the INSR was also determined by both an enzyme-linked immunosorbent assay and surface plasmon resonance. We also compared the distribution of 125I-radiolabeled HIR-Fab-IDS and IDS RP in the tissues and brain of cynomolgus monkeys after intravenous administration. The HIR-Fab-IDS primary structure investigation showed no significant post-translational modifications that could affect IDS activity, except for the formylglycine content, which was significantly higher for HIR-Fab-IDS compared with that for IDS RP (~76.5 vs ~67.7%). Because of this fact, the specific enzyme activity of HIR-Fab-IDS was slightly higher than that of IDS RP (~2.73 × 106U/μmol vs ~2.16 × 106U/μmol). However, differences were found in the glycosylation patterns of the compared IDS products, causing a minor reduced in vitro cellular uptake of HIR-Fab-IDS by mucopolysaccharidosis type II fibroblasts compared with IDS RP (half-maximal effective concentration ~26.0 vs ~23.0nM). The efficacy of HIR-Fab-IDS in IDS-deficient mice has demonstrated a statistically significant reduction in the level of glycosaminoglycans in the urine and tissues of the main organs to the level of healthy animals. The HIR-Fab-IDS has revealed high in vitro affinity for human and monkey insulin receptors, and the radioactively labeled product has been shown to penetrate to all parts of the brain and peripheral tissues after intravenous administration to cynomolgus monkeys. These findings indicate that HIR-Fab-IDS, a novel iduronate-2-sulfatase fusion protein, is a promising candidate for the treatment of central nervous system manifestations in neurological mucopolysaccharidosis type II.

Generation and characterization of an immunodeficient mouse model of mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (Hunter syndrome, MPS II) is an inherited X-linked recessive disease caused by deficiency of iduronate-2-sulfatase (IDS), resulting in the accumulation of the glycosaminoglycans (GAG) heparan and dermatan sulfates. Mouse models of MPS II have been used in several reports to study disease pathology and to conduct preclinical studies for current and next generation therapies. Here, we report the generation and characterization of an immunodeficient mouse model of MPS II, where CRISPR/Cas9 was employed to knock out a portion of the murine IDS gene on the NOD/SCID/Il2rγ (NSG) immunodeficient background. IDS-/- NSG mice lacked detectable IDS activity in plasma and all analyzed tissues and exhibited elevated levels of GAGs in those same tissues and in the urine. Histopathology revealed vacuolized cells in both the periphery and CNS of NSG-MPS II mice. This model recapitulates skeletal disease manifestations, such as increased zygomatic arch diameter and decreased femur length. Neurocognitive deficits in spatial memory and learning were also observed in the NSG-MPS II model. We anticipate that this new immunodeficient model will be appropriate for preclinical studies involving xenotransplantation of human cell products intended for the treatment of MPS II.

Gene therapy for cross-correction of somatic organs and the CNS in mucopolysaccharidosis II in rodents and non-human primates

Mucopolysaccharidosis II (MPS II) is a rare lysosomal storage disease characterized by deficient activity of iduronate-2-sulfatase (I2S), leading to pathological accumulation of glycosaminoglycans (GAGs) in tissues. We used iduronate-2-sulfatase knockout (Ids KO) mice to investigate if liver-directed recombinant adeno-associated virus vectors (rAAV8-LSP-hIDSco) encoding human I2S (hI2S) could cross-correct I2S deficiency in Ids KO mouse tissues, and we then assessed the translation of mouse data to non-human primates (NHPs). Treated mice showed sustained hepatic hI2S production, accompanied by normalized GAG levels in somatic tissues (including critical tissues such as heart and lung), indicating systemic cross-correction from liver-secreted hI2S. Brain GAG levels in Ids KO mice were lowered but not normalized; higher doses were required to see improvements in brain histology and neurobehavioral testing. rAAV8-LSP-hIDSco administration in NHPs resulted in sustained hepatic hI2S production and therapeutic hI2S levels in cross-corrected somatic tissues but no hI2S exposure in the central nervous system, perhaps owing to lower levels of liver transduction in NHPs than in mice. Overall, we demonstrate the ability of rAAV8-LSP-hIDSco to cross-correct I2S deficiency in mouse somatic tissues and highlight the importance of showing translatability of gene therapy data from rodents to NHPs, which is critical for supporting translation to clinical development.

Sex differences in hippocampal structural plasticity and glycosaminoglycan disaccharide levels after neonatal handling.

In this study we investigated the effects of a neonatal handling protocol that mimics the handling of sham control pups in protocols of neonatal exposure to brain insults on dendritic arborization and glycosaminoglycan (GAG) levels in the developing brain. GAGs are long, unbranched polysaccharides, consisting of repeating disaccharide units that can be modified by sulfation at specific sites and are involved in modulating neuronal plasticity during brain development. In this study, male and female Sprague-Dawley rats underwent neonatal handling daily between post-natal day (PD)4 and PD9, with brains analyzed on PD9. Neuronal morphology and morphometric analysis of the apical and basal dendritic trees of CA1 hippocampal pyramidal neurons were carried out by Golgi-Cox staining followed by neuron tracing and analysis with the software Neurolucida. Chondroitin sulfate (CS)-, Hyaluronic Acid (HA)-, and Heparan Sulfate (HS)-GAG disaccharide levels were quantified in the hippocampus by Liquid Chromatography/Mass Spectrometry analyses. We found sex by neonatal handling interactions on several parameters of CA1 pyramidal neuron morphology and in the levels of HS-GAGs, with females, but not males, showing an increase in both dendritic arborization and HS-GAG levels. We also observed increased expression of glucocorticoid receptor gene Nr3c1 in the hippocampus of both males and females following neonatal handling suggesting that both sexes experienced a similar stress during the handling procedure. This is the first study to show sex differences in two parameters of brain plasticity, CA1 neuron morphology and HS-GAG levels, following handling stress in neonatal rats.

Decreased Levels of Chaperones in Mucopolysaccharidoses and Their Elevation as a Putative Auxiliary Therapeutic Approach

Mucopolysaccharidoses (MPS) are rare genetic disorders belonging to the lysosomal storage diseases. They are caused by mutations in genes encoding lysosomal enzymes responsible for degrading glycosaminoglycans (GAGs). As a result, GAGs accumulate in lysosomes, leading to impairment of cells, organs and, consequently, the entire body. Many of the therapies proposed thus far require the participation of chaperone proteins, regardless of whether they are therapies in common use (enzyme replacement therapy) or remain in the experimental phase (gene therapy, STOP-codon-readthrough therapy). Chaperones, which include heat shock proteins, are responsible for the correct folding of other proteins to the most energetically favorable conformation. Without their appropriate levels and activities, the correct folding of the lysosomal enzyme, whether supplied from outside or synthesized in the cell, would be impossible. However, the baseline level of nonspecific chaperone proteins in MPS has never been studied. Therefore, the purpose of this work was to determine the basal levels of nonspecific chaperone proteins of the Hsp family in MPS cells and to study the effect of normalizing GAG concentrations on these levels. Results of experiments with fibroblasts taken from patients with MPS types I, II, IIIA, IIIB, IIIC, IID, IVA, IVB, VI, VII, and IX, as well as from the brains of MPS I mice (Idua-/-), indicated significantly reduced levels of the two chaperones, Hsp70 and Hsp40. Interestingly, the reduction in GAG levels in the aforementioned cells did not lead to normalization of the levels of these chaperones but caused only a slight increase in the levels of Hsp40. An additional transcriptomic analysis of MPS cells indicated that the expression of other genes involved in protein folding processes and the cell response to endoplasmic reticulum stress, resulting from the appearance of abnormally folded proteins, was also modulated. To summarize, reduced levels of chaperones may be an additional cause of the low activity or inactivity of lysosomal enzymes in MPS. Moreover, this may point to causes of treatment failure where the correct structure of the enzyme supplied or synthesized in the cell is crucial to lower GAG levels.

Chondroprotective Effects of Grapefruit (Citrus paradisi Macfad.) Juice in a Complete Freund's Adjuvant Rat Model of Knee Osteoarthritis.

Osteoarthritis (OA) is a common disorder that can affect any joint in the human body. This study aimed to examine the anti-arthritic properties of high and low doses of grapefruit juice (GFJ), as grapefruit appears to contain anti-inflammatory biochemicals. Forty male Sprague-Dawley rats weighing 170-180 g were divided into five groups. These groups comprised the untreated control group and osteoarthritic (Osteo) rats administered intra-articular injections of Freund's complete adjuvant (CFA; 0.5 mL; 1 mg/mL) as follows: OA rats administered low doses of GFJ (Osteo+GFJ (low); 5 mL/kg body weight (BW)); OA rats administered high doses of GFJ (Osteo+GFJ (high); 27 mL/kg BW); and OA rats administered diclofenac sodium (Osteo+Diclo) as a reference drug. Injections of CFA induced OA, as indicated by a significant increase in the serum levels of the inflammatory biomarkers C-reactive protein (CRP), interleukin-1β (IL-1β), and (prostaglandin (PGE2), as well as matrix metalloproteinases (MMP-1) and cathepsin K. The synovial levels of glycosaminoglycans (GAGs), tumor necrosis factor (TNF-α), and interleukin 6 (IL-6) also increased, with a concomitant reduction in osteocalcin levels. The administration of either high or low doses of GFJ reduced CRP, IL-1β, PGE2, MMP-1, cathepsin K, and osteocalcin while increasing the synovial levels of GAGs, TNF-α, and IL-6, slowing cartilage degradation and boosting joint function. The results showed comparable histopathological and biochemical responses. A comparison of the treatments showed that high-dose GFJ had a greater chondroprotective effect than low-dose GFJ.

Results of 14-year-long Enzyme Replacement Therapy in a Patient with Mucopolysaccharidosis Type II: Clinical Case

Background. Mucopolysaccharidosis type II (MPS II) is a rare hereditary disease from the group of lysosomal storage diseases, with progressive course. There is effective enzyme replacement therapy (ERT) for this disease, it prevents the development of severe complications and improves patients’ quality of life. Long-term follow-up of health changes in individuals on ERT is required for evaluating the treatment impact on disease progression and eventually on the quality of life of the patient and his family.Clinical case description. Results of 14-year-long follow-up of the patient with MPS II who was the first patient on ERT with idursulfase in Russia are presented. Improvement of growth, decrease in ENT-organs infections frequency, liver and spleen sizes decrease, general stabilization, no progression in cardiovascular and respiratory events, normal levels of glycosaminoglycans in urine are shown.Conclusion. Long-term therapy with idursulfase in severe MPS II stabilizes the patient’s somatic condition, prevents the development of severe complications in cardiovascular and respiratory systems, improves the quality of life of the patient and his family. Urinary glycosaminoglycans level decrease is the important indicator of the therapy efficacy along with overall patient's somatic state.

Effects of Different Perfusing Routes through The Portal Vein, Hepatic Vein, and Biliary Duct on Whole Rat Liver Decellularization.

Organ transplantation is the last therapeutic choice for end-stage liver failure, which is limited by the lack of sufficient donors. Decellularized liver can be used as a suitable matrix for liver tissue engineering with clinical application potential. Optimizing the decellularization procedure would obtain a biological matrix with completely removed cellular components and preserved 3-dimensional structure. This study aimed to evaluate the decellularization efficacy through three anatomical routes. In this experimental study, rat liver decellularization was performed through biliary duct (BD), portal vein (PV), and hepatic vein (HV); using chemical detergents and enzymes. The decellularization efficacy was evaluated by measurement of DNA content, extracellular matrix (ECM) total proteins, and glycosaminoglycans (GAGs). ECM preservation was examined by histological and immunohistochemical (IHC) staining and scanning electron microscopy (SEM). Scaffold biocompatibility was tested by the MTT assay for HepG2 and HUVEC cell lines. Decellularization through HV and PV resulted in a transparent scaffold by complete cell removal, while the BD route produced an opaque scaffold with incomplete decellularization. H and E staining confirmed these results. Maximum DNA loss was obtained using 1% and 0.5% sodium dodecyl sulfate (SDS) in the PV and HV groups and the DNA content decreased faster in the HV group. At the final stages, the proteins excreted in the HV and PV groups were significantly less than the BD group. The GAGs level was diminished after decellularization, especially in the PV and HV groups. In the HV and PV groups the collagen amount was significantly more than the BD group. The IHC and SEM images showed that the ECM structure was preserved and cellular components were entirely removed. MTT assay showed the biocompatibility of the decellularized scaffold. The results revealed that the HV is a more suitable route for liver decellularization than the PV and BD.

The Relevance of Thrombo-Inflammatory Biomarkers and Their Relationship with Circulating Glycosaminoglycans in End-Stage Renal Disease Patients

En-stage renal disease (ESRD) is a growing public health problem. The atherosclerotic cardiovascular complications are the leading causes of mortality and morbidity in the ESRD. In this study, we sought to quantify the levels of thrombo-inflammatory biomarkers in an ESRD patients in comparison to healthy controls to determine their relevance in thrombo-inflammation and adverse outcomes. The levels of D-Dimer, C-reactive protein (CRP), plasminogen activator inhibitor 1 (PAI-1) antigen, functional PAI-1, thrombin activatable fibrinolysis inhibitor, tissue plasminogen activator, von Willebrand factor, and anti-PF4 IgG and microparticle (MP) activity were quantified by using commercially available ELISA immunoassays for each of the ESRD (n = 73) and control plasma samples (n = 10). The levels of endogenous glycosaminoglycans (GAGs) were quantified by utilizing a Heparin Red Probe (Redprobes UG, Germany). The collected data were analyzed to demonstrate the relationship between various parameters. All the tested biomarkers were increased in ESRD patients in comparison to healthy controls (p < 0.05). These biomarkers have shown significant correlations within each other except for anti-PF4 Ig G and MPs. The CRP levels were significantly higher in patients who had coronary artery disease (CAD) (p < 0.05), but there was no significant difference in other biomarkers according to the cardiovascular outcomes. In the multivariate analysis, the CRP (odds ratio: 1.19; 95% confidence interval: 1.01-1.41; p: 0.03) value was an independent predictor of CAD. In this study, we demonstrated increased levels of 10 different biomarkers in ESRD patients. The CRP levels can be a good predictor of CAD in ESRD patients.

Urinary Glycosaminoglycans: Characterization and Quantification.

The growing body of evidence supports the potential of using urinary glycosaminoglycans (uGAGs) levels as biomarkers to guide diagnosis and as predictive biomarkers of treatment efficacy. Recently, studies have shown that, in addition to MPS, the prognosis and treatment of cancers and viral infections, including COVID-19, are enabled by characterization and/or traits by GAGs. Reliable and accessible detection and assay protocols of urinary GAGs are therefore of great support for laboratory workers and clinicians. Here we describe a semiquantitative and quantitative urinary glycosaminoglycans determination using 1,9-dimethylmethylene blue (DMB) and the characterization of uGAGs using thin layer chromatography (TLC).

The Impact of Glycemic Control in Thrombo-Inflammatory Biomarkers in Diabetic Pulmonary Embolism Patients

The Impact of Glycemic Control in Thrombo-inflammatory Biomarkers in Diabetic Pulmonary Embolism Patients. Bulent Kantarcioglu, Alexander O'Hara, Jacob Pozin, Roumika Patil, Maddie Allen, Emily Krupa, Fakiha Siddiqui, Debra Hoppensteadt, Atul Laddu, Amir Darki, Jawed Fareed. Introduction: Venous thromboembolism (VTE) is estimated to affect between 350,000-600,000 individuals annually in the United States. The mortality rate is approximately 100,000 people each year. Diabetes mellitus (DM) is one of the most common metabolic disorders resulting from dysregulated glucose metabolism and is a very important risk factor for cardiovascular disease. Several different factors contribute to thrombotic events in DM; these include inflammatory states, primary hemostatic changes, increased levels of various clotting factors, impaired fibrinolysis, increased oxidative stress, and endothelial dysfunction. However, the results of previous studies were inconsistent to explain the complex relationship between the poor glycemic control in DM and development of PE. HbA1c, which is used as an indicator for glycemic control, may also be an important measurement to quantify the level of thromboinflammatory response that is observed in diabetic patients. Thrombo-inflammatory biomarkers such as D-Dimer, CRP, PAI-1, tPA, TAFI, vWF and endogenous glycosaminoglycans (GAGs) were investigated in several previous studies, however, the relevance of these biomarkers in complex relationships of DM and PE is not completely understood. This study aimed to investigate the role of DM in modulating thromboinflammatory biomarkers in PE patient. Materials & Methods: The diabetic PE patient samples were collected following the ongoing IRB and PERT protocols at Loyola University Medical Center and affiliated hospitals. The levels of thrombo-inflammatory markers (D-Dimer, CRP, PAI-1, tPA, TAFI and vWF) were tested by ELISA methods, endogenous GAGs with heparin red (Red Probes, Muenster, Germany) method. The clinical information of 113 diabetic PE patients, including the HbA1c levels within 3 months of PE diagnosis were collected through chart review of the electronically medical records of the patients. After exclusion of patients with active cancer, circulating levels of each biomarker in diabetic PE patient plasma (n:90) were compared to healthy controls (n:30) and according to HbA1c levels (n:45). Results: The levels of all the tested biomarkers were elevated in diabetic PE patients in comparison to healthy controls (P< 0.05). D-dimer (25.06-fold) and CRP (42.01-fold) have shown the most prominent increases in comparison to healthy controls (Table 1). We did not observe any significant difference in the levels of D-dimer, CRP, PAI-1, t-PA and endogenous GAGs according to HbA1c levels (p< 0.05). The levels of TAFIa (p<0.01) and vWF (p: 0.03) were significantly higher in diabetic patients that had a HbA1c ≥ 7. Conclusion: Our findings support that the increased levels of thromboinflammatory biomarkers and dysregulation of hemostatic pathways are important pathophysiological mechanisms in diabetic PE patients. Increased levels of TAFI and vWF may be the potential contributors of impaired fibrinolysis and endothelial dysregulation in diabetic PE patients who have poor glycemic control. These results underscore the importance of glycemic regulation in diabetic PE patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A novel whole “Joint-in-Motion” device reveals a permissive effect of high glucose levels and mechanical stress on joint destruction

Osteoarthritis (OA) has recently been suggested to be associated with diabetes. However, this association often disappears when accounting for body mass index (BMI), suggesting that mechanical stress may be a confounding factor. We investigated the combined influence of glucose level and loading stress on OA progression using a novel whole joint-in-motion (JM) culture system. Whole mouse knee joints were placed in an enclosed chamber with culture media and actuated to recapitulate leg movement, with a dynamic stress regimen of 0.5Hz, 8h/day for 7 days. These joints were treated with varying levels of glucose and controlled for osmolarity and diffusion. Joint movement and joint space were examined by X-ray fluoroscopy and microCT. Cartilage matrix levels were quantified by blinded Mankin scoring and immunohistochemistry. Culturing in the JM device facilitated proper leg extension and flexion movements, and adequate mass transport for analyzing the effect of glucose on cartilage. Treatment with higher levels of glucose either via media supplementation or intra-articular injection caused a significant decrease in levels of glycosaminoglycan (GAG) and an increase in aggrecan neoepitope in articular cartilage, but only under dynamic stress. Additionally, collagen II level was slightly reduced by high glucose levels. High levels of glucose and dynamic stress have permissive effects on articular cartilage GAG loss and aggrecan degradation, implicating that mechanical stress confounds the association of diabetes with OA. The JM device supports novel investigation of mechanical stress on the integrity of an intact living mouse joint to provide insights into OA pathogenesis.

Plasma and Urine Levels of Glycosaminoglycans in Patients with Systemic Sclerosis and Their Relationship to Selected Interleukins and Marker of Early Kidney Injury.

Systemic sclerosis (SSc) is a chronic connective tissue disease characterized by immune system dysfunction, vasculopathy, and progressive fibrosis of the skin and internal organs, resulting from excessive accumulation of extracellular matrix (ECM) elements, including collagen and proteoglycans (PGs). An uncontrolled PG proliferation, caused by disturbances in their metabolism in tissues, is most likely reflected in the quantitative changes of their components, i.e., glycosaminoglycans (GAGs), in body fluids. Therefore, the aim of this study was to quantify the different types of GAGs in the blood and urine of systemic sclerosis patients. Chondroitin/dermatan sulfates (CS/DS) and heparan sulfates/heparin (HS/H) were quantified by hexuronic acid assay and electrophoretic fractionation, while hyaluronic acid (HA) and keratan sulfates were evaluated using ELISA tests. In turn, individual urinary GAGs were determined using the Blyscan™ Sulfated Glycosaminoglycan Assay Kit. Our results showed that the plasma concentrations of CS/DS, HS/H, HA, and KS in systemic sclerosis patients were significantly higher compared with those in healthy subjects. In the case of urine measurements, we have found that in SSc patients, CS/DC concentrations were significantly higher, while HA concentrations were significantly lower compared with the values observed in the urine of healthy subjects. Importantly, the found by us correlations between plasma keratan sulfate levels and both the duration of the disease and the severity of skin lesions, as expressed by the Rodnan scale, seems to suggest this GAG as a potential marker in assessing disease progression and activity. In addition, a level of urinary excretion of all types of GAGs due to their high positive correlation with uACR, may be a valuable complementary test in the diagnosis of early renal dysfunction in the course of SSc.

Bioactive composite hydrogels as 3D mesenchymal stem cell encapsulation environment for bone tissue engineering: in vitro and in vivo studies.

Although decellularized bone matrix (DBM) has often been used in scaffold form for osteogenic applications, its use as a stem cell encapsulation matrix adaptable to surgical shaping procedures has been neglected. This study aimed to investigate the feasibility of utilizing solubilized DBM and nanohydroxyapatite (nHAp)-incorporated DBM hydrogels as encapsulation matrix for bone marrow-derived MSCs (BM-MSCs). First, DBM and DBM/nHAp hydrogels were assessed by physical, chemical, turbidimetric, thermal, and mechanical methods; then, in vitro cytocompatibility and in vitro hemocompatibility were investigated. An in vivo study was performed to evaluate the osteogenic properties of hydrogels alone or with BM-MSCs encapsulated in them. The findings revealed that hydrogels retained high levels of collagen and glycosaminoglycans after successful decellularization. They were found to be cytocompatible and hemocompatible in vitro, and were able to gel with sufficient mechanical stability at physiological temperature. BM-MSCs survived in culture for at least 2 weeks as metabolically active when encapsulated in both DBM and DBM/nHAp. Preliminary in vivo study showed that DBM-nHAp has higher osteogenicity than DBM. Moreover, BM-MSC encapsulated DMB/nHAp showed predominant bone-like tissue formation at 30 days in the rat ectopic site compared to its cell-free form.

The Prospective Effect of Cinnamon and Chia on Submandibular Salivary Glands After Ciprofloxacin Administration in Albino Rats (Histological, Histochemical, and Ultrastructural Study).

Ciprofloxacin (CPFX®) is potent fluoroquinolone but has severe side effects. Cinnamon (CIN) and chia seeds are potent antioxidants. The current work aimed to compare the effect of CIN extract and chia seeds on CPFX®-treated submandibular salivary glands (SMGs). Thirty-two male albino rats were divided into four groups: Group 1: received saline. Group 2: received CPFX®. Group 3: received CIN extract after 4 h of CPFX® administration. Group 4: received ground chia seeds after 4 h of CPFX® administration. After 10 days, histological, histochemical, and ultrastructural examinations were done. Different examinations illustrated normal features of SMG in Groups 1 and 3. Group 2 showed degenerative signs. Group 4 showed normal features in some areas. Statistical results illustrated that Group 2 had highest mean vacuolation area%. Highest mean of PAS optical density (OD) was for Group 2. Concerning mercuric bromophenol blue stain OD; Group 1 showed highest mean OD. CPFX® has the deteriorative effect on SMG structure and ultrastructure. It leads to increased levels of glycosaminoglycans (GAGs) and decreased levels of total proteins. CIN extract showed more ameliorative effect compared to chia seeds.

THE EFFECT OF COMBINATIONS OF GROWTH FACTORS AND HYALURONIC ACID ON THE PROLIFERATION AND CHONDROGENIC DIFFERENTIATION OF HUMAN SYNOVIUM DERIVED MESENCHYMAL STEM CELLS

Growth factors and hyaluronic acid (HA) have been shown to stimulate functional activity of mesenchymal stem cells (MSCs) and thus may influence damaged tissue healing, including cartilage. In this study we investigated the effects of individual and combinatorial application of HA, platelet derived growth factor–BB (PDGF-BB), transforming growth factor β1 (TGF-β1), bone morphogenetic protein-4 (BMP-4) and fibroblast growth factor-2 (FGF-2) on the proliferation and chondrogenic differentiation of human synovium derived MSCs (SD MSCs). Isolated SD MSCs were expanded in α-MEM medium and characterized using immunocytochemistry, multilineage differentiation assay and CFU-assay. Proliferation activity of MSCs was examined with MTT-assay. Chondrogenic differentiation and glycosaminoglycan level in micro-pellet culture of MSCs were evaluated with histochemical and biochemical analysis. Our results showed that the individual use of FGF-2 and PDGF-BB significantly increased the rate of MSC proliferation compared to the control. At the same time, TGF-β1 did not affect cell growth. Combinations of growth factors: TGF-β1+FGF-2, TGF-β1+PDGF-BB, and FGF-2+PDGF-BB were able to significantly increase the proliferative activity of MSCs. The greatest synergistic effect was found with combination of all three factors TGF-β1, FGF-2, and PDGF-BB. Histochemical and biochemical analysis revealed that combination of TGF-β1 and BMP-4 have a significant effect on chondrogenic differentiation and the synthesis of glycosaminoglycans in micro-pellet culture of MSCs. The addition of HA to growth factors has minor effect on the proliferation and does not affect the chondrogenic differentiation of MSCs. This study demonstrated that combination of growth factors can promote proliferation and chondrogenic differentiation of SD MSCs and may be effective in enhancing cartilage healing.

CRISPR/nCas9-Based Genome Editing on GM2 Gangliosidoses Fibroblasts via Non-Viral Vectors.

The gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the β-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the β-hexosaminidase B. Monogenic mutations on HEXA, HEXB, or GM2A genes arise in the Tay–Sachs (TSD), Sandhoff (SD), and AB variant diseases, respectively. In this work, we validated a CRISPR/Cas9-based gene editing strategy that relies on a Cas9 nickase (nCas9) as a potential approach for treating GM2 gangliosidoses using in vitro models for TSD and SD. The nCas9 contains a mutation in the catalytic RuvC domain but maintains the active HNH domain, which reduces potential off-target effects. Liposomes (LPs)- and novel magnetoliposomes (MLPs)-based vectors were used to deliver the CRISPR/nCas9 system. When LPs were used as a vector, positive outcomes were observed for the β-hexosaminidase activity, glycosaminoglycans levels, lysosome mass, and oxidative stress. In the case of MLPs, a high cytocompatibility and transfection ratio was observed, with a slight increase in the β-hexosaminidase activity and significant oxidative stress recovery in both TSD and SD cells. These results show the remarkable potential of CRISPR/nCas9 as a new alternative for treating GM2 gangliosidoses, as well as the superior performance of non-viral vectors in enhancing the potency of this therapeutic approach.

Spatial patterning of phenotypically distinct microtissues to engineer osteochondral grafts for biological joint resurfacing

Modular biofabrication strategies using microtissues or organoids as biological building blocks have great potential for engineering replacement tissues and organs at scale. Here we describe the development of a biofabrication strategy to engineer osteochondral tissues by spatially localising phenotypically distinct cartilage microtissues within an instructive 3D printed polymer framework. We first demonstrate that immature cartilage microtissues can spontaneously fuse to form homogeneous macrotissues, and that combining less cellular microtissues results in superior fusion and the generation of a more hyaline-like cartilage containing higher levels of sulphated glycosaminoglycans and type II collagen. Furthermore, temporally exposing developing microtissues to transforming growth factor-β accelerates their volumetric growth and subsequent capacity to fuse into larger hyaline cartilage grafts. Next, 3D printed polymeric frameworks are used to further guide microtissue fusion and the subsequent self-organisation process, resulting in the development of a macroscale tissue with zonal collagen organisation analogous to the structure seen in native articular cartilage. To engineer osteochondral grafts, hypertrophic cartilage microtissues are engineered as bone precursor tissues and spatially localised below phenotypically stable cartilage microtissues. Implantation of these engineered grafts into critically-sized caprine osteochondral defects results in effective defect stabilisation and histologically supports the restoration of a more normal articular surface after 6 months in vivo. These findings support the use of such modular biofabrication strategies for biological joint resurfacing.