Glycosaminoglycan Levels Research Articles

Glycosaminoglycans as Biomarkers for Mucopolysaccharidoses and Other Disorders.

Glycosaminoglycans (GAGs) are present in proteoglycans, which play critical physiological roles in various tissues. They are known to be elevated in mucopolysaccharidoses (MPS), a group of rare inherited metabolic diseases in which the lysosomal enzyme required to break down one or more GAG is deficient. In a previous study, we found elevation of GAGs in a subset of patients without MPS. In the current study, we aim to investigate serum GAG levels in patients with conditions beyond MPS. In our investigated samples, the largest group of patients had a clinical diagnosis of viral or non-viral encephalopathy. Clinical diagnoses and conditions also included epilepsy, fatty acid metabolism disorders, respiratory and renal disorders, liver disorders, hypoglycemia, developmental disorders, hyperCKemia, myopathy, acidosis, and vomiting disorders. While there was no conclusive evidence across all ages for any disease, serum GAG levels were elevated in patients with encephalopathy and some patients with other conditions. These preliminary findings suggest that serum GAGs are potential biomarkers in MPS and other disorders. In conclusion, we propose that GAGs elevated in blood can be used as biomarkers in the diagnosis and prognosis of various diseases in childhood; however, further designed experiments with larger sample sizes are required.

Degradation of endothelial glycocalyx in Tanzanian children with falciparum malaria.

A layer of glycocalyx covers the vascular endothelium serving important protective and homeostatic functions. The objective of this study was to determine if breakdown of the endothelial glycocalyx (eGC) occurs during malaria infection in children. Measures of eGC integrity, endothelial activation, and microvascular reactivity were prospectively evaluated in 146 children: 44 with moderately severe malaria (MSM), 42 with severe malaria (SM), and 60 healthy controls (HC). Biochemical measures of eGC integrity included plasma syndecan-1 and total urinary glycosaminoglycans (GAG). Side-stream dark field imaging was used to quantitatively assess integrity of eGC. Plasma angiopoietin-2 (Ang-2) was measured as a marker of endothelial activation and also as a possible mediator of eGC breakdown. Our results show that urinary GAG, syndecan-1, and Ang-2 were elevated in patients with MSM and SM compared with HC. Syndecan-1 and GAG levels correlated significantly with each other and with plasma Ang-2. The eGC breakdown products also inversely correlated significantly with hemoglobin and platelet count. In the MSM group, imaging results provided further evidence for eGC degradation. Although not correlated with markers of eGC degradation, vascular function (assessed by non-invasive near infrared spectroscopy [NIRS]) demonstrated reduced microvascular reactivity, particularly affecting the SM group. Our findings provide further evidence for breakdown of eGC in falciparum malaria that may contribute to endothelial activation and adhesion of parasitized red blood cells, with reduced nitric oxide formation, and vascular dysfunction.

Wang-Bi Tablet Ameliorates DMM-Induced Knee Osteoarthritis through Suppressing the Activation of p38-MAPK and NF-κB Signaling Pathways in Mice.

Background Traditional Chinese medicine (TCM) exhibits outstanding therapeutic effects on the treatment of osteoarthritis (OA). Wang-Bi tablets (WBTs) have been used in clinics to treat knee osteoarthritis (KOA) by alleviating joint swelling and paining, and thus, the quality of life in patients with KOA was improved. However, its underlying molecular mechanism of anti-inflammatory response remains unclear. Therefore, further investigation is required. Purpose This study aimed to explore the function of WBT in KOA mice and uncover the possible molecular mechanisms. Study Design. A KOA model was constructed by destabilizing the medial meniscus (DMM). IL-1β-treated chondrocytes were used to investigate the precise mechanism in vitro. Methods (1) C57BL/6 male mice (8-week-old) were divided into Model, Sham, WBT-L, WBT-M, and WBT-H groups. After intragastric administration of 0.5% CMC-Na or WBT for 4 weeks, inflammation and pathological change were analyzed by ELISA, RT-qPCR, hematoxylin and eosin (H & E) and safranine O staining. (2) Isolated chondrocytes were stimulated with IL-1β followed by WBT-containing serum treatment, and then, the expression of inflammatory cytokines was analyzed by ELISA and RT-qPCR. (3) The effects of WBT on inflammatory signaling cascades in mice knee joint and chondrocytes were detected by WB. Results The results indicated that WBT could alleviate inflammation and prevent cartilage injury in KOA mice. Compared with 0.5% CMC-Na-treated mice, the serum glycosaminoglycans (GAG) level in WBT-treated mice was notably increased, while the proinflammatory cytokine interleukin- (IL-) 6 level was decreased. In addition, WBT treatment suppressed the activation of NF-κB and p38 signaling pathways both in vivo and in vitro. Conclusion WBT can effectively inhibit articular cartilage injury and inflammatory response in KOA mice. The protective role of WBT in mice KOA was a result of the downregulation of NF-κB and p38-MAPK signal pathways.

Cyclooxygenase-2 regulates PTHrP transcription in human articular chondrocytes and is involved in the pathophysiology of osteoarthritis in rats.

BackgroundCyclooxygenase-2 (COX-2) inhibitors are prescribed for the management of osteoarthritis (OA)-associated pain and inflammation. However, the role of COX-2 in normal and osteoarthritic articular chondrocytes has not been well investigated. We hypothesize that COX-2 plays a role in articular chondrocytes under normal conditions and during OA progression.MethodsIn vivo COX-2 levels in articular cartilage of normal and papain-induced osteoarthritic rats were compared. The role of COX-2 in human articular chondrocytes (HACs) was tested in vitro by COX-2 overexpression or activity inhibition. The levels of COX-2 and marker gene for normal function or articular cartilage degeneration were evaluated: mRNA by qRT-PCR; proteins by western blotting or immunohistochemistry; and glycosaminoglycan (GAG) by Safranin O–fast green staining. Parathyroid hormone-related protein (PTHrP) promoter activity was detected with luciferase reporter assays.ResultsIn the OA rat study, COX-2 and PTHrP were simultaneously increased in osteoarthritic rat chondrocytes, while increased PTHrP levels were reduced by celecoxib, a COX-2 selective inhibitor. The levels of normal cartilage matrices, GAG and type II collagen decreased, while markers of degeneration, collagen type X and MMP13 were elevated in osteoarthritic articular chondrocytes. Celecoxib rescued the loss of GAG and the increased collagen type X and MMP13 levels. In vitro, COX-2 overexpression in HACs significantly increased Col2a1, Col10a1, PTHrP and MMP13 mRNA expression, which was decreased when COX-2 activity was suppressed. More importantly, COX-2 overexpression upregulated the PTHrP transcription, mRNA expression and protein levels.ConclusionCOX-2 plays a pathophysiological role by preventing terminal differentiation of articular chondrocytes by upregulating PTHrP expression at the early stage of OA progression.The Translational potential of this articleCOX2 up-regulates PTHrP expression in normal and OA articular chondrocytes.

Mucopolysaccharidoses diagnosis in the era of enzyme replacement therapy in Egypt

BackgroundUndegraded glycosaminoglycans (GAGs) induced by deficiency of enzymes are the primary cause of mucopolyscchardoses. Mucopolysacchardoses (MPS) are a group of rare lysosomal storage diseases (LSD). The quantification of a specific enzymatic activity is needed for accurate diagnosis. The objectives of this work were: first, to continue the study of mucopolysacchardoses disease in Egypt after the start of using the enzyme replacement therapy (ERT). Second, to define the commonest types among our population after 18 years experience with the disease. Third, to compare the different MPS types’ distribution, diagnosed after the start of the ERT, to identify the impact of using ERT on the number and type of diagnosed patients. MethodUrinary GAGs were measured for all referred cases followed by two-dimensional electrophoretic separation for cases with high levels of GAGs; the specific enzyme activity was assayed for each type depending on the abnormal electrophoretic pattern obtained. Clinically suspected cases of Morquio syndrome were directly subjected to measuring the specific enzyme. ResultsOut of 1448 suspected cases, 622 (42.9%) MPS patients were diagnosed revealing the following distribution: MPS I (172, 27.7%), MPS II (57, 9.1%), MPS III [(177, 28.5%: 134 type B and 43 types A, C or D)], MPS IVA (124, 19.9%), MPS VI (90, 14.5%) and MPS VII (2, 0.3%). MPS III was the most commonly diagnosed type followed by MPS I and MPS IVA. MPS IVA represented the most common type receiving treatment, followed by MPS I, MPS II and MPS VI. ConclusionThe presence of treatment encouraged the affected families and physicians to seek diagnosis. MPS III was the commonest type among our studied group after 7 years of diagnosis, while MPS IVA was the commonest type receiving treatment.

Bromodomain-containing protein 7 regulates matrix metabolism and apoptosis in human nucleus pulposus cells through the BRD7-PI3K-YAP1 signaling axis

Intervertebral disc degeneration (IDD) results from dysregulated metabolism of the extracellular matrix of the nucleus pulposus (NP) and involves the participation of inflammatory factors such as TNF-α. Bromodomain-containing protein 7 (BRD7) shows considerable potential for anti-inflammatory applications. Herein, we investigated the role of BRD7 in IDD. The immunohistochemistry results demonstrated decreased BRD7 expression in severely degenerated human NP tissues compared to those showing mild degeneration. Lentiviruses and adenoviruses were used to knock down or overexpress BRD7 and YAP1, respectively. Our results revealed that BRD7 knockdown promoted matrix degradation and suppressed PI3K and YAP1 expression, while BRD7 overexpression alleviated matrix degradation and promoted YAP1 and PI3K expression. In addition, PI3K inhibition augmented matrix degradation, enhanced apoptosis, and reduced YAP1 expression, whereas YAP1 overexpression promoted matrix synthesis, suppressed apoptosis and promoted PI3K expression. Besides, BRD7 overexpression reversed the reductions in sulfated glycosaminoglycan levels induced by TNF-α, but this effect was blocked by PI3K or YAP1 inhibitors. Moreover, YAP1 and PI3K were shown to interact through coimmunoprecipitation analysis. In summary, our results demonstrate that BRD7 can regulate matrix metabolism and apoptosis in human NP cells through the BRD7-PI3K-YAP1 signaling axis. This study might provide new insights into the prevention and treatment of IDD.

Comparative Effectiveness of Intracerebroventricular, Intrathecal, and Intranasal Routes of AAV9 Vector Administration for Genetic Therapy of Neurologic Disease in Murine Mucopolysaccharidosis Type I.

Mucopolysaccharidosis type I (MPS I) is an inherited metabolic disorder caused by deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA). The two current treatments [hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT)], are insufficiently effective in addressing neurologic disease, in part due to the inability of lysosomal enzyme to cross the blood brain barrier. With a goal to more effectively treat neurologic disease, we have investigated the effectiveness of AAV-mediated IDUA gene delivery to the brain using several different routes of administration. Animals were treated by either direct intracerebroventricular (ICV) injection, by intrathecal (IT) infusion into the cerebrospinal fluid, or by intranasal (IN) instillation of AAV9-IDUA vector. AAV9-IDUA was administered to IDUA-deficient mice that were either immunosuppressed with cyclophosphamide (CP), or immunotolerized at birth by weekly injections of human iduronidase. In animals treated by ICV or IT administration, levels of IDUA enzyme ranged from 3- to 1000-fold that of wild type levels in all parts of the microdissected brain. In animals administered vector intranasally, enzyme levels were 100-fold that of wild type in the olfactory bulb, but enzyme expression was close to wild type levels in other parts of the brain. Glycosaminoglycan levels were reduced to normal in ICV and IT treated mice, and in IN treated mice they were normalized in the olfactory bulb, or reduced in other parts of the brain. Immunohistochemical analysis showed extensive IDUA expression in all parts of the brain of ICV treated mice, while IT treated animals showed transduction that was primarily restricted to the hind brain with some sporadic labeling seen in the mid- and fore brain. At 6 months of age, animals were tested for spatial navigation, memory, and neurocognitive function in the Barnes maze; all treated animals were indistinguishable from normal heterozygous control animals, while untreated IDUA deficient animals exhibited significant learning and spatial navigation deficits. We conclude that IT and IN routes are acceptable and alternate routes of administration, respectively, of AAV vector delivery to the brain with effective IDUA expression, while all three routes of administration prevent the emergence of neurocognitive deficiency in a mouse MPS I model.

Pectus carinatum as the key to early diagnosis of Morquio A syndrome: a case report

BackgroundA 20-month-old Asian boy with normal growth presented with genu valgum, kyphosis, and pectus carinatum, with no neurological symptoms. No other symptoms suggestive of mucopolysaccharidoses, for example joint contracture and peculiar facies, were present.Case presentationAs part of our differential diagnosis we found elevated urine glycosaminoglycans, which triggered further investigation. Detailed examination showed flattening of the ribs, kyphoscoliosis and ovalization of the thoracolumbar vertebral body, strikingly short metacarpals, and very slight cardiac regurgitation. N-Acetylgalactosamine-6-sulfatase levels in the blood and dermal fibroblasts were very low, thus confirming diagnosis of Morquio A within 2 months of presentation. The patient was placed on elosulfase alfa enzyme replacement therapy and followed for 3 years.ConclusionsThis case exemplifies the importance of considering mucopolysaccharidoses as part of the initial differential diagnosis of pediatric patients with skeletal deformities; urine glycosaminoglycan levels and a blood enzyme mucopolysaccharidoses panel are simple screening tests that could lead to early definitive diagnosis.

Characterisation of key proteoglycans in the cranial cruciate ligaments (CCLs) from two dog breeds with different predispositions to CCL disease and rupture

Cranial cruciate ligament disease and rupture (CCLD/R) is one of the most common orthopaedic conditions in dogs, eventually leading to osteoarthritis of the stifle joint. Certain dog breeds such as the Staffordshire bull terrier have an increased risk of developing CCLD/R. Previous studies into CCLD/R have found that glycosaminoglycan levels were elevated in cranial cruciate ligament (CCL) tissue from high-risk breeds when compared to the CCL from a low-risk breed to CCLD/R. Our objective was to determine specific proteoglycans/glycosaminoglycans in the CCL and to see whether their content was altered in dog breeds with differing predispositions to CCLD/R. Disease-free CCLs from Staffordshire bull terriers (moderate/high-risk to CCLD/R) and Greyhounds (low-risk to CCLD/R) were collected and key proteoglycan/glycosaminoglycans were determined by semi-quantitative Western blotting, quantitative biochemistry, quantitative reverse transcription polymerase chain reaction, and immunohistochemistry.Gene expression of fibromodulin (P = 0.03), aggrecan (P = 0.0003), and chondroitin-6-sulphate stubs (P = 0.01) were significantly increased, and for fibromodulin this correlated with an increase in protein content in Staffordshire bull terriers compared to Greyhound CCLs (P = 0.02). Decorin (P = 0.03) and ADAMTS-4 (P = 0.04) gene expression were significantly increased in Greyhounds compared to Staffordshire bull terrier CCLs. The increase of specific proteoglycans and glycosaminoglycans within the Staffordshire bull terrier CCLs may indicate a response to higher compressive loads, potentially altering their risk to traumatic injury. The higher decorin content in the Greyhound CCLs is essential for maintaining collagen fibril strength, while the increase of ADAMTS-4 indicates a higher rate of turnover helping to regulate normal CCL homeostasis in Greyhounds.

Gene Expression-Related Changes in Morphologies of Organelles and Cellular Component Organization in Mucopolysaccharidoses.

Mucopolysaccharidoses (MPS) are inherited metabolic diseases characterized by accumulation of incompletely degraded glycosaminoglycans (GAGs) in lysosomes. Although primary causes of these diseases are mutations in genes coding for enzymes involved in lysosomal GAG degradation, it was demonstrated that storage of these complex carbohydrates provokes a cascade of secondary and tertiary changes affecting cellular functions. Potentially, this might lead to appearance of cellular disorders which could not be corrected even if the primary cause of the disease is removed. In this work, we studied changes in cellular organelles in MPS fibroblasts relative to control cells. All 11 types and subtypes of MPS were included into this study to obtain a complex picture of changes in organelles in this group of diseases. Two experimental approaches were employed, transcriptomic analyses and electron microscopic assessment of morphology of organelles. We analyzed levels of transcripts of genes grouped into two terms included into the QuickGO database, ‘Cellular component organization’ (GO:0016043) and ‘Cellular anatomical entity’ (GO:0110165), to find that number of transcripts with significantly changed levels in MPS fibroblasts vs. controls ranged from 109 to 322 (depending on MPS type) in GO:0016043, and from 70 to 208 in GO:0110165. This dysregulation of expression of genes crucial for proper structures and functions of various organelles was accompanied by severe changes in morphologies of lysosomes, nuclei, mitochondria, Golgi apparatus, and endoplasmic reticulum. Interestingly, some observed changes occurred in all/most MPS types while others were specific to particular disease types/subtypes. We suggest that severe changes in organelles in MPS cells might arise from dysregulation of expression of a battery of genes involved in organelles’ structures and functions. Intriguingly, normalization of GAG levels by using recombinant human enzymes specific to different MPS types corrected morphologies of some, but not all, organelles, while it failed to improve regulation of expression of selected genes. These results might suggest reasons for inability of enzyme replacement therapy to correct all MPS symptoms, particularly if initiated at advanced stages of the disease.

Combination of chondrocytes and chondrons improves extracellular matrix production to promote the repairs of defective knee cartilage in rabbits.

BackgroundChondrons are composed of chondrocytes and the surrounding pericellular matrix (PCM) and function to enhance chondrocyte-mediated cartilage tissue engineering. This study aimed at investigating the potential effect of combined chondrocytes with chondrons on the production of proteoglycan and collagen-II (Col-2) and the repair of defective knee cartilage in rabbits.MethodsChondrocytes and chondrons were isolated from the knee cartilage of rabbits, and cultured alone or co-cultured for varying periods in vitro. Their morphology was characterized by histology. The levels of aggrecan (AGG), Col-2 and glycosaminoglycan (GAG) expression were quantified by qRT-PCR, Alcian blue-based precipitation and ELISA. The effect of combined chondrocytes with chondrons in alginate spheres on the repair of defective knee cartilage was examined in rabbits.ResultsThe isolated chondrocytes and chondrons displayed unique morphology and began to proliferate on day 3 and 6 post culture, respectively, accompanied by completely degenerated PCM on day 6 post culture. Evidently, chondrocytes had stronger proliferation capacity than chondrons. Longitudinal analyses indicated that culture of chondrons, but not chondrocytes, increased AGG mRNA transcripts and GAG levels with time and Col-2 mRNA transcripts only on day 3 post culture. Compared with chondrocytes or chondrons alone, co-culture of chondrocytes and chondrons significantly up-regulated AGG and Col-2 expression and GAG production, particularly at a ratio of 1:1. Implantation with chondrocytes and chondrons at 1:1 significantly promoted the repair of defective knee cartilage in rabbits, accompanied by reduced the Wakiteni scores with time.ConclusionCombined chondrons with chondrocytes promoted the production of extracellular matrix and the repair of defective knee cartilage in rabbits.The translational potential of this articleThis study explores that the combination of chondrons and chondrocytes may be new therapeutic strategy for cartilage tissue engineering and repair of defective cartilage.

Characterization of Glycosaminoglycan Disaccharide Composition in Astrocyte Primary Cultures and the Cortex of Neonatal Rats.

Astrocytes are major producers of the extracellular matrix (ECM), which is involved in the plasticity of the developing brain. In utero alcohol exposure alters neuronal plasticity. Glycosaminoglycans (GAGs) are a family of polysaccharides present in the extracellular space; chondroitin sulfate (CS)- and heparan sulfate (HS)-GAGs are covalently bound to core proteins to form proteoglycans (PGs). Hyaluronic acid (HA)-GAGs are not bound to core proteins. In this study we investigated the contribution of astrocytes to CS-, HS-, and HA-GAG production by comparing the makeup of these GAGs in cortical astrocyte cultures and the neonatal rat cortex. We also explored alterations induced by ethanol in GAG and core protein levels in astrocytes. Finally, we investigated the relative expression in astrocytes of CS-PGs of the lectican family of proteins, major components of the brain ECM, in vivo using translating ribosome affinity purification (TRAP) (in Aldh1l1-EGFP-Rpl10a mice. Cortical astrocytes produce low levels of HA and show low expression of genes involved in HA biosynthesis compared to the whole developing cortex. Astrocytes have high levels of chondroitin-0-sulfate (C0S)-GAGs (possibly because of a higher sulfatase enzyme expression) and HS-GAGs. Ethanol upregulates C4S-GAGs as well as brain-specific lecticans neurocan and brevican, which are highly enriched in astrocytes of the developing cortex in vivo. These results begin to elucidate the role of astrocytes in the biosynthesis of CS- HS- and HA-GAGs, and suggest that ethanol-induced alterations of neuronal development may be in part mediated by increased astrocyte GAG levels and neurocan and brevican expression.

Study of Urinary Glycosaminoglycans among Essential Hypertensive Patients

Introduction: Essential hypertension is a systemic disease which affects endothelial basement membrane. Changes in Glycosaminoglycans (GAG) distribution pattern on glomerular basement membrane has been noted in hypertension. Hence it seems likely that an increase excretion of GAG levels may be an indicator of reduction in renal function. The qualitative or quantitative determination of urinary GAGs may be of value and could constitute a non invasive marker to assess the renal damage in essential hypertension. Aim: Differential analysis of urinary GAGs in essential hypertensive patients. Materials and Methods: This analytical case-control observational study which was conducted from November 2014 to June 2016 in Department of Biochemistry, SDM College of Medical Sciences and Hospital, Dharwad, Karnataka, India. The study group included 50 male patients with age group of 30-60 years, clinically diagnosed with essential hypertension. Control group included 50 healthy male individuals with age group of 30-60 years, blood donors visiting to hospital blood bank. Random urine sample was collected. Urine creatinine was estimated by Jaffe’s method, urine GAGs by Dimethyl methylene blue (DMMB) dye method and urine microalbumin by particle enhanced Turbidimetric inhibition immunoassay method. Correlation was tested with Spearman’s correlation coefficient. Level of significance was set for p-value <0.05 with confidence interval of 95%. Results: In present study, urinary GAGs levels in essential hypertensive study group was 14.57±10.16 mg/dL and in control group was 10.09±6.04 mg/dL. Urinary GAGs levels in essential hypertensive group was significantly high (p-value=0.890) when compared with normotensive control group. But no statistical significant correlation was found between urine GAGs and urine microalbumin in hypertensive study group. Conclusion: Estimation of urine glycosaminoglycans in essential hypertensive patients is a simple, rapid and cost effective test which asseses the glomerular function. It can be used as one of the early marker for diagnosis of nephropathy before microalbuminuria sets in.

Protective effects of ginsenoside Rh1 on intervertebral disc degeneration through inhibition of nuclear factor kappa-B signaling pathway

Background: In this study, we aim to explore the protective effect of ginsenoside Rh1 against intervertebral disc degeneration (IDD) and the related mechanism. Materials and Methods: IDD model in Sprague-Dawley rats was established and the animals were treated with different concentrations of ginsenoside Rh1 for 4 weeks, after this, the animals were sacrificed and the intervertebral disc of was collected for analysis using quantitative polymerase chain reaction. Western blot analysis was performed for quantifying the expression levels of glycosaminoglycans (GAGs) and Types I and Type II collagen. Moreover, serum samples were collected and the expression levels of some of the inflammatory cytokines such as interleukin (IL)-1 β and IL-6 were evaluated. Next, we collected the nucleus pulposus (NP) cells from the animals and were divided into five groups: control, IDD, treatment groups with different concentrations of ginsenoside Rh1 (10, 20, and 50 μg/mL). After treatment, the levels of IL-1 β and IL-6 in the cell culture supernatant were examined. Then, we performed western blot analysis to quantify the levels of B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-extra-large (BCL-xL), and nuclear factor kappa-B (NF-κB) in different groups. Results: We observed that ginsenoside Rh1 significantly downregulated the expression of type I collagen and upregulated the expression of type II collagen and GAG under in vivo conditions. Moreover, the expression levels of IL-1 β and IL-6 in the serum samples of IDD rats and cell culture supernatant of NP cells isolated from the IDD rats were significantly increased. However, ginsenoside Rh1 significantly increased the levels of Bcl-2 and Bcl-xL and decreased the levels NF-κB both under in vitro and in vivo conditions. Conclusion: Ginsenoside Rh1 demonstrated protective effect against the IDD via regulation of IL-1 β/NF-κB signaling pathway.

Hypoxia Regulates the Extracellular Matrix via Mitogen-Activated Protein Kinases Pathway in Cells Retrieved from the Human Intervertebral Disc.

PurposeThe present study aimed to identify the physiological characteristics of cells by investigating the change in gene expression and protein levels during extracellular matrix (ECM) synthesis in the intervertebral disc (IVD) under hypoxic conditions.Materials and MethodsTo test the effect of oxygen on cell growth and ECM synthesis of chondrocyte-like cells, the cells from IVD were separated and cultured in two hypoxia-mimicking systems: chemical hypoxic conditions using deferoxamine (DFO), and physiological hypoxic conditions using a hypoxic chamber for 7 days. Chondrocyte like cells cultured without DFO and under the normal oxygen concentration (21% O2 and 5% CO2, 37°C) served as the controls.ResultsChondrocyte-like cells cultured in the presence of 6% oxygen demonstrated a 100% increase in cellular proliferation compared to the control. The cells treated with chemical hypoxic conditions demonstrated a dose-dependent increase in the mRNA expression of glucose transporter-1, GAPDH, aggrecan, and type II collagen on Day 1. When treated with 100 µM DFO, the cells showed a 50% increase in the levels of proteoglycan protein on Day 7. The cells treated with chemical hypoxic condition demonstrated increase in sulfated glycosaminoglycan (GAG) protein levels on Day 7. Moreover, the cells cultured in the presence of 6% oxygen showed a 120% increase in sulfated GAG levels on Day 7.ConclusionThe oxygen concentration had an important role in the viability, proliferation, and maturation of chondrocyte-like cells in IVD. In addition, chondrocyte-like cells are sensitive to the concentration of oxygen.

Construction of bioengineered corneal stromal implants using an allogeneic cornea-derived matrix

The bioengineering of corneal scaffolds that mimic native human cornea has attracted interest owing to the scarcity of donor corneas for the transplantation-based treatment of corneal blindness. However, an optimally engineered corneal tissue for clinical use has yet to emerge. Herein, human corneal tissues discarded during allogeneic corneal transplantation surgery were used to construct allogeneic cornea-derived matrix (ACM) scaffolds with favorable optical properties and structural strength. During scaffold fabrication, collagen and glycosaminoglycan levels were well preserved, while DNA decreased significantly. Scanning electron microscopy revealed the presence of fiber-like structures on the scaffold surface and specific structures featuring multiple interlaced lamellae in cross-sections. Moreover, corneal epithelial cells grown on the ACM formed a continuous multi-stratified epithelium with a strong expression of the corneal epithelial differentiation marker CK3/12, gap junction marker Connexin43, and stem-cell-specific marker p63α, while corneal stromal cells expressed the keratocyte-specific marker KERA and the adhesion marker integrin β1. When the ACM was implanted into rabbit corneal stromal pockets, the rabbit cornea remained transparent throughout the follow-up period. These results indicate that the construction of corneal stromal implants from discarded human corneal tissues may pave the way for the generation of high-quality corneal tissue for transplantation.

Idursulfase pharmacokinetics, cellular uptake, and pharmacodynamics: Effect of sialylation and manufacturing process

AbstractRecombinant iduronate‐2‐sulfatase (idursulfase) is available for the treatment of mucopolysaccharidosis II (MPS II). The effectiveness of this glycoprotein may depend on post‐translational modifications such as glycosylation and sialylation during manufacture. We assessed the effects of sialylation level and the scale‐up of idursulfase production on the pharmacokinetics, cellular uptake and pharmacodynamics of idursulfase in Sprague Dawley rats and mice (knock‐out model of MPS II and wild type). Serum clearance in rats decreased with an increase in idursulfase sialylation, from 2.23 mL/min/kg with 7.4 mol sialic acid (SA)/mol idursulfase to 0.57 mL/min/kg with 16.9 mol SA/mol idursulfase. In mice, 31%‐52% of the infused idursulfase dose was detected in the liver. The idursulfase uptake into the liver was not affected by sialylation, but high sialylation led to lower uptake in the spleen and greater uptake in the kidney and heart in both wild‐type and MPS II mice. The pharmacodynamics study revealed a greater reduction of glycosaminoglycan levels with high idursulfase sialylation in the kidney, which reflects the higher idursulfase uptake into this organ with higher sialylation. There was no effect of manufacturing process on any idursulfase characteristic, suggesting that production scale‐up would have no impact on clinical effectiveness.

Urine and serum glycosaminoglycan levels in the diagnosis of urological diseases and conditions: A narrative review of the literature.

Glycosaminoglycans (GAGs) are sulfated, negatively charged polysaccharides produced in almost every cell of the human body. As GAGs are extracellularly localized, the changes in body fluids such as blood and urine may reflect pathological changes in the urinary system as observed in other pathologies. In this review, we determined the potential of urinary and/or serum GAG levels as a marker for kidney and urothelial system diseases. We performed a search in the PubMed, MEDLINE, and ScienceDirect databases until September 30, 2019. A number of studies reported changes in the urinary and/or plasma GAG levels or composition in urological diseases and conditions, such as renal cell carcinoma, kidney stone, bladder carcinoma, and overactive bladder. GAGs were found to have a predictive biomarker potential that could be limited by generalizability concerns.

Experimental study on reconstruction of anterior labrum of shoulder joint by chemical extraction of allogeneic tendon and allogeneic chondrocytes

To study the effect of chemical extraction of allogeneic tendon and allogeneic chondrocytes for reconstruction of anterior labrum of shoulder joint in rabbits. The body weight of 45 adult New Zealand white rabbits ranged from 2.5 to 3.0 kg. The Achilles tendons of 15 rabbits were taken and the allogeneic tendons were prepared by chemical extraction with antigen inactivation. The extracted tendons were compared with untreated tendons by HE and Masson stainings. Chondrocytes were isolated and cultured by trypsin method and identified by immunohistochemical staining of collagen type Ⅱ. The remaining 30 rabbits were used to prepare the model of anterior labrum defect of shoulder joint. After the allogeneic tendon was transplanted to the damaged labrum, the rabbits was randomly divided into two groups (15 in each group). In group A, the allogeneic chondrocytes were injected into the joint immediately after transplantation, while in group B, no treatment was made. At 4, 6, and 8 weeks after operation, 5 transplanted tendons of each group were taken. After general observation, HE staining was used to observe the number of nuclei, Masson staining was used to observe the expression of collagen fibers in muscle fiber tissues, and AB staining was used to detect the glycosaminoglycan level after transplantation, to evaluate the cell growth in the tissues of the two groups of allogeneic tendon. By HE and Masson stainings, the allogeneic tendon antigen prepared by chemical extraction method was inactivated and the fibrous tissue structure was intact; collagen type Ⅱ immunohisto-chemistry staining showed that the cultured cells were chondrocytes. After tendon transplantation, the content of glycosaminoglycan in group A was significantly higher than that in group B ( P<0.05). At 6 weeks after operation, HE staining showed that the nuclear in tendon tissue of group A was significantly more than that of group B ( t=20.043, P=0.000). Masson staining showed that the number of nuclei in tendon tissue of group A was significantly increased, the muscle fibers and collagen fibers were interlaced, the tissue structure was more compact, and the tendon tissue was mainly blue stained; while the number of nuclei in group B was less, mainly collagen fibers of the original graft. The allogeneic tendon inactivated by chemical extraction can be used to reconstruct the defect of anterior labrum of shoulder joint in rabbits, and the combination of allogeneic chondrocytes can promote the healing of tendon transplantation.

Different types of cartilage neotissue fabricated from collagen hydrogels and mesenchymal stromal cells via SOX9, TGFB1 or BMP2 gene transfer

ObjectiveAs native cartilage consists of different phenotypical zones, this study aims to fabricate different types of neocartilage constructs from collagen hydrogels and human mesenchymal stromal cells (MSCs) genetically modified to express different chondrogenic factors.DesignHuman MSCs derived from bone-marrow of osteoarthritis (OA) hips were genetically modified using adenoviral vectors encoding sex-determining region Y-type high-mobility-group-box (SOX) 9, transforming growth factor beta (TGFB) 1 or bone morphogenetic protein (BMP) 2 cDNA, placed in type I collagen hydrogels and maintained in serum-free chondrogenic media for three weeks. Control constructs contained unmodified MSCs or MSCs expressing GFP. The respective constructs were analyzed histologically, immunohistochemically, biochemically, and by qRT-PCR for chondrogenesis and hypertrophy.ResultsChondrogenesis in MSCs was consistently and strongly induced in collagen I hydrogels by the transgenes SOX9, TGFB1 and BMP2 as evidenced by positive staining for proteoglycans, chondroitin-4-sulfate (CS4) and collagen (COL) type II, increased levels of glycosaminoglycan (GAG) synthesis, and expression of mRNAs associated with chondrogenesis. The control groups were entirely non-chondrogenic. The levels of hypertrophy, as judged by expression of alkaline phosphatase (ALP) and COL X on both the protein and mRNA levels revealed different stages of hypertrophy within the chondrogenic groups (BMP2>TGFB1>SOX9).ConclusionsDifferent types of neocartilage with varying levels of hypertrophy could be generated from human MSCs in collagen hydrogels by transfer of genes encoding the chondrogenic factors SOX9, TGFB1 and BMP2. This technology may be harnessed for regeneration of specific zones of native cartilage upon damage.