Chondroitin 6 sulfate

Manufactured by Merck Group

Sourced in United States

Chondroitin-6-sulfate is a chemical compound that is a structural component of cartilage. It is a type of glycosaminoglycan that is involved in the formation and maintenance of cartilage and other connective tissues. Chondroitin-6-sulfate is commonly used as a laboratory reagent for research and testing purposes.

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Lab products found in correlation

Calcium hydroxide, by Merck Group (11 mentions) Calcium nitrate hydrate, by Merck Group (8 mentions) Papain, by Merck Group (6 mentions) Picogreen assay, by Thermo Fisher Scientific (5 mentions) Quant it picogreen dsdna assay, by Thermo Fisher Scientific (3 mentions) Collagen type 1, by Merck Group (2 mentions) Gelatin, by Merck Group (2 mentions) Dimethylaminobenzoaldehyde, by Merck Group (2 mentions) Hydroxyproline, by Merck Group (2 mentions) Papain, by Thermo Fisher Scientific (2 mentions) Picogreen dna assay, by Thermo Fisher Scientific (2 mentions) Proteinase k, by Thermo Fisher Scientific (1 mentions) Hyaluronidase, by Merck Group (1 mentions) Digital camera, by Sony (1 mentions) Cell culture plate, by Corning (1 mentions) Hyaluronic acid, by Merck Group (1 mentions) N hydroxysuccinimide, by Merck Group (1 mentions) Freeze dryer, by Martin Christ (1 mentions) Sodium borate decahydrate, by Merck Group (1 mentions) Ascorbate, by Merck Group (1 mentions)

40 protocols using chondroitin 6 sulfate

Fabrication of Mineralized Collagen Scaffolds

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Mineralized collagen scaffolds were fabricated using previously described procedures.^{10,11,21,27,40–45 (link)} In a cooled, jacketed vessel, 1.9 w/v% type I bovine collagen (Sigma-Aldrich, Missouri, USA), 0.84 w/v% chondroitin-6-sulfate (Sigma-Aldrich), and Ca(OH)₂, H₃PO₄, and Ca(NO₃)₂·4H₂O were thoroughly homogenized, making sure to prevent collagen clumping. The mineralized collagen suspension was transferred to 7.62 × 7.62 cm aluminum molds to create isotropic scaffolds. Alternatively, the suspension was pipetted into Teflon molds with a copper base to create anisotropic scaffolds.^{46 (link)} The suspensions were cooled at a constant rate of 1 °C min⁻¹ from 20 °C to −10 °C. Once frozen, the suspension was held at this temperature for 2 hours then lyophilized (0.2 torr pressure, 0 °C) in a VirTis Genesis 25XL Lyophilizer (SP Industries, Inc., Pennsylvania, USA).
Anisotropic mineralized collagen scaffolds with disparate GAG content were fabricated following the same method. However initial mineralized collagen suspensions were created using one of three potential glycosaminoglycans: chondroitin-6-sulfate (chondroitin sulfate sodium salt from shark cartilage, Sigma-Aldrich), chondroitin-4-sulfate (sodium chondroitin sulfate A, Toronto Research Chemicals Inc., Ontario, Canada), or heparin sulfate (Heparin sodium salt from porcine intestinal mucosa, Sigma-Aldrich).

Dewey M.J., Nosatov A.V., Subedi K, & Harley B. (2020). Anisotropic mineralized collagen scaffolds accelerate osteogenic response in a glycosaminoglycan-dependent fashion. RSC Advances, 10(26), 15629-15641.

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Glycosaminoglycan Quantification in Mouse IVD

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For each mouse, nucleus pulposus tissue isolated from four lumbar IVDs of each mouse were pooled and digested using papain at 60 °C for 2 h. GAG content was measured in duplicates by the DMMB procedure using chondroitin-6-sulfate (Millipore Sigma C-8529) as a standard^{41 (link)}. The DNA concentration of each sample was measured using the Pico-Green assay (Molecular Probes) and used to normalize the glycosaminoglycan values.

Yousefzadeh M.J., Flores R.R., Zhu Y., Schmiechen Z.C., Brooks R.W., Trussoni C.E., Cui Y., Angelini L., Lee K.A., McGowan S.J., Burrack A.L., Wang D., Dong Q., Lu A., Sano T., O’Kelly R.D., McGuckian C.A., Kato J.I., Bank M.P., Wade E.A., Pillai S.P., Klug J., Ladiges W.C., Burd C.E., Lewis S.E., LaRusso N.F., Vo N.V., Wang Y., Kelley E.E., Huard J., Stromnes I.M., Robbins P.D, & Niedernhofer L.J. (2021). An aged immune system drives senescence and ageing of solid organs. Nature, 594(7861), 100-105.

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Glycosaminoglycan Quantification in NP Cells

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Proteoglycan content of NP cells was measured using 1,9‐dimethylmethylene blue buffer (DMMB) assay for total glycosaminoglycan (GAG). GAG was released by papain digestion of cell extract at 60°C for 2 h. Concentration of GAG was measured according to the DMMB procedure using Chondroitin‐6‐sulfate (C6737, Millipore Sigma) as the standard and normalized to DNA as measured by the Pico green assay (P11496, Thermo Fisher).

Kritschil R., Li V., Wang D., Dong Q., Silwal P., Finkel T., Lee J., Sowa G, & Vo N. (2023). Impact of autophagy inhibition on intervertebral disc cells and extracellular matrix. JOR Spine, 7(1), e1286.

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Fabrication of Mineralized Collagen Scaffolds

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Mineralized collagen-glycosaminoglycan scaffolds were fabricated via lyophilization from a mineralized collagen precursor suspension as described before.^{47,65,66 (link)} The precursor suspension was created by homogenizing type I collagen (1.9 weight per volume, Sigma Aldrich, St. Louis, Missouri USA), chondroitin-6-sulfate (0.84 weight per volume, Sigma Aldrich), and calcium salts (calcium hydroxide and calcium nitrate, Sigma Aldrich) in a mineral buffer solution (0.1456 M phosphoric acid/0.037 M calcium hydroxide). The precursor suspension was stored at 4 °C and degassed prior to lyophilization.
Mineralized collagen scaffolds were fabricated via lyophilization using a Genesis freeze-dryer (VirTis, Gardener, New York USA) as described before.⁶⁶ Briefly, 100 μL of precursor suspension was pipetted into a custom 144-well polysulfone mold (6 mm diameter, 7 mm tall wells). The precursor solution was frozen by cooling from 20 °C to −10 °C at a constant rate of 1 °C per minute followed by a temperature hold at −10 °C for 2 hours. The frozen suspension was then sublimated at 0 °C and 0.2 Torr, resulting in a porous scaffold network.
All scaffolds were hydrated for 2 hours in ethanol, crosslinked for 2 hours in EDC-NHS, and washed in phosphate buffered saline (PBS) for 48 hours prior to use in experiments.

Tiffany A.S., Dewey M.J, & Harley B.A. (2020). Sequential sequestrations increase the incorporation and retention of multiple growth factors in mineralized collagen scaffolds. RSC Advances, 10(45), 26982-26996.

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Quantifying Sulfated Glycosaminoglycans in Engineered Tissues

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At days 14 and 21 of differentiation, the electrospun scaffolds were collected, washed with PBS, and digested in a 125 μg/mL papain enzyme (from papaya latex, Sigma-Aldrich) solution (50 mM sodium phosphate, 2 mM N-acetyl cysteine, 2 mM EDTA, all from Sigma-Aldrich, pH 6.5) at 60°C overnight (16–18 h). The sulfated glycosaminoglycans (sGAG) produced by cells on the electrospun scaffolds were quantified using 1,9-dimethylmethylene blue (DMMB, Sigma-Aldrich) assay. The digested samples were mixed with a DMMB solution (16 mg DMMB in 0.3% w/v glycine, 0.27% sodium chloride in distilled water, pH 3.0) in 96-well plates and the absorbance was measured at 525 nm. The sGAG amounts were extrapolated from a calibration curve generated using chondroitin 6-sulfate (sodium salt from shark cartilage, Sigma-Aldrich) standards and normalized to the number of cells present in each scaffold. Three scaffolds (n = 3) were used for each experimental group and the absorbance values were measured in triplicate. Cell-free electrospun scaffolds for each experimental group were used as blank controls.

Silva J.C., Udangawa R.N., Chen J., Mancinelli C.D., Garrudo F.F., Mikael P.E., Cabral J.M., Ferreira F.C, & Linhardt R.J. (2019). Kartogenin-loaded coaxial PGS/PCL aligned nanofibers for cartilage tissue engineering. Materials science & engineering. C, Materials for biological applications, 107, 110291.

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Intervertebral Disc GAG Quantification

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NP tissue was isolated and pooled from six lumbar IVDs of each mouse. The pooled tissue sample was digested using papain at 60°C for two hours. GAG content was measured in duplicate by DMB procedure using chondroitin-6-sulfate (Sigma, Milwaukee, WI, C-8529) as a standard. The DNA concentration of each sample was measured using the PicoGreen assay (Molecular Probes, Sunnyvale, CA) and used to normalize the GAG values. Average values from six reaction samples (two duplicates × three mice per group) were calculated +1 SE.

Lei C., Colangelo D., Patil P., Li V., Ngo K., Wang D., Dong Q., Yousefzadeh M.J., Lin H., Lee J., Kang J., Sowa G., Wyss-Coray T., Niedernhofer L.J., Robbins P.D., Huffman D.M, & Vo N. (2020). Influences of circulatory factors on intervertebral disc aging phenotype. Aging (Albany NY), 12(12), 12285-12304.

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Quantitative Glycosaminoglycan and DNA Measurement

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GAG content was measured quantitatively using the dimethylmethylene blue (DMMB, Sigma) assay and a standard curve of chondroitin-6-sulfate (Sigma) and the ratio of absorbance at 525 nm to 590 nm. For HA/HA-MA retention experiments the assay was performed at pH 3.0, and for cell culture experiments pH 1.5 was used, since unsulfated GAGs bind with DMMB at a pH of 3.0 but not 1.5 [19] (link). For the retention experiments, cell-free gels were incubated in PBS at 37°C for up to two weeks. At each time point, four gels were removed from PBS, weighed, and digested with papain (250 µg/mL, Sigma) at 60°C overnight. GAG content was measured in cell-laden constructs after 1 and 28 days culture. The constructs weighed, lyophilised, weighed again, and digested with 0.5% hyaluronidase (Sigma) in PBS at 37°C for 48 hours, followed by digestion with proteinase K (Invitrogen) overnight at 56°C. DNA content in the digests was measured using the Quant-iT PicoGreen dsDNA assay (Invitrogen).

Levett P.A., Hutmacher D.W., Malda J, & Klein T.J. (2014). Hyaluronic Acid Enhances the Mechanical Properties of Tissue-Engineered Cartilage Constructs. PLoS ONE, 9(12), e113216.

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Biodegradable Collagen-Glycosaminoglycan Matrix

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A biodegradable collagen matrix of a 1% collagen and 0.02% glycosaminoglycan copolymer was produced as previously described with several modifications [5 (link)–7 (link)]. In brief, a coprecipitate of type I porcine tendon (SPF, Animal Technology Institute, Taiwan) and chondroitin 6-sulfate (Sigma Chemical Company, St. Louis, MO) in 0.05 M acetic acid was freeze-dried to yield a highly porous sheet 4 mm in thickness. The freeze-drying process yielded a network of the CG copolymer with approximately 95% pore volume fraction and average pore diameter of 140 ± 20 mm. The sheets of the copolymer were cross-linked by dehydrothermal treatment consisting of exposure to a vacuum at a temperature of 105°C for 24 h, followed by exposure to UV light [8 (link)]. Disks of the CG copolymer, 6 × 4 × 3 mm (72 mm³) in size, were cut by trephination and further cross-linked by immersion in 0.25% aqueous glutaraldehyde for 24 h. Residual glutaraldehyde was removed by exhaustive rinsing in multiple changes of phosphate-buffered saline (PBS) over 48 h. There is no hyaluronic acid within the scaffold. The matrix was designated with an optimal degradation time of ~28 days, in line with the time course of endogenous neural stem cell proliferation and differentiation [9 (link)].

Chen J.H., Hsu W.C., Huang K.F, & Hung C.H. (2019). Neuroprotective Effects of Collagen-Glycosaminoglycan Matrix Implantation following Surgical Brain Injury. Mediators of Inflammation, 2019, 6848943.

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Collagen-GAG Scaffold Fabrication and Crosslinking

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Col-GAG and MC-GAG scaffolds were prepared using lyophilization, as described previously (37 (link)–39 (link)). Briefly, microfibrillar, type I collagen (Collagen Matrix, Oakland, NJ) and chondroitin-6-sulfate (Sigma-Aldrich, St. Louis, MO) were combined in suspension in the absence and presence of calcium salts (calcium nitrate hydrate, Ca(NO₃)₂·4H₂O; calcium hydroxide, Ca(OH)₂; Sigma-Aldrich, St. Louis, MO) in an acetic acid (Col-GAG) or phosphoric acid (MC-GAG) solution. Using a constant cooling rate technique at a rate of 1°C/min, the solution was frozen from room temperature to −10°C using a freeze dryer (Genesis, VirTis). Following sublimation of the ice phase, scaffolds were sterilized via ethylene oxide and cut into 8-mm disks in diameter and 4 mm in height for culture.
Cross-linking of scaffolds was performed after rehydration in phosphate-buffered saline (PBS) for 4 hours using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC; Sigma-Aldrich) and N-hydroxysuccinimide (NHS; Sigma-Aldrich) at a molar ratio of 5:2:1 EDAC:NHS:COOH, where COOH represents the amount of collagen in the scaffold as we previously described (40 (link)). Scaffolds were washed with PBS to remove any of the residual chemical.

Ren X., Zhou Q., Foulad D., Tiffany A.S., Dewey M.J., Bischoff D., Miller T.A., Reid R.R., He T.C., Yamaguchi D.T., Harley B.A, & Lee J.C. (2019). Osteoprotegerin reduces osteoclast resorption activity without affecting osteogenesis on nanoparticulate mineralized collagen scaffolds. Science Advances, 5(6), eaaw4991.

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Quantitative Chondroitin Sulfate Assay

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Proteoglycan recovered by ethanol precipitation and dissolved in the keratanase buffer was assayed for chondroitin sulfate content by the dimethylmethylene blue (DMMB) colorimetric assay using chondroitin 6-sulfate (Sigma) as a standard (12.5 μg/ml to 200 μg/ml) [22 (link)]. 20 μl of the proteoglycan sample was combined with 180 μl of DMMB solution (46 mM DMMB (Aldrich), 40 mM glycine, and 40 mM NaCl, pH 3.0) in a 96-well plate, and the absorbance at 530 nm was monitored.

Mort J.S., Geng Y., Fisher W.D, & Roughley P.J. (2016). Aggrecan heterogeneity in articular cartilage from patients with osteoarthritis. BMC Musculoskeletal Disorders, 17, 89.

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