Release of O-sulfate groups under mild acid hydrolysis conditions used for estimation of N-sulfate content

The treatment of chondroitin sulfate isolated from cultured B16 mouse melanoma cells with 0.04 M HCl at 100°C for 90 min released up to 45% of O-sulfate residues as free inorganic sulfate. In addition to the release of inorganic sulfate, extensive degradation of this polysaccharide as well as of cartilage chondroitin sulfate, pig rib cartilage proteoglycan, heparin and hyaluronic acid was also evident under these conditions. The above hydrolysis conditions are used for characterizing ³⁵S-labeled heparan sulfates synthesized by cultured cells and to calculate ratio of N- and O-sulfates in these molecules. Our results suggest that caution in necessary in interpreting the results of mild acid hydrolysis of glycosaminoglycans.     

THE EFFECTS OF FLUORIDE FEEDING ON THE ORGANIC MATRIX OF BONES AND TEETH OF PIGS AS OBSERVED BY AUTORADIOGRAPHY AFTER IN VITRO UPTAKE OF CA45 AND S35

Demineralized sections of fluorinated bones and teeth have been studied by autoradiography following in vitro uptake of Ca⁴⁵ or S³⁵O₄. The portions of tissue which do not become mineralized (cartilage, prebone, predentine, and precementum) show an increased Ca⁴⁵ uptake apparently related to an increase in chondroitin sulfate content in fluorosis. The tissues from the fluoride-fed animals show an increase of in vitro uptake of sulfur in the tissues which become mineralized (bone, dentine, cementum).     

Environmental enhancement of in vitro chondrogenesis. 3. The influence of external potassium ions and chondrogenic differentiation

A simple manipulation, altering the potassium concentration of the nutrient medium, has a pronounced effect upon the in vitro chondrogenic differentiation of chick somites, as measured by chondroitin sulfate synthesis and cartilage formation. Medium containing K⁺ ions in the balanced salt solution at a concentration of 2.69 mM promotes chondrogenesis over the 48-hr period studied. By increasing the K⁺ concentration to 4.68 mM there is a striking enhancement of initial chondroitin sulfate synthesis during the first 24 hr only. If the somite explants in a high K⁺ environment are transferred after 24 hr to a lower K⁺ concentration, the chondrogenic stimulation (chondroitin sulfate synthesis) continues. These effects can be obtained by altering only one variable in the nutrient medium, the K⁺ concentration.     

AUTORADIOGRAPHIC STUDIES OF THE UTILIZATION OF S35-SULFATE BY THE CHICK EMBRYO

From studies of autoradiograms of various developmental stages of the chick embryo containing S³⁵ given us sulfate it was determined that as early as Stages 3+ and 4 there is a selective utilization or accumulation of sulfate by the various parts. The earliest accumulation site is the axial portion of the primitive streak and the floor of the groove. Later S³⁵ was found in the head process, Hensen's node, notochord, amniocardiac vesicle, wall of the omphalomesenteric vein, endocardium, subendocardial jelly, mesenchyme destined to become cartilage, basement membrane area of the gut, and a mucopolysaccharide layer formed on the free surface of the stomach. The early notochordal localizations of S³⁵ coincide with the region in which a thin ring of chondroitin sulfate is subsequently laid down. However, it is apparent that there is an intracellular accumulation of inorganic sulfate by the chondroitin-forming cells prior to the time they produce sufficient chondroitin sulfate to be demonstrable histochemically. It was interesting to note that the endocardium appears to concentrate sulfate that later apparently finds its way into the subendocardial jelly. The fact that those mesenchymal cells which later form chondroblasts begin to utilize sulfate selectively before histological differentiation is apparent was determined. In addition, the presence of sulfate-containing substances in the forming basement membrane of the gut would seem to indicate that sulfate is important in the histological differentiation of this membrane.     

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.     

Analysis of cartilage differentiation from skeletal muscle grown on bone matrix: II. Chondroitin sulfate synthesis and reaction to exogenous glycosaminoglycans

In the first paper in this series (Nathanson, M. A., and Hay, E. D. (1980). Develop. Biol. 78, 301–331), we described the ultrastructural alterations that take place when embryonic skeletal muscle is induced to form hyaline cartilage by demineralized bone matrix in vitro. In this paper, we analyze the pattern of appearance of chondroitin sulfates and dermatan sulfate in injured muscle in situ and in explants of muscle cultured either on bone matrix or on collagen gel. We also investigate the effects of exogenous glycosaminoglycans on the cultures to determine whether chondroitin sulfate (Ch-S) and hyaluronic acid (HA) can enhance or inhibit the biochemical differentiation of cartilage under these conditions. Our results indicate that during the first morphological phase, 1–3 days in vitro, there is an increased sulfate uptake, a shift in the relative abundance of Ch-S, and an increase in the ratio of chondroitin-4-sulfate (Ch-4-S) to chondroitin-6-sulfate (Ch-6-S); this change is correlated with the transformation of myoblasts to fibroblast-like cells in both types of cultures. A similar increase in the $\text{Ch-4-S}\text{Ch-6-S}$ ratio occurs in injured muscle in situ, suggesting that phase I is a regenerative response. Explants on bone matrix sustain Ch-4-S levels between 4 and 5 days (phase II) and show a large increase in Ch-4-S and sulfate incorporation when they form cartilage at 6–10 days (phase III). Explants on collagen gels regenerate muscle at 4–10 days with decreasing $\text{Ch-4-S}\text{Ch-6-S}$ ratios and decreasing sulfate incorporation. The data demonstrate that an environmental influence, such as trauma, is sufficient to alter the biosynthetic expression of skeletal muscle and that under appropriate conditions (such as the presence of bone matrix) this response may be augmented, leading to the synthesis of extracellular matrix components at ratios characteristic of cartilage. Exogenous Ch-S and HA did not significantly effect this overall pattern. These results are discussed in relation to the morphological observations presented in the preceding paper.     

Chondroitin sulfate proteoglycans from salmon nasal cartilage inhibit angiogenesis

Because cartilage lacks nerves, blood vessels, and lymphatic vessels, it is thought to contain factors that inhibit the growth and development of those tissues. Chondroitin sulfate proteoglycans (CSPGs) are a major extracellular component in cartilage. CSPGs contribute to joint flexibility and regulate extracellular signaling via their attached glycosaminoglycan, chondroitin sulfate (CS). CS and CSPG inhibit axonal regeneration; however, their role in blood vessel formation is largely unknown. To clarify the function of CSPG in blood vessel formation, we tested salmon nasal cartilage proteoglycan (PG), a member of the aggrecan family of CSPG, for endothelial capillary-like tube formation. Treatment with salmon PG inhibited endothelial cell adhesion and in vitro tube formation. The anti-angiogenic activity was derived from CS in the salmon PG but not the core protein. Salmon PG also reduced matrix metalloproteinase expression and inhibited angiogenesis in the chick chorioallantoic membrane. All of these data support an anti-angiogenic role for CSPG in cartilage.     

THE OCCURRENCE OF INTRACELLULAR CHONDROITIN SULFATE

Suspensions of chondrocytes were prepared by treatment with trypsin of the epiphyses of tibias and femurs of 13-day-old chick embryos. After washing to remove the matrix, such suspensions readily incorporate radioactive sulfate into both intracellular and extracellular chondroitin sulfate. Following disruption of the cells, the cell constituents were fractionated by centrifugation. Fractions obtained from cells incubated for 10 minutes showed a concentration of radioactivity in the material which sediments at 10,000 to 20,000 g. At this time the radioactivity of the extracellular chondroitin sulfate is low, but at 1 hour the radioactivity of the intracellular material is relatively unchanged, while that of the extracellular polysaccharide is markedly increased. Following incubation of the chondrocyte suspensions in a tissue culture medium, the intracellular chondroitin sulfate was isolated. This was compared with chondroitin sulfate isolated from the cartilage matrix. Chemical analysis and infrared spectroscopy indicated that both the intracellular and extracellular polysaccharides consist of a mixture of chondroitin sulfuric acids A and C. A portion of the chondroitin sulfate is not sulfated.     

Environmental enhancement of in vitro chondrogenesis. IV. Stimulation of somite chondrogenesis by exogenous chondromucoprotein

Proteoglycan complex extracted from embryonic cartilage (chondromucoprotein) with 4.0 M guanidinium chloride greatly stimulates in vitro somite chondrogenesis. In the presence of exogenous chondromucoprotein (CMP) which consists predominantly of proteochondroitin sulfate, there is a large increase in the amount of differentiating cartilage which can be detected visually in somite explants. There is a 2–3-fold increase in the amount of sulfated glycosaminoglycans (including chondroitin 4- and 6-sulfate) accumulated by somite explants supplied with exogenous CMP complex. These results are of potential significance, since during the period of interaction between the notochord or spinal cord and somitic mesoderm, the notochord and spinal cord synthesize and secrete proteoglycan.     

INTRACELLULAR SYNTHESIS OF CHONDROITIN SULFATE

In autoradiograms of slices of costal cartilage, incubated for 4 hours in a salt solution containing S³⁵-sulfate and then washed extensively and dehydrated, about 85 per cent of the radioactivity was assignable to the chondrocytes. From alkaline extracts of similarly prepared slices of cartilage, 64 to 83 per cent of the total sulfur-35 in the slices was isolated as chondroitin sulfate by chromatography on an anion-exchange resin. In view of the estimate that only about 15 per cent of the radioactivity was in the matrix, the isolation of 64 to 83 per cent of the total sulfur-35 as chondroitin sulfate is a strong argument that the chondrocytes are the loci in which chondroitin sulfate(s) is synthesized.     

Protamine increases the affinity of 3′-phosphoadenosine 5′-phosphosulfate toward a sulfotransferase from chicken embryo epiphyseal cartilage

A 3′-phosphoadenosine 5′-phosphosulfate (PAPS):chondroitin sulfate sulfotransferase from chicken embryo epiphyseal cartilage, which was partially purified, exhibited a molecular mass of 150 kDa. The enzymatic sulfation of totally desulfated chondroitin was activated up to 12-fold by protamine while the sulfation of partially sulfated chondroitin was activated only 3-fold. Protamine increased the affinity of the enzyme for PAPS about 4-fold when partially desulfated chondroitin was used as sulfate acceptor. The S_0.5 for the totally desulfated chondroitin was not affected by protamine, while high PAPS concentration slightly increased the affinity of the enzyme for the same sulfate acceptor. The possible role of these substances in the regulation of the sulfation of chondroitin sulfate is discussed.     

Separation and characterization of chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase from chick embryo cartilage

Two distinct sulfotransferases (chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase), which catalyzed transfer of sulfate to position 6 and position 4 of acetylgalactosamine residues of chondroitin, were extracted from epiphyseal cartilage of 14-day-old chick embryos and separated by gel chromatography on Sephacryl S-200 in the presence of 3 M guanidine-HCl. When the enzyme solutions containing 3 M guanidine-HCl were dialyzed against 0.02 M Tris-HCl, pH 7.2, containing 10% glycerol, chondroitin 4-sulfotransferase became almost insoluble, whereas chondroitin 6-sulfotransferase remained soluble. Endogenous acceptors for sulfate transfer were completely removed from both enzyme preparations. Addition of basic proteins and polyamines as well as Mn²⁺ to the incubation medium caused a stimulation of both sulfotransferases; the stimulation of chondroitin 6-sulfotransferase with these cations was higher than that of chondroitin 4-sulfotransferase. The K_m values for 3′-phosphoadenylyl sulfate of both enzymes were much smaller in the presence of protamine or spermine than in the presence of Mn²⁺. The two sulfotransferases differed in the requirement for sulfhydryl compounds; in the absence of sulfhydryl compounds, the activity of chondroitin 4-sulfotransferase was very low, whereas the activity of chondroitin 6-sulfotransferase was essentially unaffected. These observations indicate that at least two sulfotransferases are involved in the biosynthesis of chondroitin sulfate, and suggest that the production of the isomers of chondroitin sulfate in chondrocytes is affected by various factors such as the intracellular concentration of sulfhydryl compounds and basic substances.     

Impact of donor binding on polymerization catalyzed by KfoC by regulating the affinity of enzyme for acceptor

BackgroundCurrently marketed chondroitin sulfate isolated from animal sources and structurally quite heterogeneous. Synthesis of structurally defined chondroitin sulfate is highly desired. The capsular polysaccharide from Escherichia coli strain K4 is similar to chondroitin, and its biosynthesis requires a chondroitin polymerase (KfoC). The essential step toward de novo enzymatic synthesis of chondroitin sulfate, synthesis of chondroitin, could be achieved by employing this enzyme.MethodsStructurally defined acceptors and donor-sugars were prepared by chemoenzymatic approaches. In addition, surface plasmon resonance was employed to determine the binding affinities of individual substrates and donor–acceptor pairs for KfoC.ResultsKfoC has broad donor substrate specificity and acceptor promiscuity, making it an attractive tool enzyme for use in structurally-defined chimeric glycosaminoglycan oligosaccharide synthesis in vitro. In addition, the binding of donor substrate molecules regulated the affinity of KfoC for acceptors, then influenced the glycosyl transferase reaction catalyzed by this chondroitin polymerase.Conclusion and general significanceThese results assist in the development of enzymatic synthesis approaches toward chimeric glycosaminoglycan oligosaccharides and designing future strategies for directed evolution of KfoC in order to create mutants toward user-defined goals.     

Studies on glycosaminoglycans of regenerating rabbit ear cartilage

Cartilage regeneration in the adult rabbit ear was examined with respect to glycosaminoglycan (GAG) synthesis at various stages of the regeneration process. Increased hyaluronic acid and chondroitin sulfate synthesis was first seen 31 days after wounding, when a metachromatic cartilage matrix could be distinguished from blastemal cells. Analysis of cartilage and the overlying skin separately showed that 90% of the labeled chondroitin sulfate was found in the cartilage being regenerated. DEAE-cellulose chromatography of GAG preparations from 35-day regenerating cartilages showed hyaluronic acid and chondroitin sulfate peaks eluting in the same position as those isolated from normal cartilages. The identity of the hyaluronic acid and chondroitin sulfate peaks was confirmed by their susceptibility to Streptomyces hyaluronidase and chondroitinase ABC, respectively. Although the degree of sulfation in normal and regenerated cartilages was similar, the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate was increased in regenerated cartilages. GAG preparations from unlabeled cartilages were digested with chondroitinase ABC and the disaccharide digestive products were identified and quantitiated. Normal cartilage had a $\text{Δ}\text{Di-6S}\text{Δ}\text{Di-4S}$ ratio of 0.27; the same ratio for the regenerated cartilage was 1.58.     

A new potential spreading factor: Streptomyces koganeiensis hyaluronidase. A comparative study with bovine testes hyaluronidase and recombinant human hyaluronidase of the HA degradation in ECM

BackgroundRecombinant human hyaluronidase has been used in the interstitial matrix to promote the dispersion of therapeutics. The production and isolation of an extracellular hyaluronidase from Streptomyces koganeiensis (rHyal_Sk) has recently been described.MethodsThe specificity of rHyal_Sk has been assessed against heparan sulfate, chondroitin sulfates and sulfated HAs. The oligomers generated by HA degradation have been investigated by MALDI-TOF MS analysis. rHyal_Sk has been compared with BTH and PH20 in vitro, against cross-linked HA (ACP) and HA–aggrecan complex, and in vivo, by means of a diffusion assay in nude mice.ResultsDepolymerization of HA by rHyal_Sk gave tetra-, hexa- and octasaccharides in high yields. The reaction mechanism and the high HA specificity were demonstrated. The in vivo diffusion assay, supported by the in vitro tests, evidenced an initially enhanced enzymatic activity of rHyal_Sk compared to BTH and PH20.ConclusionsrHyal_Sk, compared to BTH and PH20, showed higher substrate specificity and no inhibition from GAGs sulfate, together with a superior performance for HA depolymerization in ECM. As better predictive tests for the in vivo activity of hyaluronidase we developed two assays based on the degradation of ACP or of the HA–aggrecan complex.General significancerHyal_Sk is a new potential spreading factor for intradermal drug administration. Hyaluronidases of distinct classes, that show equivalent activities in a common turbidimetric assay, could have different potencies and dose-efficacies in vivo which influences the therapeutic effect. The new proposed in vitro tests are designed to obtain a predictive characterization of the enzyme activity in vivo.     

Secretion of proteoglycans by chondrocytes. Influence of colchicine, cytochalasin B, and beta-D-xyloside.

Chondrocytes obtained from epiphyseal cartilage of fetal guinea pigs or ear cartilage of young rabbits were cultured in monolayer. The influence of colchicine, cytochalasin B, and p-nitrophenyl-β-d-xylopyranoside on secretion of proteoglycans was investigated. Radioactive sulfate was used as a precursor. As observed previously in other systems, β-d-xylosides initiated the synthesis of free chondroitin sulfate chains, competing with the endogenous proteoglycan core protein acceptor. The molecular weights of the chondroitin sulfate chains synthesized both on the xyloside and on the core-protein acceptor in maximally stimulated cells were similar and significantly lower than in proteoglycans synthesized in the absence of xyloside. The size of the chondroitin sulfate chains synthesized on the xyloside was inversely related to the concentration of this compound. This finding suggests that the chain length is dependent on the ratio between available acceptor and chain-lengthening enzymes or precursors. Cytochalasin B, a microfilament-modifying agent, inhibited proteoglycan synthesis, without any effect on secretion. Cells treated with cytochalasin B could be stimulated with β-d-xyloside to synthesize free chondroitin sulfate chains to the same relative degree as cells with intact microfilaments. Colchicine, an antimicrotubular agent, partially inhibited synthesis and secretion of proteoglycan. However, cells treated with colchicine could be stimulated with β-d-xyloside to synthesize and secrete free chondroitin sulfate chains to about the same relative degree as cells with intact microtubules. The data suggest that microtubules may have a facilitatory rather than an obligatory role in the secretion of proteoglycans and that at least part of the effect of colchicine is located at or after the site of glycosaminoglycan synthesis.     

Use of human induced pluripotent stem cells for cartilage regeneration in vitro and within chondral defect models of knee joint cartilage in vivo: a Preferred Reporting Items for Systematic Reviews and Meta-Analyses systematic literature review

Background aimsArticular cartilage has limited regenerative ability when damaged through trauma or disease. Failure to treat focal chondral lesions results in changes that inevitably progress to osteoarthritis. Osteoarthritis is a major contributor to disability globally, which results in significant medical costs and lost wages every year. Human induced pluripotent stem cells (hiPSCs) have long been considered a potential autologous therapeutic option for the treatment of focal chondral lesions. Although there are significant advantages to hiPSCs over other stem cell options, such as mesenchymal and embryonic stem cells, there are concerns regarding their ability to form bona fide cartilage and their tumorgenicity in vivo.MethodsThe authors carried out a systematic literature review on the use of hiPSCs to produce differentiated progeny capable of producing high-quality cartilage in vitro and regenerate cartilage in osteochondral defects in vivo in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Eight studies were included in the review that used hiPSCs or their derived progeny in xenogeneic transplants in animal models to regenerate cartilage in osteochondral defects of the knee joint. The in vitro-differentiated, hiPSC-derived and in vivo defect repair ability of the hiPSC-derived progeny transplants were assessed.ResultsMost studies reported the generation of high-quality cartilage-producing progeny that were able to successfully repair cartilage defects in vivo. No tumorigenicity was observed.ConclusionsThe authors conclude that hiPSCs offer a valuable source of cartilage-producing progeny that show promise as an effective cell-based therapy in treating focal chondral lesions.     

Synthesis of sulfated glycosaminoglycans by embryonic corneal epithelium

The primary corneal stroma is produced by the overlying epithelium. The endothelium appears between 4 and 5 days, fibroblasts at 6 days, and at 12 days the epithelium stratifies. We investigated the synthesis of glycosaminoglycan (GAG) by the epithelium during this developmentally significant period. The sulfated GAG synthesized by isolated 4–6-day-old corneal epithelia during the first 24 hr in vitro are entirely accountable for as chondroitin sulfates and heparan sulfates. Nearly 50% of the total sulfated GAG synthesized by epithelia on Millipore filters is lost to the medium, but only 30–40% is lost when frozen killed lens capsule or stroma is the substratum. Retention of isotope by the tissue is correlated with visible matrix polymerization. The relative amount of heparan sulfate synthesized by the developing epithelium 24 hr in vitro decreases from about 50% of the total sulfated GAG for 4-day-old epithelium to 12% for 12-day-old epithelium. A similar decrease in heparan sulfate synthesis occurs with time in culture. The relative amount of GAG identified as chondroitin sulfate and heparan sulfate is the same when ³H-glucosamine is used to label GAG as when ³⁵SO₄ is used. We conclude that the corneal epithelium produces only sulfated polysaccharides. Since hyaluronate is synthesized by whole 5-day-old corneas, it must be the product of the endothelium.     

The nature and origin of the glycosaminoglycans of the embryonic chick vitreous body

Glycosaminoglycans of the embryonic chicken vitreous were characterized and then were used as markers to establish which tissues synthesize the vitreous humor during development. The glycosaminoglycans are predominantly chondroitin sulfates by several criteria. They are resistant to streptomyces hyaluronidase, an enzyme which degrades only hyaluronate, and are digested by testicular hyaluronidase and chondroitinase AC, enzymes which degrade hyaluronate plus chondroitin 4- and 6-sulfates. On electrophoresis on cellulose acetate in 0.15 M phosphate buffer, pH 6.7, the vitreous glycosaminoglycans migrate slightly slower than authentic chondroitin sulfate, but, in 0.1 N HCl, they migrate very close to chondroitin sulfate standards. Finally, the disaccharides produced by digestion of these radioactively labeled glycosaminoglycans with chondroitinases AC and ABC were identified as Δdi-4S and Δdi-6S, as expected for chondroitin 4- and 6-sulfate. By using incorporation of radioactive precursors into chondroitin sulfates in vitro, we than determined which tissues synthesize the vitreous humor in the developing eye. Late in development, on Day 12–13, the isolated vitreous is very active in chondroitin sulfate synthesis, while the neural retina, the lens, and the pecten are less active and produce a high proportion of enzyme-resistant GAG. The eye tissues isolated from embryos labeled in ovo retain similar amounts and types of glycosaminoglycans, indicating that cells within the vitreous synthesize the vitreous humor glycosaminoglycans at this time. Earlier in development, from Days 6 to 8, the isolated vitreous incorporates very low levels of radioactivity into GAG, but the neural retina incorporates high levels of radioactivity into chondroitin sulfate. When the embryos are labeled in ovo and the same tissues are isolated following incorporation, the vitreous retains more radioactive chondroitin sulfate than does the neural retina. Thus, the vitreous humour glycosaminoglycan is initially synthesized by the neural retina and is secreted into the vitreous space.