Studies on hyaluronidase, chondroitin sulphatase, proteinase and phospholipase secreted by Candida species

We studied the ability of different Candida species to produce,at the same time, hyaluronidase, chondroitin sulphatase, proteinase, and phospholipase to assess whether they could be related to Candida pathogenicity. Only C. albicans was able to produce the four enzymes tested (73%) and was highly virulent to mice. Strains, that lack the capacity to produce one or more of the enzymes assayed, seemed less virulent or avirulent, similarly to the spontaneous hyaluronidase, chondroitin sulphatase, phospholipase and proteinase-deficient C. albicans strain FCF 14, 1 which was non-pathogenic to mice. Among the other Candida species tested, none of them produced the four enzymes simultaneously, being less virulent in intravenously inoculated mice.

Hyaluronidase and chondroitin sulphatase production by different species of Candida

The production of hyaluronidase and chondroitin sulphatase by Candida albicans, Candida tropicalis, Candida parapsilosis, Candida guilliermondii and Candida krusei was investigated using a complex culture medium (Sabouraud glucose agar) and a chemically defined medium. Among the 63 C. albicans isolates tested, 61 (97.8%) were found to be hyaluronidase and chondroitin sulphatase producers; one isolate produced only chondroitin sulphatase and one other was unable to produce either enzyme. The second major hyaluronidase and chondroitin sulphatase producing species was C. tropicalis followed by C. guilliermondii, C. parapsilosis and C. krusei. Among the C. albicans isolates tested no relation between the source of isolation and the amount of hyaluronidase and chondroitin sulphatase produced was found.

A biomimetic extracellular matrix for cartilage tissue engineering centered on photocurable gelatin, hyaluronic acid and chondroitin sulfate

The development of hydrogels tailored for cartilage tissue engineering has been a research and clinical goal for over a decade. Directing cells towards a chondrogenic phenotype and promoting new matrix formation are significant challenges that must be overcome for the successful application of hydrogels in cartilage tissue therapies. Gelatin-methacrylamide (Gel-MA) hydrogels have shown promise for the repair of some tissues, but they have not been extensively investigated for cartilage tissue engineering. We encapsulated human chondrocytes in gel-MA based hydrogels, and show that with the incorporation of small quantities of photo-crosslinkable hyaluronic acid methacrylate (HA-MA), and to a lesser extent chondroitin sulfate methacrylate (CS-MA), chondrogenesis and mechanical properties can be enhanced. The addition of HA-MA to Gel-MA constructs resulted in more rounded cell morphologies, enhanced chondrogenesis as assessed by gene expression and immunofluorescence, and increased quantity and distribution of the newly synthesised ECM throughout the construct. Consequently, while the compressive moduli of control Gel-MA constructs increased by 26 kPa after 8 weeks culture, constructs with HA-MA and CS-MA increased by 96 kPa. The enhanced chondrogenic differentiation, distribution of ECM, and improved mechanical properties make these materials potential candidates for cartilage tissue engineering applications.

Mesoporous silica-chondroitin sulphate hybrid nanoparticles for targeted and bio-responsive drug delivery

This work proposes the fabrication of a novel targeted drug delivery system based on mesoporous silica-biopolymer hybrids that can release drugs in response to biological stimuli present in cancer cells. The proposed system utilizes mesoporous silica nanoparticles as a carrier to host the drug molecules. A bio-polymer cap is attached onto these particles which serves the multiple functions of drug retention, targeting and bio-responsive drug release. The biopolymer chondroitin sulphate used here is a glycosaminoglycan that can specifically bind to receptors over-expressed in cancer cells. This molecule also possesses the property of disintegrating upon exposure to enzymes over-expressed in cancer cells. When these particles interact with cancer cells, the chondroitin sulphate present on the surface recognizes and attaches onto the CD44 receptors facilitating the uptake of these particles. The phagocytised particles are then exposed to the degradative enzymes, such as hyaluronidase present inside the cancer cells, which degrade the cap resulting in drug release. By utilizing a cervical cancer cell line we have demonstrated the targetability and intracellular delivery of hydrophobic drugs encapsulated in these particles. It was observed that the system was capable of enhancing the anticancer activity of the hydrophobic drug curcumin. Overall, we believe that this system might prove to be a valuable candidate for targeted and bioresponsive drug delivery.

Gene cloning and functional analysis of glycosaminoglycan-degrading enzyme chondroitin AC lyase from Flavobacterium columnare G(4)

The chondroitin AC lyase gene, cslA, was cloned for the first time from the fish bacterial pathogen F. columnare G(4). From the first transcription initiation site, the cslA extends 2620 nucleotides to the end of the 3' region. The open reading frame of cslA transcript has 2286 nucleotides encoding 762 amino acids with a 16 residues long signal peptide at the N-terminus. The gene, cslA was then successfully expressed in Escherichia coli and recombinant chondroitin AC lyase, rChonAC was purified, with its lytic activity analyzed. Zymography analysis copolymerized with chondroitin sulphate revealed the lytic activity of rChonAC and also the crude native ChonAC isolated from periplamic space of cultured F. columnare G(4). The low level of lytic activity observed in crude native ChonAC may be due possibly to the low level of expression of this gene in the cultured condition. The expression and the role of this virulence factor is of interest for further research on the pathogenesis of F. columnare.

The Mobility of Chondroitin Sulfate in Articular and Artificial Cartilage Characterized by (13)C Magic-Angle Spinning NMR Spectroscopy

We have studied the molecular dynamics of one of the major macromolecules in articular cartilage, chondroitin sulfate. Applying (13)C high-resolution magic-angle spinning NMR techniques, the NMR signals of all rigid macromolecules in cartilage can be suppressed, allowing the exclusive detection of the highly mobile chondroitin sulfate. The technique is also used to detect the chondroitin sulfate in artificial tissue-engineered cartilage. The tissue-engineered material that is based on matrix producing chondrocytes cultured in a collagen gel should provide properties as close as possible to those of the natural cartilage. Nuclear relaxation times of the chondroitin sulfate were determined for both tissues. Although T(1) relaxation times are rather similar, the T(2) relaxation in tissue-engineered cartilage is significantly shorter. This suggests that the motions of chondroitin sulfate in data:rat and artificial cartilage different. The nuclear relaxation times of chondroitin sulfate in natural and tissue-engineered cartilage were modeled using a broad distribution function for the motional correlation times. Although the description of the microscopic molecular dynamics of the chondroitin sulfate in natural and artificial cartilage required the identical broad distribution functions for the correlation times of motion, significant differences in the correlation times of motion that are extracted from the model indicate that the artificial tissue does not fully meet the standards of the natural ideal. This could also be confirmed by macroscopic biomechanical elasticity measurements. Nevertheless, these results suggest that NMR is a useful tool for the investigation of the quality of artificially engineered tissue. (C) 2010 Wiley Periodicals, Inc. Biopolymers 93: 520-532, 2010.

Histopathological evaluation of treatment with chondroitin sulphate for osteoarthritis induced by continuous immobilization in rabbits

The aim of this study was to evaluate histologically the action of chondroitin sulphate in osteoarthritis experimentally induced by continuous immobilization. Fourteen young female Norfolk rabbits aged 2.5-3 months at the beginning of the experiment were divided into two equitable groups submitted to immobilization of the right knee for a period of 12 weeks. The treated group received 1.0 ml/animal/s.c. of 12% chondroitin sulphate, once a week for 12 weeks, and the untreated group did not receive any treatment. Two additional animals were not submitted to knee immobilization (sham group). Microscopical examination of knee preparations stained with haematoxylin-eosin and Masson trichrome showed lesions of both joints in treated and untreated groups, with no significant difference between the scores obtained for the right and left knees. Examination of preparations stained with picrosirius red showed collagen fibre alignment and misalignment in the right and left knees of the animals of all groups, but statistic analysis could not be performed. It was not possible to differentiate the proteoglycan concentration between limbs or groups (treated and untreated) by safrtanin O or toluidine blue staining. It was possible to conclude that the chondroitin sulphate was not able to reduce the histological changes induced by this osteoarthritis experimental model.

Diabetes induces stromal remodelling and increase in chondroitin sulphate proteoglycans of the rat ventral prostate

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Chondroitin sulfate proteoglycans are structural renewable constituents of the rabbit vitreous body

Purpose: To characterize the vitreous intrinsic proteoglycans, investigate their dynamics, and examine their role in the supramolecular organization of the vitreous. Methods: Vitreous from normal rabbits was collected and processed for observation with the transmission electron microscope after treatment with glycosidases. Also, rabbits were injected intravitreally with [S-35]-sodium sulfate and sacrificed at several time intervals after the injection. Proteoglycans (PGs) were assayed in the vitreous supernatant or in whole samples extracted with guanidine hydrochloride by polyacrylamide or agarose gel electrophoresis, followed respectively by fluorography or autoradiography, and ion-exchange chromatography and gel-filtration chromatography, combined with glycolytic treatment of the samples. The sulfated glycosaminoglycans (GAGs) were characterized by agarose gel electrophoresis after treating vitreous samples with protease and specific glycosidases. Results: the electron microscopic study revealed a network with hyaluronic acid ( HA) as thin threads coating and connecting collagen fibrils. The elimination of the HA coat showed chondroitin sulfate granules (8-25 nm) arranged at regular intervals on the fibril surface. The chondroitinase ABC digestion, besides removing the granules, also caused the formation of thicker bundles of the collagen fibrils. The PG and GAG analysis indicated that there are three renewable PGs in the vitreous ( e. g., one heparan-and two chondroitin-sulfate ones). Conclusions: At least one of the chondroitin sulfate PGs is involved in the interactions that occur in the vitreous structure, mainly by providing adequate spacing between the collagen fibrils, a condition that is probably required for the transparency of the vitreous.

INCREASED CALCIUM AFFINITY OF A FUCOSYLATED CHONDROITIN SULFATE FROM SEA-CUCUMBER

Calcium binding and charge distribution on a fucosylated chondroitin sulfate and a standard chondroitin 6-sulfate have been studied using a metallochromic indicator and conductimetric titrations. The fucosylated chondroitin sulfate has a similar to 5-fold greater affinity for calcium ions than the standard chondroitin 6-sulfate. Possibly, this increased affinity for calcium ions is due to the branches on the fucosylated chondroitin sulfate, since the calcium affinity of an unbranched, sulfated fucan is similar to that of the standard chondroitin 6-sulfate. More charged groups per disaccharide unit (and a shorter distance between these groups) also distinguish the fucosylated chondroitin sulfate from standard chondroitin 6-sulfate. Comparison between native and chemically modified (desulfated or carboxyl-reduced) polysaccharides suggests that the sulfate esters are responsible for the increased charge density of the fucosylated chondroitin sulfate and that the presence of the fucose branches does not alter the length of the repetitive units which compose the central core of chondroitin from sea cucumber. These results are consistent with the chemical studies of these two polysaccharides.

Biocompatibility of Ferrara intracorneal ring segment with and without chondroitin sulfate coating: Clinical and histopathological evaluation in rabbits

PURPOSE: To investigate and compare the biocompatibility of two types of Ferrara intracorneal ring segment: with and without chondroitin sulfate coating by clinical and histopathological evaluation. METHODS: A randomized experimental study was carried out on thirty right-eye corneas from 30 Norfolk albino rabbits allocated into two experimental groups: Group G1 - implanted with Ferrara intracorneal ring segment without coating (FICRS) and Group G2 - implanted with Ferrara intracorneal ring segment with chondroitin sulfate coating (FICRS-CS). Left eyes formed the control group. Clinical parameters analyzed were: presence of edema, vascularization, infection and ring extrusion one, 30, and 60 days after surgery. Histopathological parameters analyzed were: number of corneal epithelial layers over and adjacent to the ring, presence of spongiosis, hydropic degeneration, basement membrane thinning, inflammatory cells, neovascularization and pseudocapsule formation. RESULTS: At clinical examination 60 days after implant, edema, vascularization and extrusion were observed respectively in 20%, 26.7%, 6.7% of FICRS corneas and in 6.7%, 6.7%, and 0% of FICRS-CS corneas. Histopathological evaluation showed epithelial-layer reduction from 5 (5;6) to 3 (3;3) with FICRS and from 5 (5;5) to 4 (3;5) with FICRS-CS in the region over the ring. Epithelial spongiosis, hydropic degeneration, and basement membrane thinning were present in 69.2%, 53.8%, and 69.2% of FICRS and in 73.3%, 73.3%, and 46.7% with FICRS-CS, respectively. Vascularization was present in 38.5% of FICRS and 13.3% with FICRS-CS, inflammatory cells in 75% of FICRS and 33.3% with FICRS-CS, and pseudocapsule in 66.7% of FICRS and 93.3% with FICRS-CS. Giant cells occurred only in the FICRS-CS group (20%). CONCLUSION: Ferrara intracorneal rings coated with chondroitin sulfate (FICRS-CS) caused lower frequency of clinical and histopathological alterations than Ferrara intracorneal rings without the coating (FICRS), demonstrating higher biocompatibility of the FICRS-CS.