Dimethylmethylene Blue Research Articles

Evaluation of alginate modification effect on cell-matrix interaction, mechanotransduction and chondrogenesis of encapsulated MSCs.

Mesenchymal stem cells (MSCs) are promising cell candidates for cartilage regeneration. Furthermore, it is important to control the cell-matrix interactions that have a direct influence on cell functions. Providing an appropriate microenvironment for cell differentiation in response to exogenous stimuli is a critical step towards the clinical utilization of MSCs. In this study, hydrogels consisted of different proportions of alginates that were modified using gelatin, collagen type I and arginine-glycine-aspartic acid (RGD) and were evaluated regarding their effects on mesenchymal stem cells. The effect of applying hydrostatic pressure on MSCs encapsulated in collagen-modified alginate with and without chondrogenic medium was evaluated 7, 14 and 21days after culture, which is a comprehensive evaluation of chondrogenesis in 3D hydrogels with mechanical and chemical stimulants. Alcian blue, safranin O and dimethyl methylene blue (DMMB) staining showed the chondrogenic phenotype of cells seeded in the collagen- and RGD-modified alginate hydrogels with the highest intensity after 21days of culture. The results of real-time PCR for cartilage-specific extracellular matrix genes indicated the chondrogenic differentiation of MSCs in all hydrogels. Also, the synergic effects of chemical and mechanical stimuli are indicated. The highest expression levels of the studied genes were observed in the cells embedded in collagen-modified alginate by loading after 14days of exposure to the chondrogenic medium. The effect of using IHP on encapsulated MSCs in modified alginate with collagen type I is equal or even higher than using TGF-beta on encapsulated cells. The results of immunohistochemical assessments also confirmed the real-time PCR data.

Alterations in characteristics of canine articular chondrocytes in non-passaged long-term monolayer culture: Matter of differentiation, dedifferentiation and redifferentiation.

This study investigated the effects of culture time on phenotype stability of canine articular chondrocytes (CACs) in non-passaged long-term monolayer culture. Third passage (P3) CACs isolated from four cartilage samples were seeded at three different initial seeding densities (0.2 × 104, 1.0 × 104 and 5.0 × 104 cells/cm2) and maintained in monolayer condition up to 8 weeks without undergoing subculture after confluence. The characteristic changes of chondrocytes during the culture period were evaluated based on the cell morphology, cell proliferation, glycosaminoglycans (GAGs) content, DNA quantification, mRNA expression and ultrastructure of chondrocytes. Chondrocytes maintained under post-confluence condition exhibited a capability to grow and proliferate up to 4 weeks. Alcian blue staining and Dimethylmethylene blue (DMMB) assay revealed that the extracellular matrix (ECM) synthesis was increased in a time-dependent manner from 2 to 8 weeks. The chondrocyte mRNA expression profile was dramatically affected by prolonged culture time, with a significant downregulation of collagen type I, whereas the expression of collagen type II, aggrecan, Sox9 and matrix metalloproteinase 13 (MMP-13) were significantly upregulated. In addition, transmission electron microscopy (TEM) result indicated dilation of rough endoplasmic reticulum (RER) in these long-term monolayer cultured chondrocytes. These findings demonstrate that the chondrocytes phenotype could be partially redifferentiated through the spontaneous redifferentiation process in long-term cultures using standard culture medium without the addition of chondrogenic supplements or tissue-culture scaffolds.

Interleukin-1 receptor antagonist (IL-1Ra) is more effective in suppressing cytokine-induced catabolism in cartilage-synovium co-culture than in cartilage monoculture

BackgroundMost in vitro studies of potential osteoarthritis (OA) therapies have used cartilage monocultures, even though synovium is a key player in mediating joint inflammation and, thereby, cartilage degeneration. In the case of interleukin-1 (IL-1) inhibition using its receptor antagonist (IL-1Ra), like chondrocytes, synoviocytes also express IL-1 receptors that influence intra-articular IL-1 signaling and IL-1Ra efficacy. The short residence time of IL-1Ra after intra-articular injection requires the application of frequent dosing, which is clinically impractical and comes with increased risk of infection; these limitations motivate the development of effective drug delivery strategies that can maintain sustained intra-articular IL-1Ra concentrations with only a single injection. The goals of this study were to assess how the presence of synovium in IL-1-challenged cartilage-synovium co-culture impacts the time-dependent biological response of single and sustained doses of IL-1Ra, and to understand the mechanisms underlying any co-culture effects.MethodsBovine cartilage explants with or without synovium were treated with IL-1α followed by single or multiple doses of IL-1Ra. Effects of IL-1Ra in rescuing IL-1α-induced catabolism in cartilage monoculture and cartilage-synovium co-culture were assessed by measuring loss of glycosaminoglycans (GAGs) and collagen using DMMB (dimethyl-methylene blue) and hydroxyproline assays, respectively, nitric oxide (NO) release using Griess assay, cell viability by fluorescence staining, metabolic activity using Alamar blue, and proteoglycan biosynthesis by radiolabel incorporation. Day 2 conditioned media from mono and co-cultures were analyzed by mass spectrometry and cytokine array to identify proteins unique to co-culture that contribute to biological crosstalk.ResultsA single dose of IL-1Ra was ineffective, and a sustained dose was necessary to significantly suppress IL-1α-induced catabolism as observed by enhanced suppression of GAG and collagen loss, NO synthesis, rescue of chondrocyte metabolism, viability, and GAG biosynthesis rates. The synovium exhibited a protective role as the effects of single-dose IL-1Ra were significantly enhanced in cartilage-synovium co-culture and were accompanied by release of anti-catabolic factors IL-4, carbonic anhydrase-3, and matrilin-3. A total of 26 unique proteins were identified in conditioned media from co-cultures, while expression levels of many additional proteins important to cartilage homeostasis were altered in co-culture compared to monocultures; principal component analysis revealed distinct clustering between co-culture and cartilage and synovium monocultures, thereby confirming significant crosstalk.ConclusionsIL-1Ra suppresses cytokine-induced catabolism in cartilage more effectively in the presence of synovium, which was associated with endogenous production of anti-catabolic factors. Biological crosstalk between cartilage and synovium is significant; thus, their co-cultures should better model the intra-articular actions of potential OA therapeutics. Additionally, chondroprotective effects of IL-1Ra require sustained drug levels, underscoring the need for developing drug delivery strategies to enhance its joint residence time following a single intra-articular injection.

Glycocalyx breakdown is increased in African children with cerebral and uncomplicated falciparum malaria.

Cerebral malaria (CM) from Plasmodium falciparum infection is associated with endothelial dysfunction and parasite sequestration. The glycocalyx (GCX), a carbohydrate-rich layer lining the endothelium, is crucial in vascular homeostasis. To evaluate the role of its loss in the pathogenesis of pediatric CM, we measured GCX degradation in Tanzanian children with World Health Organization-defined CM (n = 55), uncomplicated malaria (UM; n = 20), and healthy controls (HCs; n = 25). Urine GCX breakdown products [glycosaminoglycans (GAGs)] were quantified using dimethylmethylene blue (DMMB) and liquid chromatography-tandem mass spectrometry assays. DMMB-GAG and mass spectrometry (MS)-GAG (g/mol creatinine) were increased in CM and UM compared with HCs (P < 0.001), with no differences in DMMB-GAG and MS-GAG between CM and UM children or between those with and without a fatal outcome. In CM survivors, urinary GCX DMMB-GAG normalized by d 3. After adjusting for disease severity, DMMB-GAG was significantly associated with parasitemia [partial correlation coefficient (Pcorr) = 0.34; P = 0.01] and plasma TNF (Pcorr = 0.26; P = 0.04) and inversely with plasma and urine NO oxidation products [Pcorr = -0.31 (P = 0.01) and Pcorr = -0.26 (P = 0.03), respectively]. GCX breakdown is increased in children with falciparum malaria, with similar elevations in CM and UM. Endothelial GCX degradation may impair endothelial NO production, exacerbate adhesion-molecule expression, exposure, and parasite sequestration, and contribute to malaria pathogenesis.-Yeo, T. W., Bush, P. A., Chen, Y., Young, S. P., Zhang, H., Millington, D. S., Granger, D. L., Mwaikambo, E. D., Anstey, N. M., Weinberg, J. B. Glycocalyx breakdown is increased in African children with cerebral and uncomplicated falciparum malaria.

An At-Risk Population Screening Program for Mucopolysaccharidoses by Measuring Urinary Glycosaminoglycans in Taiwan.

Background: The mucopolysaccharidoses (MPSs) are a group of rare lysosomal storage disorders characterized by the accumulation of glycosaminoglycans (GAGs) and which eventually cause progressive damage to various tissues and organs. We developed a feasible MPS screening algorithm and established a cross-specialty collaboration platform between medical geneticists and other medical specialists based on at-risk criteria to allow for an earlier confirmative diagnosis of MPS. Methods: Children (<19 years of age) with clinical signs and symptoms compatible with MPS were prospectively enrolled from pediatric clinics between July 2013 and June 2018. Urine samples were collected for a non-specific total GAG analysis using the dimethylmethylene blue (DMB) spectrophotometric method, and the quantitation of three urinary GAGs (dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS)) was performed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). The subjects with elevated urinary GAG levels were recalled for leukocyte enzyme activity assay and genetic testing for confirmation. Results: Among 153 subjects enrolled in this study, 13 had a confirmative diagnosis of MPS (age range, 0.6 to 10.9 years—three with MPS I, four with MPS II, five with MPS IIIB, and one with MPS IVA). The major signs and symptoms with regards to different systems recorded by pediatricians at the time of the decision to test for MPS were the musculoskeletal system (55%), followed by the neurological system (45%) and coarse facial features (39%). For these 13 patients, the median age at the diagnosis of MPS was 2.9 years. The false negative rate of urinary DMB ratio using the dye-based method for these 13 patients was 31%, including one MPS I, two MPS IIIB, and one MPS IVA. However, there were no false negative results with urinary DS, HS and KS using the MS/MS-based method. Conclusions: We established an at-risk population screening program for MPS by measuring urinary GAG fractionation biomarkers using the LC-MS/MS method. The program included medical geneticists and other medical specialists to increase awareness and enable an early diagnosis by detecting MPS at the initial onset of clinical symptoms.

The Influence of bFGF on the Fabrication of Microencapsulated Cartilage Cells under Different Shaking Modes.

Cell encapsulation in hydrogels has been extensively used in cytotherapy, regenerative medicine, 3D cell culture, and tissue engineering. Herein, we fabricated microencapsulated cells through microcapsules loaded with C5.18 chondrocytes alginate/chitosan prepared by a high-voltage electrostatic method. Under optimized conditions, microencapsulated cells presented uniform size distribution, good sphericity, and a smooth surface with different cell densities. The particle size distribution was determined at 150–280 μm, with an average particle diameter of 220 μm. The microencapsulated cells were cultured under static, shaking, and 3D micro-gravity conditions with or without bFGF (basic fibroblast growth factor) treatment. The quantified detection (cell proliferation detection and glycosaminoglycan (GAG)/type II collagen (Col-II)) content was respectively determined by cell counting kit-8 assay (CCK-8) and dimethylmethylene blue (DMB)/Col-II secretion determination) and qualitative detection (acridine orange/ethidium bromide, hematoxylin-eosin, alcian blue, safranin-O, and immunohistochemistry staining) of these microencapsulated cells were evaluated. Results showed that microencapsulated C5.18 cells under three-dimensional microgravity conditions promoted cells to form large cell aggregates within 20 days by using bFGF, which provided the possibility for cartilage tissue constructs in vitro. It could be found from the cell viability (cell proliferation) and synthesis (content of GAG and Col-II) results that microencapsulated cells had a better cell proliferation under 3D micro-gravity conditions using bFGF than under 2D conditions (including static and shaking conditions). We anticipate that these results will be a benefit for the design and construction of cartilage regeneration in future tissue engineering applications.

PHEMA Hydrogels Obtained by Infrared Radiation for Cartilage Tissue Engineering

Although the exposure of polymeric materials to radiation is a well-established process, little is known about the relationship between structure and property and the biological behavior of biomaterials obtained by thermal phenomena at 1070 nm wavelength. This study includes results concerning the use of a novel infrared radiation source (ytterbium laser fiber) for the synthesis of poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel in order to produce medical devices. The materials were obtained by means of free radical polymerization mechanism and evaluated regarding its cross-linking degree, polymer chain mobility, thermal, and mechanical properties. Their potential use as a biomaterial toward cartilage tissue was investigated through incubation with chondrocytes cells culture by dimethylmethylene blue (DMMB) dye and DNA quantification. Differential scanning calorimetry (DSC) results showed that glass transition temperature (Tg) was in the range 103°C–119°C, the maximum degree of swelling was 70.8%, and indentation fluency test presented a strain of 56%–85%. A significant increase of glycosaminoglycans (GAGs) concentration and DNA content in cells cultured with 40 wt% 2-hydroxyethyl methacrylate was observed. Our results showed the suitability of infrared laser fiber in the free radicals formation and in the rapid polymer chain growth, and further cross-linking. The porous material obtained showed improvements concerning cartilage tissue regeneration.

LncRNA TUG1 promotes osteoarthritis-induced degradation of chondrocyte extracellular matrix via miR-195/MMP-13 axis.

Osteoarthritis is a degenerative disease characterized by articular cartilage degradation. Long non-coding ribonucleic acid (lncRNA) plays important roles in a series of biological processes, but its role in osteoarthritis is still not quite clear. This study aims to investigate the regulatory role of taurine upregulated gene 1 (TUG1) in osteoarthritis. The expression level of lncRNA-TUG1 in cartilages of patients with osteoarthritis and those of normal people was compared using Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Primary chondrocytes were induced by interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α), followed by expression detection of lncRNA-TUG1, microRNA-195 (miR-195), and matrix metalloproteinase-13 (MMP-13). In addition, in vitro regulatory roles of lncRNA-TUG1 and miR-195 in osteoarthritis were verified by transfection of lncRNA-TUG1 and miR-195 plasmids. The dimethylmethylene blue (DMMB) assay was performed to analyze the secretion and formation of soluble sulfated glycosaminoglycan (sGAG). The expression levels of lncRNA-TUG1 and MMP-13 in cartilages of patients with osteoarthritis were higher than those in cartilages of normal people, while the level of miR-195 decreased in cartilages of patients with osteoarthritis. After chondrocytes were induced by IL-1β and TNF-α, the expression of lncRNA-TUG1 increased. Overexpression of lncRNA-TUG1 decreased the expressions of miR-195, collagen, and aggrecan, but increased the expression of MMP-13. LncRNA-TUG1 knockdown obtained the opposite results. LncRNA-TUG1 regulates the degradation of extracellular matrix in osteoarthritis via lncRNA-TUG1/miR-195/MMP-13 axis.

Fabrication of Nanofibrous PVA/Alginate-Sulfate Substrates for Growth Factor Delivery.

Providing affinity sites on alginate (ALG) matrix enables specific binding of growth factors to the polymer backbone and allows their release in a controlled fashion. In this study, we used a blend of alginate sulfate (ALG-S) and polyvinyl alcohol (PVA) to fabricate electrospun scaffolds capable of delivering a heparin-like growth factor, transforming growth factor-beta1 (TGF-β1). The alginate was sulfated with different degrees of sulfation (DS, from 0.8, 3.4 to 12.4%) by a simple process. The success of sulfation was determined by Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR), elemental analysis, ultraviolet (UV) spectroscopy and staining with dimethylmethylene blue. The physical-mechanical properties of nanofibrous mats were characterized by scanning electron microscopy (SEM), FTIR, energy-dispersive X-ray spectroscopy (EDX), tensile strength and mass loss analysis. Additionally, the release kinetics of transforming growth factor-β1 (TGF-β1) from PVA/ALG-S and PVA/ALG scaffolds were compared. The results showed that the binding and entrapment of TGF-β1 to the nanofibrous scaffolds are improved by the addition of sulfate group to alginate. In conclusion, our results support that nanofibrous scaffold based on PVA/ALG-S can deliver growth factors in tissue engineering application. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 403-413, 2019.

The relationships between urinary glycosaminoglycan levels and phenotypes of mucopolysaccharidoses.

BackgroundThe aim of this study was to use the liquid chromatography/tandem mass spectrometry (LC‐MS/MS) method to quantitate levels of three urinary glycosaminoglycans (GAGs; dermatan sulfate [DS], heparan sulfate [HS], and keratan sulfate [KS]) to help make a correct diagnosis of mucopolysaccharidosis (MPS).MethodsWe analyzed the relationships between phenotypes and levels of urinary GAGs of 79 patients with different types of MPS.ResultsThe patients with mental retardation (n = 21) had significantly higher levels of HS than those without mental retardation (n = 58; 328.8 vs. 3.2 μg/ml, p < 0.001). The DS levels in the patients with hernia, hepatosplenomegaly, claw hands, coarse face, valvular heart disease, and joint stiffness were higher than those without. Twenty patients received enzyme replacement therapy (ERT) for 1–12.3 years. After ERT, the KS level decreased by 90% in the patients with MPS IVA compared to a 31% decrease in the change of dimethylmethylene blue (DMB) ratio. The DS level decreased by 79% after ERT in the patients with MPS VI compared to a 66% decrease in the change of DMB ratio.ConclusionsThe measurement of GAG fractionation biomarkers using the LC‐MS/MS method is a more sensitive and reliable tool than the DMB ratio for MPS high‐risk screening, diagnosis, subclass identification, and monitoring the efficacy of ERT.

Chondroprotective effects of aqueous extract of Anthriscus sylvestris leaves on osteoarthritis in vitro and in vivo through MAPKs and NF-κB signaling inhibition

Osteoarthritis (OA) is a common degenerative joint disease, characterized by cartilage degradation and inflammation, in the elderly population. Anthriscus sylvestris has been used in Korean traditional medicine and contains many polyphenolic compounds such as cynaroside and chlorogenic acid, which are major active components responsible for its antioxidant effect. In this study, we aimed to evaluate the chondroprotective effect of an aqueous extract of A. sylvestris leaves (AE-ASL) on OA, both in vitro and in vivo. Rat primary chondrocytes were pretreated with AE-ASL for 1 h before interleukin-1β (20 ng/mL) stimulation. The production of nitrite, PGE2, aggrecan, and collagen type II were detected by Griess reagent and ELISAs. The mRNA levels of iNOS, COX-2, MMP-3, and MMP-13 were measured by RT-PCR. In addition, protein levels of iNOS, COX-2, MMP-3, MMP-13, ADAMTS-4, MAPKs, and NF-κB p65 subunit were measured by western blot analysis. Sulfated glycosaminoglycan (sGAGs) were detected by dimethylmethylene blue (DMMB) assay. During in vivo study, the effects of AE-ASL were evaluated for 8 weeks in a rat model of destabilization of the medial meniscus (DMM) surgery-induced OA. AE-ASL significantly inhibited expression of nitrite, iNOS, PGE2, COX-2, MMP-3, MMP-13, and ADAMTS-4 in IL-1β-stimulated chondrocytes. Moreover, it decreased the IL-1β-induced degradation of aggrecan, collagen type II, and proteoglycan. In addition, AE-ASL suppressed IL-1β-induced phosphorylation of MAPKs and NF-κB p65 subunit translocation to nucleus. In vivo, AE-ASL inhibited DMM surgery-induced cartilage destruction and proteoglycan loss. Taken together, these results suggest that AE-ASL may be a potential therapeutic agent for the alleviation of OA progression.

Simultaneous release of melatonin and methylprednisolone from an injectable in situ self-crosslinked hydrogel/microparticle system for cartilage tissue engineering.

Recently, injectable hydrogel/microparticle systems have so considered for tissue engineering and regenerative medicine. In this study, we produced an injectable in situ self-crosslinked hydrogel/microparticle system for simultaneous dual drug delivery. First, melatonin conjugated chitosan microparticle loaded with methylprednisolone (MCC-MP) microparticle was fabricated by the covalent linkage of melatonin to chitosan by N-hydroxysuccinimide (NHS)1-Ethyl-3-(3-dimethylamino propyl)-carbodiimide (EDC) followed by an ionic gelation of MCC and MP using tripolyphosphate (TPP). Second, the hydrogel was prepared by the connection between the aldehyde group of alginate oxide (AD) and the amine group belonging to carboxymethyl chitosan (CMC) via Schiff base reaction. Finally, microparticle was incorporated into the AD-CMC hydrogel to produce a hydrogel/microparticle system. Hydrogel/microparticle was assessed by many techniques including microscopy, spectroscopy, particle size measurements, mechanical analysis, injectability, rheological analyses to ascertain hydrogel/microparticle properties. The biological assays of mesenchymal stem cells (MSCs) culture, 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), acridine orange/propidium iodide (AO/PI), and 4, 6-diamidino-2-phenylindole (DAPI) to assess cell viability and dimethylmethylene blue (DMMB) to evaluate proteoglycan content were done. The release profiles of melatonin and MP showed acceptable release after 60 and 20 days, respectively. The hydrogel/microparticle system has the ability to sustain cells alive. A higher rate of proteoglycan content was observed in hydrogel/microparticle as compared with hydrogel. With appropriate biocompatibility and adequate properties, this system can be a proper alternative for cartilage tissue engineering. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1932-1940, 2018.

A novel organ culture model of a joint for the evaluation of static and dynamic load on articular cartilage.

ObjectivesThe purpose of this study was to create a novel ex vivo organ culture model for evaluating the effects of static and dynamic load on cartilage.MethodsThe metatarsophalangeal joints of 12 fresh cadaveric bovine feet were skinned and dissected aseptically, and cultured for up to four weeks. Dynamic movement was applied using a custom-made machine on six joints, with the others cultured under static conditions. Chondrocyte viability and matrix glycosaminoglycan (GAG) content were evaluated by the cell viability probes, 5-chloromethylfluorescein diacetate (CMFDA) and propidium iodide (PI), and dimethylmethylene blue (DMMB) assay, respectively.ResultsChondrocyte viability in the static model decreased significantly from 89.9% (sd 2.5%) (Day 0) to 66.5% (sd 13.1%) (Day 28), 94.7% (sd 1.1%) to 80. 9% (sd 5.8%) and 80.1% (sd 3.0%) to 46.9% (sd 8.5%) in the superficial quarter, central half and deep quarter of cartilage, respectively (p < 0.001 in each zone; one-way analysis of variance). The GAG content decreased significantly from 6.01 μg/mg (sd 0.06) (Day 0) to 4.71 μg/mg (sd 0.06) (Day 28) (p < 0.001; one-way analysis of variance). However, with dynamic movement, chondrocyte viability and GAG content were maintained at the Day 0 level over the four-week period without a significant change (chondrocyte viability: 92.0% (sd 4.0%) (Day 0) to 89.9% (sd 0.2%) (Day 28), 93.1% (sd 1.5%) to 93.8% (sd 0.9%) and 85.6% (sd 0.8%) to 84.0% (sd 2.9%) in the three corresponding zones; GAG content: 6.18 μg/mg (sd 0.15) (Day 0) to 6.06 μg/mg (sd 0.09) (Day 28)).ConclusionDynamic joint movement maintained chondrocyte viability and cartilage GAG content. This long-term whole joint culture model could be of value in providing a more natural and controlled platform for investigating the influence of joint movement on articular cartilage, and for evaluating novel therapies for cartilage repair.Cite this article: Y-C. Lin, A. C. Hall, A. H. R. W. Simpson. A novel organ culture model of a joint for the evaluation of static and dynamic load on articular cartilage. Bone Joint Res 2018;7:205–212. DOI: 10.1302/2046-3758.73.BJR-2017-0320.

Mutant Fibulin-3 Causes Proteoglycan Accumulation and Impaired Diffusion Across Bruch's Membrane.

PurposeThe mutation R345W in EFEMP1 (fibulin-3) causes macular degeneration. This study sought to determine whether proteoglycan content and diffusion across Bruch's membrane are altered in Efemp1ki/ki mice carrying this mutation or in Efemp1−/− mice.MethodsProteoglycans in mouse Bruch's membranes were stained with Cupromeronic Blue (CB). Heparan sulfated proteoglycan (HSPG) and chondroitin/dermatan sulfate proteoglycan (C/DSPG) distributions were visualized following treatments with chondroitinase ABC (C-ABC) or nitrous acid. Total sulfated glycosaminoglycans (sGAGs) in Bruch's membrane/choroid (BrM/Ch) were measured with dimethylmethylene blue (DMMB). Matrix metalloprotease (MMP)-2, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-3 were examined by immunofluorescence and quantified using Image J. Molecules with different Stokes radius (Rs) were allowed simultaneously to diffuse through mouse BrM/Ch mounted in a modified Ussing chamber. Samples were quantified using gel exclusion chromatography.ResultsHSPGs and C/DSPGs were markedly increased in Efemp1ki/ki Bruch's membrane, and MMP-2 and MMP-9 were decreased, but TIMP-3 was increased. Diffusion across Efemp1ki/ki Bruch's membrane was impaired. In contrast, the proteoglycan amount in Efemp1−/− Bruch's membrane was not significantly different, but the size of proteoglycans was much larger. MMP-2, MMP-3, and TIMP-3 levels were similar to that of Efemp1+/+ mice, but they were localized diffusely in retinal pigment epithelium (RPE) cells instead of Bruch's membrane. Diffusion across Efemp1−/− Bruch's membrane was enhanced.ConclusionsMutant fibulin-3 causes proteoglycan accumulation, reduction of MMP-2 and MMP-9, but increase of TIMP-3, and impairs diffusion across Bruch's membrane. Fibulin-3 ablation results in altered sizes of proteoglycans, altered distributions of MMP-2, MMP-9, and TIMP-3, and enhances diffusion across Bruch's membrane.

Long Noncoding RNA CIR Promotes Chondrocyte Extracellular Matrix Degradation in Osteoarthritis by Acting as a Sponge For Mir-27b

Background/Aims: Osteoarthritis (OA) is a degenerative joint disease characterized by articular cartilage degradation. The degradation of the extracellular matrix (ECM) of chondrocyte is closely associated with the destruction of joints in OA patients. lncRNAs are non-coding segments of RNA that possess important regulatory functions at the cellular level and in a variety of pathophysiological processes. The present study was conducted to investigate whether lncRNA-CIR regulated the expression of MMP13 as a sponge of miR-27 in OA. Methods: Primary cultured chondrocytes were challenged by IL-1β and TNF-α to simulate OA conditions. qRT-PCR was performed to detect the miR-27, lncRNA-CIR, MMP13 mRNA expression levels. Western blot was applied to detect MMP13 protein expression. Soluble sGAG secretion/ formation was analysed by the dimethylmethylene blue (DMMB) assay. lncRNA-CIR overexpression or inhibition was performed using overexpression plasmid and small interfering RNAs (siRNAs), respectively. Results: lncRNA-CIR significantly up-regulated in OA patients, concomitantly down-regulated miR-27 and up-regulated MMP13. Bioinformatics analysis predicted miR-27 was the target of both lncRNA-CIR and MMP13. Overexpression of lncRNA-CIR significantly increased the expression of MMP13, while miR-27 remarkably suppressed the expression of MMP13, Accompanying with the increases of mRNA level, protein level and relative luciferase activity. Conclusion: The present findings indicated that lncRNA-CIR/miR-27/MMP13 axis involved in the degradation of the ECM of chondrocyte in OA.

Effect of selected flavonoids on glycosaminoglycans in human skin fibroblasts

Purpose: Glycosaminoglycans (GAGs) and proteoglycans (PG) in addition to collagen are the main components of extracellular matrix (ECM). They play an important role in intercellular communication and interactions between cells and ECM. The biological changes in ECM that occur during aging are induced by decrease in GAG biosynthesis. The purpose of this study was to evaluate the effect of selected flavonoids isolated from Cirsium palustre (L.) Scop. on GAG content in human skin fibroblasts. Materials and methods: Human skin fibroblasts were treated with eriodictyol 7-O-glucoside (C1), 6-hydroxyluteolin 7-O-glucoside (C2), scutellarein 7-O-glucoside (C3) and pedalitin (C4) at 1, 20 and 40 μM for 24 h. Concentration of GAGs in the medium was assayed using method based on their ability to bind the cationic dye 1,9- dimethylmethylene blue (DMMB). Results: C1, C2 and C4 at concentration of 20 and 40 µM significantly increased content of sulphated GAGs in the medium. In contrast, treatment of cells with compound C3 did not have a statistically significant impact on GAG level. Ascorbic acid used as a positive control at 50 µM showed no effect on GAG concentration and increased their content at 100 µM but to a much lower extent than flavonoids. Conclusion: Flavonoids C1, C2 and C4 showed greater than ascorbic acid stimulatory impact on GAGs in healthy human skin fibroblasts, demonstrating their therapeutic potential in the aging.

Screening for mucopolysaccharidoses in the Turkish population: Analytical and clinical performance of an age-range specific, dye-based, urinary glycosaminoglycan assay

Comprehensive analytical and diagnostic performance of urinary quantitative GAG analysis with dimethylmethylene blue (DMB) and the age-specific reference ranges were determined in Turkish population, which has a high incidence of MPSs. Precision, linearity, recovery and accuracy/trueness, limits, stability, and effect of interferents were tested according to CLSI guideline. Clinical performance was evaluated with ROC analyses including 45 MPS patients. Intra-day and inter-day precisions were <5% and <11% (CV), respectively. LoD was 9.12mg/L and LoQ was 23.3mg/L. The highest reference values for urinary GAG excretion were determined in an age-specific manner. In the 2–13years age cohort, a cut-off of 89.86mg/g creatinine resulted in 98.07% sensitivity and 93.33% specificity. Proteinuria and hematuria interfered with analysis in some instances. Neither leukocyturia nor pH changes affected the assay. Stability analysis indicated that freezing urine samples for transfer is unnecessary. Of the 45 MPS patient samples evaluated, only three tested negative including MPS II, IVA and VI. Despite limitations due to low levels of urinary GAG excretion in some cases, urinary GAG analysis with DMB with its technical simplicity, low cost, and precise quantitative results, is a valuable screening method, particularly in populations with a high rate of MPSs.

Quantitative measurement of urinary glycosaminoglycans using a modified DMB method facilitates the diagnosis and monitoring of mucopolysaccharidoses

Mucopolysaccharidoses (MPS) are a heterogeneous group of inherited lysosomal storage disorders (LSDs), due to impaired breakdown of glycosaminoglycans (GAGs), resulting from dysfunctional lysosomal acid hydrolases. Determination of urinary GAG content is a first-line screening assay for MPS. We modified and evaluated a quantitative colorimetric method based on 1,9 Dimethyl-Methylene Blue (DMB) and were able to identify seven new pediatric cases of MPS within a year and subsequently confirmed them by enzymatic analysis. In addition, three cases with borderline urinary GAG content were subsequently diagnosed with other LSDs. We monitored a child with MPS III (Sanfilippo) type C treated with genistein at a dose of 5 mg/kg/day for over a year and we found no change in urine GAG content. In conclusion: the modified DMB colorimetric method is an, accurate and convenient method for measuring urinary GAGs content and facilitates the diagnosis and monitoring of MPS.

UPLC-MS/MS detection of disaccharides derived from glycosaminoglycans as biomarkers of mucopolysaccharidoses

Mucopolysaccharidoses (MPSs) are a group of disorders resulting from primary defects in lysosomal enzymes involved in the degradation of glycosaminoglycans (GAGs). Depending on the specific enzyme defect, the catabolism of one or more GAGs is blocked leading to accumulation in tissues and biological fluids. GAG measurements are important for high-risk screening, diagnosis, monitoring treatment efficacy, and patient follow up. The dimethylmethylene blue (DMB) spectrophotometric method commonly used in most biochemical genetics laboratories relies on a non-specific total GAG analysis which has led to false positive results, and even false negative results (mainly for MPS III and IV patients). The main objective of our project was to devise and validate a reliable tandem mass spectrometry multiplex analysis for the urine quantitation of four GAGs (dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and chondroitin sulfate (CS)) for an eventual technological transfer to the clinic. The developed methodology is rapid (7 min) and our results showed good intraday and interday precision (RSDs ≤ 8.7%) and accuracy (Biases range: −12.0%–18.4%). Linearity was good (r2 > 0.995) for DS, HS, CS, and KS calibration curves. In comparison with the DMB spectrophotometric method, this multiplex tandem mass spectrometry method allows GAG fractionation, thus a differentiation of MPS types, except for MPS I and II which are characterized by the same GAG profile. The devised method is a useful and reliable tool for diagnosis of MPS patients, as well as their monitoring and follow up, as shown by longitudinal studies.

Hypoxia Potentiates Anabolic Effects of Exogenous Hyaluronic Acid in Rat Articular Cartilage.

Hyaluronic acid (HA) is used clinically to treat osteoarthritis (OA), but its pharmacological effects under hypoxic conditions remain unclear. Articular chondrocytes in patients with OA are exposed to a hypoxic environment. This study investigated whether hypoxia could potentiate the anabolic effects of exogenous HA in rat articular cartilage and whether these mechanisms involved HA receptors. HA under hypoxic conditions significantly enhanced the expression of extracellular matrix genes and proteins in explant culture, as shown by real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and dimethylmethylene blue (DMMB) assays. Staining with Safranin-O and immunohistochemical staining with antibody to type II collagen were also enhanced in pellet culture. The expression of CD44 was increased by hypoxia and significantly suppressed by transfection with siRNAs targeting hypoxia-inducible factor 1 alpha (siHIF-1α). These findings indicate that hypoxia potentiates the anabolic effects of exogenous HA by a mechanism in which HIF-1α positively regulates the expression of CD44, enhancing the binding affinity for exogenous HA. The anabolic effects of exogenous HA may increase as OA progresses.