Glycosaminoglycans Research Articles

Ovine Mesenchymal Stem Cell Chondrogenesis on a Novel 3D-Printed Hybrid Scaffold In Vitro.

This study evaluated the use of silica/poly(tetrahydrofuran)/poly(ε-caprolactone) (SiO2/PTHF/PCL-diCOOH) 3D-printed scaffolds, with channel sizes of either 200 (SC-200) or 500 (SC-500) µm, as biomaterials to support the chondrogenesis of sheep bone marrow stem cells (oBMSC), under in vitro conditions. The objective was to validate the potential use of SiO2/PTHF/PCL-diCOOH for prospective in vivo ovine studies. The behaviour of oBMSC, with and without the use of exogenous growth factors, on SiO2/PTHF/PCL-diCOOH scaffolds was investigated by analysing cell attachment, viability, proliferation, morphology, expression of chondrogenic genes (RT-qPCR), deposition of aggrecan, collagen II, and collagen I (immunohistochemistry), and quantification of sulphated glycosaminoglycans (GAGs). The results showed that all the scaffolds supported cell attachment and proliferation with upregulation of chondrogenic markers and the deposition of a cartilage extracellular matrix (collagen II and aggrecan). Notably, SC-200 showed superior performance in terms of cartilage gene expression. These findings demonstrated that SiO2/PTHF/PCL-diCOOH with 200 µm pore size are optimal for promoting chondrogenic differentiation of oBMSC, even without the use of growth factors.

Repetitive strikes loading organ culture model to investigate the biological and biomechanical responses of the intervertebral disc.

Disc degeneration is associated with repetitive violent injuries. This study aims to explore the impact of repetitive strikes loading on the biology and biomechanics of intervertebral discs (IVDs) using an organ culture model. IVDs from the bovine tail were isolated and cultured in a bioreactor, with exposure to various loading conditions. The control group was subjected to physiological loading, while the model group was exposed to either one strike loading (compression at 38% of IVD height) or repetitive one strike loading (compression at 38% of IVD height). Disc height and dynamic compressive stiffness were measured after overnight swelling and loading. Furthermore, histological morphology, cell viability, and gene expression were analyzed on Day 32. Glycosaminoglycan (GAG) and nitric oxide (NO) release in conditioned medium were also analyzed. The repetitive one strike group exhibited early disc degeneration, characterized by decreased dynamic compression stiffness, the presence of annulus fibrosus clefts, and degradation of the extracellular matrix. Additionally, this group demonstrated significantly higher levels of cell death (p < 0.05) and glycosaminoglycan (GAG) release (p < 0.05) compared to the control group. Furthermore, upregulation of MMP1, MMP13, and ADAMTS5 was observed in both nucleus pulposus (NP) and annulus fibrosus (AF) tissues of the repetitive one strike group (p < 0.05). The one strike group exhibited annulus fibrosus clefts but showed no gene expression changes compared to the control group. This study shows that repetitive violent injuries lead to the degeneration of a healthy bovine IVDs, thereby providing new insights into early-stage disc degeneration.

Multi-step semi-synthesis, structural characterization and growth factor interaction study of regiochemically sulfated diabolican polysaccharides

Diabolican is an exopolysaccharide (EPS) produced by Vibrio diabolicus HE800, a mesophilic bacterium firstly isolated from a deep-sea hydrothermal field. Its glycosaminoglycan (GAG)-like structure, consisting of a tetrasaccharide repeating unit composed of two aminosugars (N-acetyl-glucosamine and N-acetyl-galactosamine) and two glucuronic acid units, suggested to subject it to regioselective sulfation processes, in order to obtain some sulfated derivatives potentially acting as GAG mimics. To this aim, a multi-step semi-synthetic approach, relying upon tailored sequence of regioselective protection, sulfation and deprotection steps, was employed in this work. The chemical structure of the obtained sulfated diabolican derivatives was characterized by a multi-technique analytic approach, in order to define both degree of sulfation (DS) and sulfation pattern within the polysaccharide repeating unit, above all. Finally, binding affinity for some growth factors relevant for biomedical applications was measured for both starting diabolican and sulfated derivatives thereof. Collected data suggested that sulfation pattern could be a key structural element for the selective interaction with signaling proteins not only in the case of native GAGs, as already known, but also for GAG-like structures obtained by regioselective sulfation of naturally unsulfated polysaccharides.

Molecular Mechanisms in Pathophysiology of Mucopolysaccharidosis and Prospects for Innovative Therapy.

Mucopolysaccharidoses (MPSs) are a group of inborn errors of the metabolism caused by a deficiency in the lysosomal enzymes required to break down molecules called glycosaminoglycans (GAGs). These GAGs accumulate over time in various tissues and disrupt multiple biological systems, including catabolism of other substances, autophagy, and mitochondrial function. These pathological changes ultimately increase oxidative stress and activate innate immunity and inflammation. We have described the pathophysiology of MPS and activated inflammation in this paper, starting with accumulating the primary storage materials, GAGs. At the initial stage of GAG accumulation, affected tissues/cells are reversibly affected but progress irreversibly to: (1) disruption of substrate degradation with pathogenic changes in lysosomal function, (2) cellular dysfunction, secondary/tertiary accumulation (toxins such as GM2 or GM3 ganglioside, etc.), and inflammatory process, and (3) progressive tissue/organ damage and cell death (e.g., skeletal dysplasia, CNS impairment, etc.). For current and future treatment, several potential treatments for MPS that can penetrate the blood-brain barrier and bone have been proposed and/or are in clinical trials, including targeting peptides and molecular Trojan horses such as monoclonal antibodies attached to enzymes via receptor-mediated transport. Gene therapy trials with AAV, ex vivo LV, and Sleeping Beauty transposon system for MPS are proposed and/or underway as innovative therapeutic options. In addition, possible immunomodulatory reagents that can suppress MPS symptoms have been summarized in this review.

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

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

Dimerization and Crowding in the Binding of Interleukin 8 to Dendritic Glycosaminoglycans as Artificial Proteoglycans.

The interactions of glycosaminoglycans (GAG) with proteins of the extracellular matrix govern and regulate complex physiological functions including cellular growth, immune response, and inflammation. Repetitive presentation of GAG binding motifs, as found in native proteoglycans, might enhance GAG-protein binding through multivalent interactions. Here, we report the chemical synthesis of dendritic GAG oligomers constructed of nonasulfated hyaluronan tetrasaccharides for investigating the binding of the protein chemokine interleukin 8 (IL-8) to artificial, well-defined proteoglycan architectures. Binding of mutant monomeric and native dimerizable IL-8 was investigated by NMR spectroscopy and isothermal titration calorimetry. Dendritic oligomerization of GAG increased the binding affinity of both monomeric and dimeric IL-8. Monomeric IL-8 bound to monomeric and dimeric GAG with KD values of 7.3 and 0.108 μM, respectively. The effect was less pronounced for dimerizable wild-type IL-8, for which GAG dimerization improved the affinity from 34 to 5 nM. Binding of dimeric IL-8 to oligomeric GAG was limited by steric crowding effects, strongly reducing the affinity of subsequent binding events. In conclusion, the strongest effect of GAG oligomerization was the amplified binding of IL-8 monomers, which might concentrate monomeric protein in the extracellular matrix and thus promote protein dimerization under physiological conditions.

The Impact of Complex Loadings on the Structure of the L2-L3 Intervertebral Disc in a Sheep Spine Cadaver Model: A Biomechanical and Histological Evaluation.

Background The human vertebral column generates movements under versatile, dynamic loads. Understanding how the spine reacts to these movements and loads is crucial for developing new spine implants and surgical treatments for intervertebral disc injuries. Mechanically uni-axial compression models have been extensively studied. However, the spine's daily loading is not limited to compression, so it is crucial to measure its behavior in allmovements (flexion-extension, rotation, and axial compression). Methods This study utilized L1-L5 segments from 19 healthy adult sheep spines. The L2-L3 disc of the first spine underwent only histological evaluation without biomechanical testing to define basic histological parameters. The remaining 18 were divided into three groups of six and subjected to biomechanical tests. Different mechanisms for three groups of spinal segments were prepared, and tests were performed on Shimadzu AG-IS 10 KN (Universal Drawing Press, Kyoto, Japan). An axial load (800 N) was applied to the first group, an axial load with 15 degrees of flexion to the second group, and an axial load with 10 degrees of rotation plus 15 degrees of flexion to the third group. A biomechanical evaluation of the maximum elongation amounts (MEAs) was performed and compared between the groups. Then, the L2-L3 discs were removed from the sheep spines, and a histological examination of the discs was conducted usingHematoxylin-Eosin (HE), Alcian Blue (AB), and Masson's Trichrome (MT) staining. Results The mean MEA ± Standard Deviation (Range) was 1.39 ± 0.38 (0.91-1.94) for Group 1, 2.02 ± 0.75 (0.91-3.01) for Group 2, and 2.47 ± 1.09 (0.64-3.9) for Group 3. Biomechanically, although MEAs increased from Group 1 to Group 3 (meaning that the mean MEAs increased as the number of types of applied force increased), there was no statistically significant difference between the groups regarding the MEAs (P = 0.092). Histologically, no significant differences were observed between all groups after HE staining. In all groups, hypercellularity, edema in the connective tissue, separation between tissue layers, delamination, and signs of swelling and necrosis in the cells were observed similarly. For the AB staining, there was a decrease in the glycosaminoglycan (GAG) structure in the tissue samples compared to the control tissue, but no significant differences were observed between the groups. However, it was observed that the stratification in Group 3 was slightly more deteriorated than in the other groups. For the MT staining, collagen structure deterioration was observed in all groups. It was observed that the amount of collagen was significantly reduced compared to the control tissue. Conclusion As a result, when the axial load is applied biomechanically, there is more displacement of the vertebral discsin Group 3 with multidimensional movements. Furthermore, histological studies revealed deterioration between tissue layers when exposed to complex movements, and the degradation of stratification in group 3 compared to other loading combinations in groups 2 and 3 may indicate the role of complex loads in the formation of disc herniation.

Gelatin-Based Hybrid Hydrogels as Matrices for Organoid Culture.

The application of liver organoids is very promising in the field of liver tissue engineering; however, it is still facing some limitations. One of the current major limitations is the matrix in which they are cultured. The mainly undefined and murine-originated tumor matrices derived from Engelbreth-Holm-Swarm (EHS) sarcoma, such as Matrigel, are still the standard culturing matrices for expansion and differentiation of organoids toward hepatocyte-like cells, which will obstruct its future clinical application potential. In this study, we exploited the use of newly developed highly defined hydrogels as potential matrices for the culture of liver organoids and compared them to Matrigel and two hydrogels that were already researched in the field of organoid research [i.e., polyisocyanopeptides, enriched with laminin-entactin complex (PIC-LEC) and gelatin methacryloyl (GelMA)]. The newly developed hydrogels are materials that have a physicochemical resemblance with native liver tissue. Norbornene-modified dextran cross-linked with thiolated gelatin (DexNB-GelSH) has a swelling ratio and macro- and microscale properties that highly mimic liver tissue. Norbornene-modified chondroitin sulfate cross-linked with thiolated gelatin (CSNB-GelSH) contains chondroitin sulfate, which is a glycosaminoglycan (GAG) that is present in the liver ECM. Furthermore, CSNB-GelSH hydrogels with different mechanical properties were evaluated. Bipotent intrahepatic cholangiocyte organoids (ICOs) were applied in this work and encapsulated in these materials. This research revealed that the newly developed materials outperformed Matrigel, PIC-LEC, and GelMA in the differentiation of ICOs toward hepatocyte-like cells. Furthermore, some trends indicate that an interplay of both the chemical composition and the mechanical properties has an influence on the relative expression of certain hepatocyte markers. Both DexNB-GelSH and CSNB-GelSH showed promising results for the expansion and differentiation of intrahepatic cholangiocyte organoids. The stiffest CSNB-GelSH hydrogel even significantly outperformed Matrigel based on ALB, BSEP, and CYP3A4 gene expression, being three important hepatocyte markers.

2-N, 6-O sulfated chitosan evokes periosteal stem cells for bone regeneration

Musculoskeletal injuries and bone defects represent a significant clinical challenge, necessitating innovative approaches for effective bone tissue regeneration. In this study, we investigated the potential of harnessing periosteal stem cells (PSCs) and glycosaminoglycan (GAG)-mimicking materials for in situ bone regeneration. Our findings demonstrated that the introduction of 2-N, 6-O sulfated chitosan (26SCS), a GAG-like polysaccharide, enriched PSCs and promoted robust osteogenesis at the defect area. Mechanistically, 26SCS amplifies the biological effect of endogenous platelet-derived growth factor-BB (PDGF-BB) through enhancing the interaction between PDGF-BB and its receptor PDGFRβ abundantly expressed on PSCs, resulting in strengthened PSC proliferation and osteogenic differentiation. As a result, 26SCS effectively improved bone defect repair, even in an osteoporotic mouse model with lowered PDGF-BB level and diminished regenerative potential. Our findings suggested the significant potential of GAG-like biomaterials in regulating PSC behavior, which holds great promise for addressing osteoporotic bone defect repair in future applications.

Glycosaminoglycans and collagen fibril distribution at various depths of the corneal stroma of normal and CXL treated rats

Corneal collagen cross-linking (CXL) is widely used to treat keratoconus and ecstatic corneal disorders. The present studies were carried out to investigate the distribution of glycosaminoglycans (GAGs) and collagen fibril (CF) at different depths of the normal and CXL treated corneal stroma of four week old rats 7 days after standard CXL application. Ten Wistar rats’ corneas were used for the study. The epithelium of the cornea from the left eye of each rat was removed and treated with standard CXL application using riboflavin and Ultraviolet-A (UVA) (3 mW/cm2 for 30 min). The cornea from the right eye was used as the control cornea. The cornea was removed from the eye and processed for transmission electron microscopy. A bottom mounted Quemesa camera was used to capture digital images and these images were analysed using iTEM software. In the control cornea, the GAGs area size was not significantly different in the anterior, middle, and posterior stroma. In the CXL treated rats the GAGs area size gradually increased from the anterior to the posterior stroma whereas the spacing between the GAGs gradually decreased. There were very large GAGs present in the posterior stroma of the CXL treated rats. When comparing the control and CXL cornea, the GAGs area in the CXL cornea was significantly higher and inter-GAGs-spacing was smaller than in the control cornea. In the control cornea, the collagen fibrils diameter was higher in the anterior stroma and lowest in the posterior stroma. In the CXL treated cornea, the CF diameter and the interfibrillar spacing gradually decreased from the anterior to the posterior stroma. On comparison between the control and the CXL treated cornea, the interfibrillar spacing was significantly smaller in the CXL treated cornea than the control cornea in the anterior, middle, and posterior stroma but there was no difference in the diameter. The CXL treatment significantly increased the GAGs area and decreased the inter-GAGs-spacing, and inter–CF–spacing. This could be due to the gradual decline in the availability of riboflavin, UVA, and oxygen in the middle and posterior stroma. Further studies are required to investigate the role of keratan sulphate and chondroitin sulphate by using monoclonal antibodies with immunogold technique.

ANTI-INFLAMMATORY EFFECTS OF PRECONDITIONED BONE MARROW MSC-DERIVED SECRETOME ON DEGENERATED HUMAN NUCLEUS PULPOSUS CELLS IN VITRO

Invertebral disc degeneration (IDD) is a degenerative disease involving a variety of musculoskeletal and spinal disorders such as lower back pain (LBP). Secretome derived from mesenchymal stem cells (MSCs) have exerted beneficial effect on tissue regeneration. In this study, the goal was to investigate the paracrine and the anti-inflammatory effects of secretome from interleukin IL1β preconditioned Bone Marrow MSCs (BMSCs) on human nucleus pulposus cells (hNPCs) in a 3D in vitro model.Secretome was collected from BMSCs (BMSCs-sec) after preconditioning with 10 ng/mL IL1β. hNPCs were isolated from surgical specimens, culture expanded in vitro, encapsulated in alginate beads and treated with: growth medium; IL1β 10 ng/mL; IL1β 10 ng/mL for 24 hours and then BMSCs-sec. We examined: i) cell proliferation and viability (flow cytometry), ii) nitrite production (Griess assay) and ROS quantification (Immunofluorescence) iii) glycosaminoglycan (GAG) amount (DMBB) and iv) gene expression levels of extracellular matrix (ECM) components and inflammatory mediators (qPCR). One-way ANOVA analysis was used to compare the groups under exam and data were expressed as mean ± S.D.In vitro tests showed an enhancement of hNPCs proliferation after treatment with BMSCs-sec (p ≤ 0.05) compared to IL1β group. After 24 hours, the percentage of dead cells was higher in IL1β treated hNPCs compared to control group and decreased significantly in combined IL1β and BMSCs-sec sample group (p ≤ 0.01). Nitrite and ROS production were significantly mitigated and GAGs content was improved by preconditioned BMSCs-sec (p ≤ 0.05). Furthermore, gene expression levels were modulated by BMSCs-sec treatment compared to controls.Our results supported the potential use of BMSCs' secretome as a cell-free strategy for IDD, overcoming the side effects of cell-therapy. Moreover, secretome derived from IL1β preconditioned BMSCs was able to reduce hNPCs death, attenuate ECM degradation and oxidative stress counteracting IDD progression.Acknowledgements: Financial support was received from the “iPSpine” and “RESPINE” Horizon 2020 projects.

Glycosaminoglycans in mucopolysaccharidoses and other disorders.

Glycosaminoglycans (GAGs) are sulfated polysaccharides comprising repeating disaccharides, uronic acid (or galactose) and hexosamines, including chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate. Hyaluronan is an exception in the GAG family because it is a non-sulfated polysaccharide. Lysosomal enzymes are crucial for the stepwise degradation of GAGs to provide a normal function of tissues and extracellular matrix (ECM). The deficiency of one or more lysosomal enzyme(s) results in the accumulation of undegraded GAGs, causing cell, tissue, and organ dysfunction. Accumulation of GAGs in various tissues and ECM results in secretion into the circulation and then excretion in urine. GAGs are biomarkers of certain metabolic disorders, such as mucopolysaccharidoses (MPS) and mucolipidoses. GAGs are also elevated in patients with various conditions such as respiratory and renal disorders, fatty acid metabolism disorders, viral infections, vomiting disorders, liver disorders, epilepsy, hypoglycemia, myopathy, developmental disorders, hyperCKemia, heart disease, acidosis, and encephalopathy. MPS are a group of inherited metabolic diseases caused by the deficiency of enzymes required to degrade GAGs in the lysosome. Eight types of MPS are categorized based on lack or defect in one of twelve specific lysosomal enzymes and are described as MPS I through MPS X (excluding MPS V and VIII). Clinical features vary with the type of MPS and clinical severity of the disease. This chapter addresses the historical overview, synthesis, degradation, distribution, biological role, and method for measurement of GAGs.

Rapid cellular uptake of citrate-coated iron oxide nanoparticles unaffected by cell-surface glycosaminoglycans.

Citrate-coated iron oxide nanoparticles, specifically Synomag®-COOH (SynC), are promising tracers in magnetic particle imaging (MPI) due to their high magnetic moments and rapid cellular uptake. The mechanisms driving efficient SynC uptake remain unclear. Previous observations suggest a role of the extracellular glycocalyx during nanoparticle uptake. Here, we ascertain whether the cell-surface glycosaminoglycans (GAGs) regulate the uptake of SynC. Using transmission electron microscopy (TEM), we visualized SynC uptake by THP-1 cells, a human acute monocytic leukemia cell line. We investigated the interaction of SynC with GAGs in living cells using click-chemistry-based labeling. Upon treating THP-1 cells with chondroitinase or hyaluronidase and with a xylosyltransferase-deficient cell line, we quantified SynC uptake and measured interactions of SynC with cells in real time using magnetic particle spectroscopy (MPS). The THP-1 cell membrane engulfed or formed extensions around SynC, indicating uptake through pinocytosis and phagocytosis. We measured an increased MPS signal of SynC within seconds of cell contact, suggesting an interaction with extracellular components like the glycocalyx. Upon adding SynC to THP-1 cells, we could not observe disruption of fluorescently labeled GAGs or an enhanced intracellular fluorescence, implying that SynC does not accelerate the turnover of GAGs by binding. Lack of chondroitin sulfate, heparan sulfate, and hyaluronic acid did not affect the rapid magnetic behavior increase of SynC upon cell contact. Accordingly, we measured no significant differences in SynC uptake between wild type cells and our GAG-deficient models. These findings suggest that GAGs act as a permeable bandpass for SynC nanoparticles with a minor negative surface charge of -13.8 mV. This finding has significant implications for MPI-based cell tracking because it facilitates efficient tracking of cell types that lack a strong repulsion by cell-surface GAGs. It will be crucial to investigate whether the rapid uptake of SynC is cell-type specific and influenced by different extracellular matrix compositions.

Characterising glycosaminoglycans in human breastmilk and their potential role in infant health.

Human breastmilk is composed of many well researched bioactive components crucial for infant nutrition and priming of the neonatal microbiome and immune system. Understanding these components gives us crucial insight to the health and wellbeing of infants. Research surrounding glycosaminoglycans (GAGs) previously focused on those produced endogenously; however, recent efforts have shifted to understanding GAGs in human breastmilk. The structural complexity of GAGs makes detection and analysis complicated therefore, research is time consuming and limited to highly specialised teams experienced in carbohydrate analysis. In breastmilk, GAGs are present in varying quantities in four forms; chondroitin sulphate, heparin/heparan sulphate, dermatan sulphate and hyaluronic acid, and are hypothesised to behave similar to other bioactive components with suspected roles in pathogen defense and proliferation of beneficial gut bacteria. Chondroitin sulphate and heparin, being the most abundant, are expected to have the most impact on infant health. Their decreasing concentration over lactation further indicates their role and potential importance during early life.

Bone protective effect of sinomenine against monosodium iodoacetate induced knee and hip injury in rat model: an inflammatory pathway.

Osteoarthritis (OA) is a degenerative joint disease which is categorized via destruction of joint cartilage and it also affects the various joints, especially knees and hips. Sinomenine active phytoconstituents isolated from the stem of Sinomenium acutum and already proof anti-inflammatory effect against the arthritis model of rodent. In this experimental protocol, we scrutinized the anti-osteoarthritis effect of sinomenine against monosodium iodoacetate (MIA) induced OA in rats. MIA (3 mg/50 μL) was used for inducing the OA in the rats, and rats received the oral administration of sinomenine (2.5, 5 and 7.5 mg/kg body weight) up to the end of the experimental study (four weeks). The body and organs weight were estimated. Aggrecan, C-terminal cross-linked telopeptide of type II collagen (CTX-II), glycosaminoglycans (GCGs), monocyte chemoattractant protein-1 (MCP-1), Interferon gamma (IFN-γ), antioxidant, inflammatory cytokines, inflammatory mediators and matrix metalloproteinases (MMP) were analyzed. Sinomenine significantly (P < 0.001) boosted the body weight and reduced the heart weight, but the weight of spleen and kidney remain unchanged. Sinomenine significantly (P < 0.001) reduced the level of nitric oxide, MCP-1 and improved the level of aggrecan, IFN-γ and GCGs. Sinomenine remarkably upregulated the level of glutathione, superoxide dismutase and suppressed the level of malonaldehyde. It effectually modulated the level of inflammatory cytokines and inflammatory mediators and significantly (P < 0.001) reduced the level of MMPs, like MMP-1, 2, 3, 9 and 13. Sinomenine is a beneficial active agent for the treatment of OA disease.

Quantification of Glycosaminoglycans Using Harmine Extract from Peganum harmala L. in Mucopolysaccharidoses Researches

Background: Harmine is used in the quantification of glycosaminoglycans (GAGs) for the research on mucopolysaccharidoses (MPSs). Although this product is commercially available, researchers may consider preparing it under laboratory conditions when it is unavailable for various reasons. This research aims to extract harmine from Peganum harmala L. seeds and determine whether this extract can be used as a substitute for pure harmine in the quantification of GAGs and, consequently, in the research on MPS. Methods: P. harmala L. seeds were obtained from the plant and extracted using methanol. The harmine extract was then used in a spectrophotometric assay on controls, including patients known of having MPS and healthy subjects and patients suspected of having different types of MPS. Results: One milligram of harmine extract in 1 ml of ethanol is sufficient to produce the chromogen as that obtained by pure harmine. The chromogen exhibits a peak absorbance peak at 510 nm. The concordance between the two forms of harmine reaches 100%, either for abnormal values seen in GAGs from MPS patients or for normal values in those from healthy controls. Suspicion of having MPS was lifted in investigated patients. The harmine extracted from P. harmala L. seeds allowed for accurate and reproducible quantification of the GAGs. Conclusion: This study demonstrates that harmine extract can be used as a reagent for the quantification of GAGs in the research on MPS when pure harmine is unavailable in the same way as pure harmine.

VAR2HP recognizing heparin‐like epitopes in targeted therapy and diagnosis of tumors

AbstractThe synthesis of glycosaminoglycans (GAGs) in vivo occurs with high spatiotemporal specificity, and any aberrant expression of GAGs is closely related to the occurrence of diseases. In terms of tumorigenesis, the abnormally expressed GAGs have become a potential target for the diagnosis and therapy of tumors. As previously reported, VAR2HP, a protein probe that recognizes the unique heparin (Hep)‐like epitopes, interacts with a decasaccharide structure containing at least three HexA2S(1‐4)GlcNS6S disaccharides. Its recognition epitopes are overexpressed in various tumor cells and appear promising as target molecules of multiple tumors. Herein, we found that VAR2HP used as an antineoplastic carrier could promote drug enrichment in tumor sites by targeting the specific Hep‐like epitopes on tumor cells, thereby reducing the damage to normal cells in vitro and in vivo. Moreover, VAR2HP acting on cells alone could inhibit cell proliferation, indicating that VAR2HP as a drug carrier has a dual effect of antitumor activity. Additionally, we observed that VAR2HP significantly stained cancer tissues more strongly than neighboring nonmalignant tissues. The staining was competitively inhibited by added exogenous Hep, indicating that the Hep‐like epitopes recognized by VAR2HP were a potential target molecule in tumor diagnosis. Moreover, the VAR2HP‐bound Hep‐like epitopes were found to be overexpressed in the sera of patients with hepatocellular carcinoma (HCC) but not in normal persons and patients with cirrhosis. Taken together, this study shows that VAR2HP and its heparin‐like epitopes have great potential in targeted therapy of tumors and HCC diagnosis.

A pilot study for decellularizing human and porcine cornea for future use in corneal regeneration

Purpose: To effectively decellularize human and porcine corneas for future use in corneal regeneration by using an already established decellularization technique being efficient in decellularizing blood vessels.Methods: One human and 4 porcine corneas were decellularized by using various washing steps with 3‐[(3‐cholamidopropyl) dimethylammonio]‐1‐propanesulfonate (CHAPS) detergent, Benzonase, Ethylenediaminetetraacetic acid (EDTA), 5% Dextran or 5% D‐Mannitol. The quality of the decellularization process was assessed by quantitative and qualitative measurement of DNA content, glycosaminoglycans (GAGs), immunofluorescent staining for Collagen I, V and keratocan, and Transmission Electron Microscopy (TEM) to observe the structure of the collagen fibrils was used.Results: The decellularization process showed 99,94% and 99,52% DNA content reduction in the human and porcine corneas when using Dextran, respectively, while more DNA remained in the porcine corneas when D‐Mannitol was used. Similar pattern was observed in the preservation of GAGs, with the highest amount being found in the human cornea, and less in the porcine corneas. H&amp;E and Immunofluorescent staining showed no presence of cell nuclei when compared to the control native corneas, while Alcian blue staining qualitatively confirmed the presence of GAGs. Collagen I, V and keratocan were still present in the decellularized corneas when compared to the native ones, while TEM microscopy further confirmed the similar patterns of the collagen fibrils in the decellularized‐, compared to the native‐ corneas.Conclusions: This pilot study showed that our decellularization method is effective in decellularizing human and porcine corneas, with very high amount of DNA being removed from the corneas, while the GAGs were preserved to an acceptable extent, and the structure and pattern of the collagen fibrils maintained.

Screening of poly-beta amino ester coated emulsion of ketorolac for cartilage delivery.

Osteoarthritis (OA) is a prevalent chronic health condition necessitating effective treatment strategies. Globally, there were 86 million people with incident knee osteoarthritis in 2020. Pain management remains the primary approach to OA as the nature of cartilage poses challenges for drug delivery. An emulsion-based delivery system, using a class of positively charged and hydrolysable polymers (poly-beta-amino-esters) to coat oil droplets containing drugs, has been shown to enhance and prolong drug localization in ex vivo cartilage models. As the properties of the polymers used in this technology strongly depend on the monomers used in the synthesis, this study presents the screening of a wide range of PBAEs as droplet coating agents and using ketorolac as a model of nonsteroidal anti-inflammatory drugs. The emulsions prepared with this PBAE library were characterized, and drug localisation and retention were evaluated in both native and glycosaminoglycan (GAG) depleted cartilage ex vivo models. Optimal candidates were identified and tested in an ex vivo model showing the ability to protect chondrocyte cell viability and increase both GAG and collagen contents in cartilage exposed to cytokine (IL-1α) simulating acute cartilage damage. This work demonstrates the potential of PBAE coated emulsion as a delivery system for effective drug delivery in OA treatment.

Development of an Analytical Method for Unsaturated Disaccharides from Heparan Sulfate Using Dansylhydrazine Derivatization and High-Performance Liquid Chromatography with Fluorescence Detection.

Glycosaminoglycans (GAGs), such as heparan sulfate (HS), play essential roles in living organisms. Understanding the functionality of HS and its involvement in disease progression necessitates the sensitive and quantitative detection of HS-derived unsaturated disaccharides. Conventionally, fluorescence derivatization precedes the HPLC analysis of these disaccharides. However, the presence of excess unreacted derivatization reagents can inhibit rapid and sensitive analysis in chromatographic determinations. In this study, we describe analytical methods that use dansylhydrazine as a derivatization agent for the detection and determination of HS-derived unsaturated disaccharides using HPLC. In addition, we have developed a straightforward method for removing excess unreacted reagent using a MonoSpin NH2 column. This method may be employed to remove excess pre-labeling reagents, thereby facilitating the analysis of HS-derived unsaturated disaccharides with satisfactory reproducibility.