Individual Glycosaminoglycans Research Articles

B-253 Using biomarkers in recurrent renal stone formers to detect silent stones

Abstract Background Glycosaminoglycans (GAGs) protect cells from binding of calcium oxalate (CaOx) crystals to prevent CaOx stone formation. Hyaluronan (HA) is a non-sulfated GAG with crystallization-promoting activity during renal stone formation. The excretion of urinary GAGs and HA were measured in stone-formers (SF), post-treated SF, and normal controls to reveal their relationship with renal stone disease. Methods Three groups were included - active SF, post-treated SF (SF after extracorporeal shock wave lithotripsy) and normal controls with 40 subjects in each group. Early morning urine was collected for each subject. The hexuronate content of the GAGs were measured by carbazole reaction and values for GAGs were standardized against creatinine. Individual GAGs extracts were then digested sequentially with Streptomyces hyaluronidase and chondroitinase ABC to yield the HA disaccharides for analysis by high performance liquid chromatography. Results Hexuronate content of GAGs were in the order of post-treated SF < SF << normal controls. SF had an enhanced urinary excretion of HA with no increase of urinary HA in post-treated SF group. However, both the SF and post-treated SF groups had increased proportion of HA (of total GAGs) compared to normal controls. Active-SF and post-treated SF groups had lower total GAGs content but increased proportion of HA than that of the normal controls indicating that urinary GAGs and HA are probably protective/risk factors, respectively, for renal stone disease. Conclusions The higher recurrence rate of renal stone disease may be due to the presence of sub-species of GAGs, HA, that becomes an accidental participant for renal stone formation. Concomitantly, other species of charged sulfated GAGs, which are protective to the cells and prevent crystal binding, are found to be in lesser content in active SF and post-treated SF. Clinicians can use these two markers, which are easily detectable (by ELISA) for prevention and monitoring of silent renal stone formation.

A novel graphene oxide-based fluorescence method for detection of urine glycosaminoglycans.

Glycosaminoglycans (GAGs) serve as a biomarker for mucopolysaccharidoses disease. In this study, a novel fluorometric method was developed to measure total GAGs in urine. Graphene oxide (GO) and rhodamine B (RhB), a cationic fluorescent dye, were employed in the development of the method. RhB attaches to the GO surface via electrostatic attraction, leading to the quenching of its fluorescence upon the establishment of the RhB-GO complex. However, the presence of GAGs prompts a resurgence of intense fluorescence. The linear range of the method is between 5.00 and 70.00mg/L. The total GAG levels of urine samples analyzed using the method agree with the results of the biochemistry analysis laboratory (65.85 and 79.18mg/L; 73.30± 1.76 and 72.21± 2.21). The method is simple, accurate, and sensitive and may be used for both first-step diagnosis of the mucopolysaccharidoses and detection of individual GAGs for studies of GAG-related research and other biological applications.

Plasma and Urine Levels of Glycosaminoglycans in Patients with Systemic Sclerosis and Their Relationship to Selected Interleukins and Marker of Early Kidney Injury.

Systemic sclerosis (SSc) is a chronic connective tissue disease characterized by immune system dysfunction, vasculopathy, and progressive fibrosis of the skin and internal organs, resulting from excessive accumulation of extracellular matrix (ECM) elements, including collagen and proteoglycans (PGs). An uncontrolled PG proliferation, caused by disturbances in their metabolism in tissues, is most likely reflected in the quantitative changes of their components, i.e., glycosaminoglycans (GAGs), in body fluids. Therefore, the aim of this study was to quantify the different types of GAGs in the blood and urine of systemic sclerosis patients. Chondroitin/dermatan sulfates (CS/DS) and heparan sulfates/heparin (HS/H) were quantified by hexuronic acid assay and electrophoretic fractionation, while hyaluronic acid (HA) and keratan sulfates were evaluated using ELISA tests. In turn, individual urinary GAGs were determined using the Blyscan™ Sulfated Glycosaminoglycan Assay Kit. Our results showed that the plasma concentrations of CS/DS, HS/H, HA, and KS in systemic sclerosis patients were significantly higher compared with those in healthy subjects. In the case of urine measurements, we have found that in SSc patients, CS/DC concentrations were significantly higher, while HA concentrations were significantly lower compared with the values observed in the urine of healthy subjects. Importantly, the found by us correlations between plasma keratan sulfate levels and both the duration of the disease and the severity of skin lesions, as expressed by the Rodnan scale, seems to suggest this GAG as a potential marker in assessing disease progression and activity. In addition, a level of urinary excretion of all types of GAGs due to their high positive correlation with uACR, may be a valuable complementary test in the diagnosis of early renal dysfunction in the course of SSc.

The Role of Extracellular Matrix in Human Neurodegenerative Diseases.

The dense neuropil of the central nervous system leaves only limited space for extracellular substances free. The advent of immunohistochemistry, soon followed by advanced diagnostic tools, enabled us to explore the biochemical heterogeneity and compartmentalization of the brain extracellular matrix in exploratory and clinical research alike. The composition of the extracellular matrix is critical to shape neuronal function; changes in its assembly trigger or reflect brain/spinal cord malfunction. In this study, we focus on extracellular matrix changes in neurodegenerative disorders. We summarize its phenotypic appearance and biochemical characteristics, as well as the major enzymes which regulate and remodel matrix establishment in disease. The specifically built basement membrane of the central nervous system, perineuronal nets and perisynaptic axonal coats can protect neurons from toxic agents, and biochemical analysis revealed how the individual glycosaminoglycan and proteoglycan components interact with these molecules. Depending on the site, type and progress of the disease, select matrix components can either proactively trigger the formation of disease-specific harmful products, or reactively accumulate, likely to reduce tissue breakdown and neuronal loss. We review the diagnostic use and the increasing importance of medical screening of extracellular matrix components, especially enzymes, which informs us about disease status and, better yet, allows us to forecast illness.

Seeking consistency for the role of urinary macromolecules and glycosaminoglycans in calcium oxalate crystallization processes pertaining to the risk of renal stone formation using a multi-faceted basic science approach

BackgroundThe roles of urinary macromolecules (UMMs) in calcium oxalate (CaOx) renal stone formation have not been consistently established. AimTo unravel these roles using a multi-faceted, multi-technique approach employing a wide range of experimental variables on a rotational basis in strategically chosen combinations. MethodsEndogenous urinary glycosaminoglycans (GAGs) were investigated in fractions obtained after ultrafiltration of pooled human urine (HU). Exogenous GAGs (chondroitin sulphate, CS, and hyaluronic acid, HA) were studied in artificial (AU) and in individual HUs. Experiments were conducted in a batch crystallizer and in a mixed suspension, mixed product removal flow system. Crystallization was quantitatively followed using Coulter multisizer and flow cytometer techniques. Crystal aggregation in the presence and absence of exogenous CS and HA was measured by Zeta potential and crystal sedimentation. ResultsTotal UMMs (endogenous) and individual GAGs (exogenous) consistently promoted CaOx crystallization and disaggregation. Evidence of UMM-UMM and UMM-solution synergistic effects was consistently observed for achieving modulation of crystallization processes. ConclusionsTotal UMMs, the main modulatory component of which is GAGs, are promoters of CaOx crystal nucleation and inhibitors of CaOx crystal aggregation. These results allow researchers to disregard alternative roles that have been advocated in such studies.

Sprint Exercise of Juvenile Animals Does Not Impact Cartilage Glycosaminoglycan or Synovial Fluid Neopeptide Collagenase Cleavage of Type I and II Collagen Content

Short sprints performed during growth can increase bone strength, mineral density, and cortical widths, but their impact on joint health is not fully understood. Some studies have found joints of young animals are damaged by forced sprints, while others found confinement hindered joint development. This study aimed to determine the impact of short sprints on synovial fluid neopeptide collagenase cleavage of type I and II collagen (C1,2C) and cartilage glycosaminoglycan (GAG) content. Calves were used as a model for young horses in this terminal study. Twenty-four Holstein bull calves were assigned to treatment groups of 1, 3, 5, or 0 days/week of sprinting (n = 6). A sprint consisted of an individual calf being walked from its stall and down a 71-m concrete aisle, at the end of which the calf was released, and encouraged to sprint towards the barn. Calves remained on treatment for 6 weeks and were euthanized on day 42. Immediately posteuthanasia synovial fluid was sampled from the middle carpal joint and cartilage discs sampled from 9 locations on the carpal bones. Analysis was performed in SAS 9.4 with fixed effects of treatment and bone. There were no differences in C1,2C or GAG concentrations between treatments (P = .73 and P = .96, respectively), but there was a difference between individual carpal bone GAG concentration (P = .03). Short sprints for 6 weeks during growth did not impact middle carpal joint C1,2C or carpal bone GAG content, and should be permitted given the benefits to bone health.

Roles of glycosaminoglycans as regulators of ligand/receptor complexes.

Glycosaminoglycans (GAGs) play a widespread role in embryonic development, as deletion of enzymes that contribute to GAG synthesis lead to deficiencies in cell migration and tissue modelling. Despite the biochemical and structural characterization of individual protein/GAG interactions, there is no concept available that links the molecular mechanisms of GAG/protein engagements to tissue development. Here, we focus on the role of GAG polymers in mediating interactions between cell surface receptors and their ligands. We categorize several switches that lead to ligand activation, inhibition, selection and addition, based on recent structural studies of select receptor/ligand complexes. Based on these principles, we propose that individual GAG polymers may affect several receptor pathways in parallel, orchestrating a cellular response to an environmental cue. We believe that it is worthwhile to study the role of GAGs as molecular switches, as this may lead to novel drug candidates to target processes such as angiogenesis, neuroregeneration and tumour metastasis.

Perspective on computational simulations of glycosaminoglycans.

Glycosaminoglycans (GAGs) represent a formidable frontier for chemists, biochemists, biologists, medicinal chemists and drug delivery specialists because of massive structural complexity. GAGs are arguably the most complex, natural linear biopolymers with theoretical diversity orders of magnitude higher than proteins and nucleic acids. Yet, this diversity remains generally untapped. Computational approaches offer major routes to understand GAG structure and dynamics so as to enable novel applications of these biopolymers. In fact, computational algorithms, softwares, online tools and techniques have reached a level of sophistication that help understand atomistic details of conformational variation and protein recognition of individual GAG sequences. This review describes current approaches and challenges in computational study of GAGs. It presents a history of major findings since the earliest mention of GAGs (the 1960s), the development of parameters and force fields specific for GAGs, and the application of these tools in understanding GAG structure-function relationship. This review also presents a section on how to perform simulation of GAGs, which is directed toward researchers interested in entering this promising field with potential to impact therapy.

Creation of disease-inspired biomaterial environments to mimic pathological events in early calcific aortic valve disease

An insufficient understanding of calcific aortic valve disease (CAVD) pathogenesis remains a major obstacle in developing treatment strategies for this disease. The aim of the present study was to create engineered environments that mimic the earliest known features of CAVD and apply this in vitro platform to decipher relationships relevant to early valve lesion pathobiology. Glycosaminoglycan (GAG) enrichment is a dominant hallmark of early CAVD, but culture of valvular interstitial cells (VICs) in biomaterial environments containing pathological amounts of hyaluronic acid (HA) or chondroitin sulfate (CS) did not directly increase indicators of disease progression such as VIC activation or inflammatory cytokine production. However, HA-enriched matrices increased production of vascular endothelial growth factor (VEGF), while matrices displaying pathological levels of CS were effective at retaining lipoproteins, whose deposition is also found in early CAVD. Retained oxidized low-density lipoprotein (oxLDL), in turn, stimulated myofibroblastic VIC differentiation and secretion of numerous inflammatory cytokines. OxLDL also increased VIC deposition of GAGs, thereby creating a positive feedback loop to further enrich GAG content and promote disease progression. Using this disease-inspired in vitro platform, we were able to model a complex, multistep pathological sequence, with our findings suggesting distinct roles for individual GAGs in outcomes related to valve lesion progression, as well as key differences in cell-lipoprotein interactions compared with atherosclerosis. We propose a pathogenesis cascade that may be relevant to understanding early CAVD and envision the extension of such models to investigate other tissue pathologies or test pharmacological treatments.

A case study: Glycosaminoglycan profiles of autologous chondrocyte implantation (ACI) tissue improve as the tissue matures

BackgroundAutologous chondrocyte implantation (ACI) has been used to treat cartilage defects in thousands of patients worldwide with good clinical effectiveness 10–20years after implantation. Information concerning the quality of the repair cartilage is still limited because biopsies are small and rare. Glycosaminoglycan structure influences physiological function and is likely to be important in the long term stability of the repair tissue. The aim of this study was to assess glycosaminoglycans in ACI tissue over a two year period. MethodsBiopsies were taken from one patient (25years old) at 12months and 20months post-ACI-treatment and from three normal cadavers (21, 22 and 25years old). Fluorophore-assisted carbohydrate electrophoresis (FACE) was used to quantitatively assess the individual glycosaminoglycans. ResultsAt 12months the ACI biopsy had 40% less hyaluronan than the age-matched cadaveric biopsies but by 20months the ACI biopsy had the same amount of hyaluronan as the controls. Both the 12 and 20month ACI biopsies had less chondroitin sulphate disaccharides and shorter chondroitin sulphate chains than the age-matched cadaveric biopsies. However, chondroitin sulphate chain length doubled as the ACI repair tissue matured at 12months (3913Da±464) and 20months (6923Da±711) and there was less keratan sulphate as compared to the controls. ConclusionsAlthough the glycosaminoglycan composition of the repair tissue is not identical to mature articular cartilage its quality continues to improve with time.

Furosemide modifies heart hypertrophy and glycosaminoglycan myocardium content in a rat model of neurogenic hypertension

Hypertension is a major risk factor for atherogenesis and heart hypertrophy, both of which are associated with specific morphological and functional changes of the myocardium. Glycosaminoglycans (GAGs) are complex molecules involved both in tissue morphology and function. In the present study, we investigated the effects of neurogenic hypertension and subsequent antihypertensive treatment with furosemide, on heart hypertrophy and the content of GAGs in the myocardium. Neurogenic hypertension was achieved in male Wistar rats by bilateral aortic denervation (bAD). At days 2, 7 and 15 after surgery, animals were sacrificed and the hearts were dissected away, weighted, and homogenized. Total GAGs were assessed by measuring the uronic acid content colorimetrically and individual GAGs were isolated and characterized by enzymatic treatment, with GAG-degrading enzymes, using electrophoresis on polyacrylamide gradient gels and cellulose acetate membranes. In bAD-animals blood pressure, blood pressure lability, heart rate and heart weight were significantly increased 15 days postoperatively. These effects were prevented by treatment with furosemide. Major GAGs identified in the heart were chondroitin sulphates, heparin (H), heparan sulphate (HS) and hyaluronic acid. The content of uronic and the relative content of H and HS in the heart in bAD animals significantly decreased from day 2 to day 15 postoperatively. Furosemide prevented the bAD induced decrease in GAG content. Considering that H and HS are potent inhibitors of cardiomyocyte hypertrophy, our results indicate that heart hypertrophy induced by neurogenic hypertension may be associated with decreases in the relative content of heparin and heparan sulphate in the heart.

AB0060 Cross-Talk between the Adipogenic and the Chondrogenic Programs Elicited by A Glycosaminoglycan Mixture

BackgroundMesenchymal stem cells (MSCs) are multipotent cells able to differentiate into chondrocytes, osteoblasts and adipocytes in a competitively balanced manner. MSCs play a role in the homeostasis of adipose tissue...

Synthesis and Assessment of Glycosaminoglycan Priming Activity of Cluster-xylosides for Potential Use as Proteoglycan Mimetics

One of the distinct structural features of many proteoglycans (PGs) is the presence of two or more glycosaminoglycan (GAG) side chains covalently linked to a core protein. Previous studies have shown that the synergistic biological activity of multiple GAG chains, as found in the majority of PGs, cannot be accomplished by the sum of the activities of individual GAG chains. To delineate the biological significance of GAG valency, a number of cluster-xylosides carrying two, three, or four xylose residues on the same scaffold were synthesized using click chemistry. Assessment of cluster-xylosides for their GAG chain priming activity in a cellular system revealed that these cluster-xylosides prime multiple GAG chains per scaffold. Multivalent GAG chains, produced by cluster-xylosides, can better mimic PGs as they carry two or more GAG chains attached to a core protein and therefore can be used as molecular probes to examine the biological significance of GAG multivalency in model organisms.

The Structural Stability of the Endothelial Glycocalyx after Enzymatic Removal of Glycosaminoglycans

RationaleIt is widely believed that glycosaminoglycans (GAGs) and bound plasma proteins form an interconnected gel-like structure on the surface of endothelial cells (the endothelial glycocalyx layer–EGL) that is stabilized by the interaction of its components. However, the structural organization of GAGs and proteins and the contribution of individual components to the stability of the EGL are largely unknown.ObjectiveTo evaluate the hypothesis that the interconnected gel-like glycocalyx would collapse when individual GAG components were almost completely removed by a specific enzyme.Methods and ResultsUsing confocal microscopy, we observed that the coverage and thickness of heparan sulfate (HS), chondroitin sulfate (CS), hyaluronic acid (HA), and adsorbed albumin were similar, and that the thicknesses of individual GAGs were spatially nonuniform. The individual GAGs were degraded by specific enzymes in a dose-dependent manner, and decreased much more in coverage than in thickness. Removal of HS or HA did not result in cleavage or collapse of any of the remaining components. Simultaneous removal of CS and HA by chondroitinase did not affect HS, but did reduce adsorbed albumin, although the effect was not large.ConclusionAll GAGs and adsorbed proteins are well inter-mixed within the structure of the EGL, but the GAG components do not interact with one another. The GAG components do provide binding sites for albumin. Our results provide a new view of the organization of the endothelial glycocalyx layer and provide the first demonstration of the interaction between individual GAG components.

Urinary glycosaminoglycans and glycoproteins in a calcium oxalate crystallization system

This study measures the effects of total urinary glycosaminoglycans (GAGs), glycoproteins (GPs) and individual GAGs on the nucleation rates (Bo), growth rates (G) and suspension densities (Mт) of calcium oxalate (CaOx) crystallization by the mixed suspension mixed product removal (MSMPR) system. Total urinary GAGs, glycoproteins and individual GAGs including heparan sulfate (HS), chondroitin sulfate (CS) and Hyaluronic acid (HA) were added into the artificial urine (AU) and then introduced into the MSMPR test chamber and the crystal sizes and numbers were analyzed by a particle counter. The effects of added GAGs and GPs on CaOx crystallization were reflected by the changes on the crystallization indexes including the Bo, G and Mт of CaOx that were calculated based on the crystal size and numbers. Total urinary GAGs showed no statistical significance on CaOx crystallization. However, individual GAGs such as HA, CS and HS enhanced Bo and suppressed the G when measured individually. CS and HS enhanced the Mт while HA shown no significant change in the Mт of CaOx. Total urinary GPs showed an increase in the G and Mт of crystals. Although total urinary GAGs showed no statistically significant effect on CaOx crystallization, individual GAGs (CS, HS) promoted the CaOx crystallization by increasing the suspension density of smaller crystals, indicative of reduced risk of stones while HA showed no significance in the M T of CaOx formed. Urinary GPs indicated increased sizes and M T suggesting larger crystals and/or aggregates.

Single-Molecule Force Spectroscopy of Cartilage Aggrecan Self-Adhesion

We investigated self-adhesion between highly negatively charged aggrecan macromolecules extracted from bovine cartilage extracellular matrix by performing atomic force microscopy (AFM) imaging and single-molecule force spectroscopy (SMFS) in saline solutions. By controlling the density of aggrecan molecules on both the gold substrate and the gold-coated tip surface at submonolayer densities, we were able to detect and quantify the Ca 2+-dependent homodimeric interaction between individual aggrecan molecules at the single-molecule level. We found a typical nonlinear sawtooth profile in the AFM force-versus-distance curves with a molecular persistence length of l p = 0.31 ± 0.04 nm. This is attributed to the stepwise dissociation of individual glycosaminoglycan (GAG) side chains in aggrecans, which is very similar to the known force fingerprints of other cell adhesion proteoglycan systems. After studying the GAG-GAG dissociation in a dynamic, loading-rate-dependent manner (dynamic SMFS) and analyzing the data according to the stochastic Bell-Evans model for a thermally activated decay of a metastable state under an external force, we estimated for the single glycan interaction a mean lifetime of τ = 7.9 ± 4.9 s and a reaction bond length of x β = 0.31 ± 0.08 nm. Whereas the x β -value compares well with values from other cell adhesion carbohydrate recognition motifs in evolutionary distant marine sponge proteoglycans, the rather short GAG interaction lifetime reflects high intermolecular dynamics within aggrecan complexes, which may be relevant for the viscoelastic properties of cartilage tissue.

Reversible secretion of glycosaminoglycans and proteoglycans by cyclically stretched valvular cells in 3D culture.

Mitral valve leaflets and chordae have been shown to contain different amounts and proportions of glycosaminoglycans (GAGs) and proteoglycans (PGs) corresponding to in vivo normal or diseased cyclic strain patterns. To understand the effect of cyclic strains on GAG/PG synthesis by valvular interstitial cells (VICs) isolated from valve leaflet and chordae separately, porcine VICs were seeded within collagen gels and alternately stretched or relaxed for 24 h periods for one week in a custom-designed tissue engineering bioreactor. We found cyclic-stretch-induced upregulation of total GAGs and of individual GAG classes secreted into the culture medium. Leaflet cells showed a delayed response to stretching compared to chordal cells, but altered the proportions of various GAG classes they secreted during the culture duration. Decorin and biglycan PGs were slightly responsive to stretch. We demonstrated that mechanical stretch and relaxation conditions reversibly regulate GAG and PG production in a novel 3D model of valve tissues. This is the first study using cyclic strains to modulate GAG/PG synthesis by valve cells and our results may have implications for the remodeling of the mitral valve as well as other tissues.

Collagen pretzels revealed by electron microscopy

Collagen XV is a million-dalton protein with a structural role in skeletal muscle and capillaries. As with all collagens, studies of its function are hindered by the absence of good structural data: collagens are triple-helical, non-crystallizable, multidomain proteins with extensive post-translational modification that are refractory to analysis by high-resolution structural techniques. For collagen XV, this situation is compounded by the fact that it is also a proteoglycan. In this issue of the Biochemical Journal, Myers and her colleagues use rotary shadowing electron microscopy to obtain images of purified collagen XV molecules that are sufficiently detailed to show the three-lobed structure of the N-terminus and individual glycosaminoglycan side chains. Individual molecules appear as knotted strands resembling a pretzel (a pastry snack folded in a unique figure-of-eight), which contrasts with our conventional image of collagen molecules as semi-rigid rods. Importantly, collagen XV multimerizes into cruciform structures in which simpler forms have two to four molecules per complex. Immunoelectron microscopy revealed knotted collagen XV complexes bridging collagen fibrils adjacent to basement membrane. These accomplishments are made all the more impressive by the fact that collagen XV was purified from human umbilical cord, in which the protein is represented at only (1-2)x10(-4)% of dry weight!

In the Presence of Phospholipids, Glycosaminoglycans Potentiate Factor Xa-Mediated Protein C Activation by Modulating Factor Xa Activity

Although the thrombin/thrombomodulin complex is considered the physiological activator of protein C, factor Xa (f.Xa) can also activate protein C in a reaction that is potentiated by glycosaminoglycans. To explore this phenomenon, we first examined the effect of glycosaminoglycans of varying degrees of sulfation on the kinetics of protein C activation by f.Xa in the presence of Ca2+ and phosphatidylcholine-phosphatidylserine vesicles (PCPS). Heparin increased the rate of protein C activation by f.Xa by 4-fold. In contrast, N-desulfated heparin had no effect on activation, whereas dextran sulfate, which is more sulfated than heparin, increased catalytic efficiency 21-fold. These data suggest that the capacity of glycosaminoglycans to catalyze protein C activation by f.Xa depends on their degree of sulfation. The affinities of individual glycosaminoglycans for protein C and f.Xa were measured in the absence or presence of PCPS by monitoring changes in extrinsic fluorescence when fluorescein-labeled f.Xa or protein C was titrated with the various glycosaminoglycans. Heparin binds protein C with low affinity in the absence or presence of PCPS. In contrast, the affinity of heparin for f.Xa is 86-fold higher in the presence of PCPS compared to that in the absence of PCPS. Similar results were obtained using surface plasmon resonance. These findings suggest that a high affinity glycosaminoglycan binding site is exposed when f.Xa binds to PCPS. The observation that heparin promotes f.Xa-mediated activation of prethrombin 1 only in the presence of phospholipid suggests that glycosaminoglycan binding modulates the active site of f.Xa. This study reveals that when f.Xa interacts with anionic phospholipids, glycosaminoglycans bind f.Xa more tightly, allosterically modulate its active site, and enhance its capacity to activate protein C.

Composite chondroitin-6-sulfate/dermatan sulfate/chitosan scaffolds for cartilage tissue engineering

Conjugating a single glycosaminoglycan (GAG) species such as chondroitin-6-sulfate (CSC) to chitosan is beneficial to chondrocyte culture and extracellular matrix (ECM) production, but whether fabrication of 3D chitosan scaffolds with additional minor GAG species such as dermatan sulfate (DS) further improves the ECM production is unknown. In this study, Response Surface Methodology (RSM) was employed to design CSC/DS/chitosan scaffolds of various formulations for cartilage engineering and to investigate the roles of individual GAG species in cartilage formation. The CSC/DS formulation affected neither the physical properties of scaffolds nor cell adhesion, but influenced cell morphology, GAGs and collagen production and chondrocytic gene expression. The linear effects elucidated by RSM analysis suggested that within the level range higher CSC levels favored GAGs and collagen production, whereas lower DS levels were desired for these responses. Nonetheless, the quadratic effects of DS and two-way interactions between CSC and DS also contributed to the GAGs and collagen production. Accordingly, the optimal formulation, as predicted by RSM and validated by experiments, comprised 2.8 mg CSC and 0.01 mg DS per scaffold. This study confirmed the importance of DS in cartilage tissue engineering and implicated the feasibility of rational CSC/DS/chitosan scaffold design with the aid of RSM.