Distribution of small proteoglycans and glycosaminoglycans in humerus-related articular cartilage of chickens

Abstract

The expression of components present in the cartilaginous extracellular matrix is related to development, gender, and genotype, as well as to the biomechanical properties of each type of cartilage. In the present study, we analyzed small proteoglycans and glycosaminoglycans present in different cartilages of the chicken wing after extraction with guanidine hydrochloride or papain. Quantitative analysis of glycosaminoglycans showed a larger amount in humeral cartilage (around 200 mg/g tissue) than in articular cartilage of the radius and ulna, with 138 and 80 mg/g tissue, respectively. Non-collagenous proteins isolated were predominantly from cartilage in the proximal regions of the humerus and radius. D4 fractions obtained by ultracentrifugation were separated by DEAE-Sephacel and Octyl-Sepharose chromatography and analyzed by SDS-PAGE. Two bands of 57 and 70-90 kDa were observed for all samples treated with ß-mercaptoethanol. Immunoblotting of these proteins was positive for the small proteoglycans fibromodulin and decorin, respectively. Apparently, the 57-kDa protein is present in macromolecular complexes of 160 and 200 kDa. Chondroitin sulfate was detected in all regions. HPLC analysis of the products formed by chondroitinase AC and ABC digestion mainly revealed ß-D-glucuronic acid and N-acetyl ß-D-galactosamine residues. The 4-sulfation/6-sulfation ratio was close to 3, except for the proximal cartilage of the radius (2.5). These results suggest functional differences between the scapula-humerus, humerus-ulna, and humerus-radius joints of the chicken wing. This study contributes to the understanding of the physiology of cartilage and joints of birds under different types of mechanical stress.

Novel strategies for enhancing tissue integration in cartilage repair

“…It would therefore be expected that the relative amount of digestion would be highest in the superficial zone with progressively less towards the deeper zones of the tissue. However, it is also important to take into account the fact that the SLRPs decorin and biglycan, with their GAG chains, are found in relatively high concentrations within the superficial and transitional zones (Poole et al, 1996;Rodrigues et al, 2005). As expected, digestion within the superficial zone was more extensive than within other zones of the cartilage.…”

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

“…The mass ratios of CSC/DS conjugated to the scaffolds were evaluated indirectly by quantifying the hydrolyzed residues of CSC (DDi-6S) and DS (DDi-4S) in the waste PBS using SAX-HPLC [7,17] with some modifications. Briefly, the waste GAGs solutions for each reaction were collected, lyophilized and then dissolved in 0.9 ml Tris-acetate buffer (50 mM, pH 8.0).…”

“…Aside from the bulk cross-linking efficiency, we further evaluated whether CSC and DS were cross-linked to the chitosan scaffolds to the same degree. Since the disaccharide units of DS (DDi-4S) and CSC (DDi-6S) consist of 4-sulfated and 6-sulfated N-acetylgalactosamine, respectively, the D-disaccharide compositions of the chondroitin/DS chains in the native or engineered tissues can be characterized after enzyme digestion by fluorophore-assisted carbohydrate electrophoresis [22] or HPLC [7,17]. However, the cross-linking reactions during scaffold preparation could lead to structural changes of DS and CSC in the side chains, resulting in the loss of functional groups for enzyme digestion and incorrect quantification.…”

“…Because simultaneous quantification of 4 different GAGs in the 3D scaffolds is technically difficult and optimization of the scaffold formulation containing 4 GAGs requires an exceedingly large number of experiments, herein the number of GAG species was reduced to 2 to simplify the experimental design. Among the 4 GAGs we previously employed [6], little is known about the roles of DS in cartilage regeneration due to the low abundance of DS in the native articular cartilage ECM [7][8][9] and few studies have attempted to fabricate scaffolds with DS. Nonetheless, we hypothesized that DS, albeit scarce in the native ECM, plays a role in modulating chondrogenesis and conjugation of DS to scaffolds may promote chondrocyte differentiation and ECM production.…”