Chondrocyte Homeostasis and Differentiation: Transcriptional Control and Signaling in Healthy and Osteoarthritic Conditions.

Chondrocytes are the main cell type in articular cartilage. They are embedded in an avascular, abundant, and specialized extracellular matrix (ECM). Chondrocytes are responsible for the synthesis and turnover of the ECM, in which the major macromolecular components are collagen, proteoglycans, and non-collagen proteins. The crosstalk between chondrocytes and the ECM plays several relevant roles in the regulation of cell phenotype. Chondrocytes live in an avascular environment in healthy cartilage with a low oxygen supply. Although chondrocytes are adapted to anaerobic conditions, many of their metabolic functions are oxygen-dependent, and most cartilage oxygen is supplied by the synovial fluid. This review focuses on the transcription control and signaling responsible for chondrocyte differentiation, homeostasis, senescence, and cell death and the changes that occur in osteoarthritis. The effects of chondroitin sulfate and other molecules as anti-inflammatory agents are also approached and analyzed.

Chondrogenic potential of mesenchymal stem cells from horses using a magnetic 3D cell culture system.

BACKGROUND: Mesenchymal stem cells (MSCs) represent a promising therapy for the treatment of equine joint diseases, studied due to their possible immunomodulatory characteristics and regenerative capacity. However, the source of most suitable MSCs for producing cartilage for regenerative processes in conjunction with biomaterials for an enhanced function is yet to be established. AIM: To compare the chondrogenicity of MSCs derived from synovial fluid, bone marrow, and adipose tissue of horses, using the aggrecan synthesis. METHODS: MSCs from ten horses were cultured, phenotypic characterization was done with antibodies CD90, CD44 and CD34 and were differentiated into chondrocytes. The 3D cell culture system in which biocompatible nanoparticles consisting of gold, iron oxide, and poly-L-lysine were added to the cells, and they were forced by magnets to form one microspheroid. The microspheroids were exposed to a commercial culture medium for 4 d, 7 d, 14 d, and 21 d. Proteoglycan extraction was performed, and aggrecan was quantified by enzyme-linked immunosorbent assay. Keratan sulfate and aggrecan in the microspheroids were identified and localized by immunofluorescence. RESULTS: All cultured cells showed fibroblast-like appearance, the ability to adhere to the plastic surface, and were positive for CD44 and CD90, thus confirming the characteristics and morphology of MSCs. The soluble protein concentrations were higher in the microspheroids derived from adipose tissue. The aggrecan concentration and the ratio of aggrecan to soluble proteins were higher in microspheroids derived from synovial fluid than in those derived from bone marrow, thereby showing chondrogenic superiority. Microspheroids from all sources expressed aggrecan and keratan sulfate when observed using confocal immunofluorescence microscopy. All sources of MSCs can synthesize aggrecan, however, MSCs from synovial fluid and adipose tissue have demonstrated better biocompatibility in a 3D environment, thus suggesting chondrogenic superiority. CONCLUSION: All sources of MSCs produce hyaline cartilage; however, the use of synovial liquid or adipose tissue should be recommended when it is intended for use with biomaterials or scaffolds.

Hyaluronic acid has chondroprotective and joint-preserving effects on LPS-induced synovitis in horses.

The intra-articular use of hyaluronic acid (HA) for the treatment of synovitis and osteoarthritis is still controversial. As a consequence, corticosteroids remain the most frequently employed therapeutic agents, despite their potential systemic and local deleterious effects. This study examined the anti-inflammatory, antioxidant, and chondroprotective activities of low and high molecular weight hyaluronic acid (LMW-HA and HMW-HA) on lipopolysaccharide (LPS)-induced synovitis in horses compared to triamcinolone acetonide (TA). LPS was injected in the metacarpophalangeal joints, which were treated intra-articularly with either TA (as control) or LMW-HA or HMW-HA. Joint clinical evaluation and synovial fluid (SF) analysis were performed at 0, 8, 24, and 48 h. The white blood cell counts (WBC), prostaglandin E2 (PGE2), interleukin (IL)-1, IL-6, IL-10, tumor necrosis factor-α, chondroitin sulfate (CS) and HA concentrations, oxidative burst, and HA molecular weights were measured. TA reduced the lameness, swelling, and PGE2 release but increased the SF CS concentrations enormously at 24h and 48h, and decreased the SF HA modal molecular weight. These results indicate the breakdown of articular cartilage aggrecan and SF HA. In contrast, LMW-HA and HMW-HA were less effective in reducing the inflammation symptoms, but preserved the joints because only a modest increase in CS occurred at 24 h, decreasing at 48 h, and the SF HA was maintained. The HA-treatment also had anti-inflammatory actions, and LMW-HA was the most effective in reducing the release of cytokine. In summary, the HA treatment inhibited efficiently the digestion of cartilage proteoglycans and SF HA breakdown.

Does Double Centrifugation Lead to Premature Platelet Aggregation and Decreased TGF-ß1 Concentrations in Equine Platelet-Rich Plasma?

Blood-derived autologous products are frequently used in both human and equine medicine to treat musculoskeletal disorders. These products, especially the platelet-rich plasma (PRP), may contain high concentrations of growth factors (GFs), and thus improve healing in several tissues. Nevertheless, the procedures for preparation of PRP are currently non-standardized. Several protocols, which are based on distinct centrifugation patterns (rotation speed and time), result in PRPs with different characteristics, concerning platelet and GFs concentrations, as well as platelet activation. The aim of the present study was to compare two different protocols for PRP preparation: protocol (A) that is based on a single-centrifugation step; protocol (B), which included two sequential centrifugation steps (double-centrifugation). The results here reported show that the double-centrifugation protocol resulted in higher platelet concentration, while leukocytes were not concentrated by this procedure. Although platelet activation and aggregation were increased in this protocol in comparison to the single-centrifugation one, the TGF-ß1 concentration was also higher. Pearson's correlation coefficients gave a significant, positive correlation between the platelet counts and TGF-ß1 concentration. In conclusion, although the double-centrifugation protocol caused premature platelet aggregation, it seems to be an effective method for preparation of PRP with high platelet and TGF-ß1 concentrations.

Do chondroitin sulfates with different structures have different activities on chondrocytes and macrophages?

The aim of the present study was to investigate the activities of natural chondroitin sulfates (CS) with different structures on cultured chondrocytes and macrophages. CS were isolated from cartilages of bovine trachea (BT), porcine trachea (PT), chicken sternum (Ch) and skate (Sk). The preparations were 90-98% pure, with ∼1% proteins, nucleic acids and keratan sulfate contaminants. Structural analysis of these CS and of commercial chondroitin 4- and 6-sulfate (C4S, C6S) have shown that most of their disaccharides are monosulfated, with varying proportions of 4- and 6-sulfation, and 2-7% non-sulfated disaccharides. Sk-CS and C6S contained detectable amounts of disulfated disaccharides. All the CS were polydisperse, with modal molecular weights of 26-135kDa. These CS had anti-inflammatory activities on both chondrocytes and macrophages, but with different efficiencies. On horse and human chondrocytes, they reduced the IL-1ß-induced liberation of NO and PGE2, and on RAW 264.7 immortalized macrophage-like cell line, C4S, C6S, Ch and Sk-CS decreased the LPS-induced liberation of TNF-α, but did not affect IL-6. In contrast, on bone marrow derived macrophages, C4S, C6S, BT and PT-CS reduced the LPS-induced liberation of TNF-α, IL-6, IL-1ß and NO, indicating that the RAW response to CS was different from that of primary macrophages.

Impact of high glucose and AGEs on cultured kidney-derived cells. Effects on cell viability, lysosomal enzymes and effectors of cell signaling pathways.

We have previously reported decreased expression and activities of lysosomal cathepsins B and L in diabetic kidney. Relevant morphological changes were observed in proximal tubules, suggesting that these cells are implicated in the early stages of the disease. The aim of the present study was to investigate the mechanisms that lead to these changes. The effects of high glucose (HG) and advanced glycation end products (AGEs) on cell viability, lysosomal enzymes and other effectors of cell signaling of cultured kidney cells were studied. HG increased viable mesangial cells (ihMC) in 48 h, while epithelial tubular cells were not affected (LLC-PK1 and MDCK). In contrast, the number of viable cells was markedly decreased, for all cell lines, by AGE-BSA. Concerning lysosomal enzymes, the main cysteine-protease expressed by these cells was cathepsin B, and its concentration was much higher in epithelial than in mesangial cells. Exposure to HG had no effect on the cathepsin B activity, but AGE-BSA caused a marked decrease in LLC-PK1, and increased the enzyme activities in the other cell lines. The levels of nitric oxide (NO) was increased by AGE-BSA in all cell lines, suggesting oxidative stress, and Western blotting has shown that, among the investigated proteins, cathepsin B, mTOR and transcription factor EB (TFEB) were the most significantly affected by exposure to AGE-BSA. As mTOR induces anabolism and inhibits autophagy, and TFEB is a master transcription factor for lysosomal enzymes, it is possible that this pathway plays a role in the inhibition of lysosomal enzymes in proximal tubule cells.

Pharmaceutical grade chondroitin sulfate: Structural analysis and identification of contaminants in different commercial preparations.

The aim of the present study was to characterize 16 pharmaceutical grade chondroitin sulfate (CS) samples, concerning the structure and presence of contaminants, in comparison to USP and analytical grade CS. Agarose gel electrophoresis has shown that only 5 samples were >90% CS, while 11 contained less than 15% CS. FACE (fluorophore-assisted carbohydrate electrophoresis) revealed that maltodextrin was the main contaminant in nine of them, and lactose in two. Raman spectroscopy corroborated these results. Concerning the structure of the CS present in the five CS-rich samples, the ratios 4-sulfated:6-sulfated disaccharides varied from 0.9 to 1.7, and their modal molecular weight was 20-29 kDa. Also, they were all contaminated by small amounts of keratan sulfate (<1%). In conclusion, our findings indicate that the composition of CS preparations not always corresponds to the manufacturers' descriptions, and indicate that further characterization should be required for the registry and license of pharmaceutical grade CS.

Short- and long-term effects of platelet-rich plasma upon healthy equine joints: Clinical and laboratory aspects.

This study aimed to verify whether transient inflammatory reactions incited by the administration of intra-articular platelet-rich plasma (PRP) affected joint components through short- and long-term in vivo evaluation of inflammatory biomarkers and extracellular matrix degradation products in synovial fluid. The effects of PRP were analyzed in a short phase protocol (SPP) and in a prolonged phase protocol (PPP), using saline-injected joints as controls. In the SPP, higher white blood cell counts and prostaglandin E2 and total protein concentrations were observed in the synovial fluid of PRP-treated joints (P < 0.05). There were no differences between the interleukin-1ß, interleukin-1 receptor antagonist protein, tumor necrosis factor-α, chondroitin sulfate, or hyaluronic acid concentrations between PRP and saline injected joints. In the PPP, there were no differences in evaluated parameters between groups. PRP injection elicits a mild and self-limiting inflammatory response shortly after administration, without long-term deleterious effects on joint homeostasis.

Effets à court et à long terme de le plasma riche en plaquettes sur les articulations sains chez les équines : aspects cliniques et laboratoiren. Cette étude a pour but de vérifier si les réactions inflammatoires passagères induites par l'administration intra articulaire de Plasma Enrichi en Plaquettes (PRP) affectent les composants articulaires. Les bio-marqueurs de l'inflammation et les produits de dégradation de la matrice extracellulaire ont été évalués in vivo, dans le liquide synovial, à court et long terme. Les effets du PRP ont été analysés lors d'un protocole court terme et lors d'un protocole long terme et les articulations contrôles ont été injectées avec du liquide physiologique. Le protocole court terme a révélé des comptages de globules blancs et de prostaglandine E2, ainsi que des concentrations en protéines totales plus élevés dans le liquide synovial des articulations traitées au PRP (P < 0,05). Cependant, aucune différence de concentration en interleukine-1 bêta, en protéine antagoniste des récepteurs à l'interleukine-1, en facteur de nécrose tumorale alpha, en chondroïtine sulfate et en acide hyaluronique n'a été notée entre les articulations injectées au PRP et les articulations contrôles. Le protocole long terme n'a démontré aucune différence des paramètres évalués entre les deux groupes. L'injection de PRP provoque une réponse inflammatoire légère et auto-limitante rapidement après l'administration, sans effet délétère sur l'homéostasie de l'articulation à long terme.(Traduit par les auteurs).

Autologous processed plasma: cytokine profile and effects upon injection into healthy equine joints.

This experimental controlled study was performed to evaluate the composition of autologous processed plasma (APP), and the effects of APP intra-articular injection into healthy equine metacarpophalangeal joints. The effects on joints were analysed with a short-phase protocol and a prolonged-phase protocol using saline-injected joints as controls. For the short protocol, horses received one intra-articular APP injection. Synovial fluid samples were collected prior to the injection and 3, 6, 24, 48, and 16 h after treatment. For the prolonged protocol, the joints received three weekly injections of APP, and samples were collected at 0, 7, 14, 21, and 28 days before APP administration. IL1-ra level was found to be increased in APP compared to plasma. Upon intra-articular administration of APP, transient (up to 24 h) increases in white blood cell (WBC) counts along with elevated protein and prostaglandin E2 (PGE2) concentrations were observed in the treated joints. Over the 28-day observation period, APP did not elicit changes relative to baseline levels, but WBC counts, PGE2 and chondroitin sulphate concentrations were lower than those found in the control. In conclusion, APP intra-articular injection induced a mild and transitory inflammatory response but no inflammation reaction was observed over a longer period of treatment and observation.

Evaluation of chitosan-GP hydrogel biocompatibility in osteochondral defects: an experimental approach.

BACKGROUND: Articular cartilage, because of its avascular nature, has little capacity for spontaneous healing, and tissue engineering approaches, employing different biomaterials and cells, are under development. Among the investigated biomaterials are the chitosan-based hydrogels. Although thoroughly studied in other mammalian species, studies are scarce in equines. So, the aim of the present study was to investigate the biocompatibility of chitosan-GP in horse joints submitted to high mechanical loads. RESULTS: An osteochondral defect was created by arthroscopy in the medial surface of lateral trochlea of talus of left or right leg, randomly selected, from six healthy geldings. The defect was filled up with chitosan-GP. The contralateral joint received an identical defect with no implant. The chondral fragment removed to produce the defect was collected, processed and used as the "Initial" sample (normal cartilage) for histology, immunohistochemistry, and metabolic labelling of PGs. After 180 days, the repair tissues were collected, and also analyzed. At the end of the experiment (180 days after lesion), the total number of cells per field in repair tissues was equal to control, and macrophages and polymorphonuclear cells were not detected, suggesting that no significant inflammation was present. These cells were able to synthesize type II collagen and proteoglycans (PGs). Nevertheless, the cell population in these tissues, both in presence of chitosan-GP and in untreated controls, were heterogeneous, with a lower proportion of type II collagen-positives cells and some with a fibroblastic aspect. Moreover, the PGs synthesized in repair tissues formed in presence or absence of chitosan-GP were similar to those of normal cartilage. However, the chitosan-GP treated tissue had an disorganized appearance, and blood vessels were present. CONCLUSIONS: Implanted chitosan-GP did not evoke an important inflammatory reaction, and permitted cell growth. These cells were able to synthesize type II collagen and PGs similar to those synthesized in normal cartilage and in healing tissue without implant, indicating its chondrocyte nature.

Relevance of synovial fluid chondroitin sulphate as a biomarker to monitor polo pony joints.

Osteoarthritis (OA) of the metacarpophalangeal joint is the most common articular disease in polo ponies leading to early retirement. A biomarker that would discriminate between pathological and physiological changes secondary to exercise could be helpful in OA prevention. The aim of this study was to investigate the effects of polo training on synovial fluid biomarkers of inflammation and cartilage turnover in polo ponies of different skill levels. Synovial fluid samples were collected from metacarpophalangeal joints of polo ponies before and during the polo season (320 d). Nucleated cells, soluble protein, prostaglandin E2 (PGE2), glycosaminoglycans (GAG), and urea were measured. The main synovial fluid GAG are chondroitin sulphate (CS, ~25 µg/mL) and hyaluronic acid (HA, ~400 µg/mL). After a polo match, a transitory increase in protein and PGE2, but not CS and HA, occurred (expressed as urea ratio), returning to basal levels in 24 h. During the polo season, the number of synovial fluid nucleated cells was always in the normal range. Increases in protein and HA occurred during the initial 40 to 80 d, returning to basal levels afterwards. In contrast, in polo prospects the concentration of CS steadily increased during the season. Long-term follow-up revealed that the synovial fluid CS was significantly higher in polo ponies that developed joint diseases within 24 months following our study. In conclusion, CS seems to be an early marker of articular cartilage damage.

L'arthrose (OA) de l'articulation métacarpophalangienne est la maladie articulaire la plus fréquente chez les poneys de polo menant à la retraite anticipée. Un biomarqueur qui était discriminateur entre les changements pathologiques et physiologiques secondaires au exercice pourrait être utile pour la prévention de l'OA. L'objectif de la présente étude était examiner les effets de l'activité de polo sur les biomarqueurs de l'inflammation et de le métabolisme de la cartilage dans le liquide synovial des poneys de polo de différents niveaux de qualification. Le SF était obtenu à partir de les articulations métacarpophalangiennes de poneys de polo, avant et pendant la saison de polo (320 jours). Les cellules nucléés, protéine soluble, prostaglandine E2 (PGE2), glycosaminoglycanes (GAG) et l'urée ont été mesurés. Les principaux GAG de le liquide synovial sont le chondroïtine sulfate (CS, ~25 µg/mL) et l'acide hyaluronique (HA, ~400 µg/mL). Après un match de polo, ocorru une augmentation transitoire de la protéine et de la PGE2, mais pas de CS et de HA (exprimé comme le raison d'urée), qui a retourné aux niveaux basal dans 24 h. Pendant la saison de polo, le numero de cellules nucléés dans le liquide synovial était toujours normaux. La protéine et le HA augmentaient pendant les premiers 40­80 jours, mais tous les deux sont retournés aux niveaux de base plus tard. En contraste, dans le group de jeunes poneys (G1), la concentration de CS a augmenté régulièrement pendant la saison. Accompagnant à long terme avait révéle que le CS de liquide synovial était significativement plus élevée chez les poneys de polo que, dans les 24 mois suivants, avaient developpé des maladies articulaires. En conclusion, le CS du liquide synovial semble être un marqueur précoce des destructions de la cartilage articulaire.(Traduit par les auteurs).

Lysosomal enzymes are decreased in the kidney of diabetic rats.

The objective of the present study was to investigate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in diabetic rat kidney. Cathepsins, glycosidases and sulfatases were studied on the 10th (DM-10) and on the 30th (DM-30) day of streptozotocin-induced diabetes mellitus (DM). The activity of cathepsin B, the main kidney cysteine protease, was decreased both in DM-10 and DM-30. Gel filtration chromatography of urinary proteins has shown the prevalence of low molecular weight peptides in normal and DM-10 urine, in contrast to the prevalence of high molecular weight peptides and intact proteins in DM-30. These results show that the decrease in lysosomal proteases could explain, at least in part, the increased albuminuria detected by radial immunodiffusion (RID), due to the excretion of less degraded or intact albumin. Concerning sulfated polysaccharides, the activities of ß-glucuronidase, N-acetyl-ß-d-glucosaminidase, and N-acetyl-ß-d-galactosaminidase were also decreased in DM-30, while aryl sulfatases did not vary. Increased toluidine blue metachromatic staining of the tissue suggests that the lower activities of glycosidases could lead to intracellular deposition of partially digested molecules, and this could explain the decreased urinary excretion and increased tissue buildup of these molecules. The main morphological changes observed in kidney were proximal convoluted tubules with thinner walls and thinner brush border. Immunohistochemistry revealed that most of cathepsin B was located in the brush border of proximal tubular cells, highlighting the involvement of proximal convoluted tubules in diabetic nephropathy.

Changes in glycosaminoglycans and proteoglycans of normal breast and fibroadenoma during the menstrual cycle.

BACKGROUND: Fibroadenoma is the most common breast tumor in young women, and its growth and metabolism may be under hormonal control. In the present paper we described the proteoglycan (PG) composition and synthesis rate of normal breast and fibroadenoma during the menstrual cycle. METHODS: Samples of fibroadenoma and adjacent normal breast tissue were obtained at surgery. PGs were characterized by agarose gel electrophoresis and enzymatic degradation with glycosaminoglycan (GAG) lyases, and immunolocalized by confocal microscopy. To assess the synthesis rate, PGs were metabolic labeled by 35S-sulfate. RESULTS: The concentration of PGs in normal breast was higher during the secretory phase. Fibroadenoma contained and synthesized more PGs than their paired controls, but the PG concentrations varied less with the menstrual cycle and, in contrast to normal tissue, peaked in the proliferative phase. The main mammary GAGs are heparan sulfate (HS, 71%-74%) and dermatan sulfate (DS, 26%-29%). The concentrations of both increased in fibroadenoma, but DS increased more, becoming 35%-37% of total. The DS chains contained more ß-d-glucuronic acid (IdoUA/GlcUA ratios were >10 in normal breast and 2-7 in fibroadenoma). The 35S-sulfate incorporation rate revealed that the in vitro synthesis rate of DS was higher than HS. Decorin was present in both tissues, while versican was found only in fibroadenoma. CONCLUSIONS: In normal breast, the PG concentration varied with the menstrual cycle. It was increased in fibroadenoma, especially DS. GENERAL SIGNIFICANCE: PGs are increased in fibroadenoma, but their concentrations may be less sensitive to hormonal control.

Urinary glycosaminoglycans in horse osteoarthritis. Effects of chondroitin sulfate and glucosamine.

Our objectives were to characterize the urinary excretion of glycosaminoglycans (GAGs) in horse osteoarthritis, and to investigate the effects of chondroitin sulfate (CS) and glucosamine (GlcN) upon the disease. Urinary GAGs were measured in 47 athletic horses, 20 healthy and 27 with osteoarthritis. The effects of CS and GlcN were investigated in mild osteoarthritis. In comparison to normal, urinary GAGs were increased in osteoarthritis, including mild osteoarthritis affecting only one joint. Treatment with CS+GlcN led to a long lasting increase in the urinary CS and keratan sulfate (KS), and significant improvement in flexion test of tarsocrural and metacarpophalangeal joints was observed. In conclusion, urinary CS and KS seems to reflect the turnover rates of cartilage matrix proteoglycans, and the measurement of these compounds could provide objective means of evaluating and monitoring joint diseases.

Heparin affects the interaction of kininogen on endothelial cells.

In the plasma kallikrein-kinin system, it has been shown that when plasma prekallikrein (PK) and high molecular weight kininogen (HK) assemble on endothelial cells, plasma kallikrein (huPK) becomes available to cleave HK, releasing bradykinin, a potent mediator of the inflammatory response. Because the formation of soluble glycosaminoglycans occurs concomitantly during the inflammatory processes, the effect of these polysaccharides on the interaction of HK on the cell surface or extracellular matrix (ECM) of two endothelial cell lines (ECV304 and RAEC) was investigated. In the presence of Zn(+2), HK binding to the surface or ECM of RAEC was abolished by heparin; reduced by heparan sulfate, keratan sulfate, chondroitin 4-sulfate or dermatan sulfate; and not affected by chondroitin 6-sulfate. By contrast, only heparin reduced HK binding to the ECV304 cell surface or ECM. Using heparin-correlated molecules such as low molecular weight dextran sulfate, low molecular weight heparin and N-desulfated heparin, we suggest that these effects were mainly dependent on the charge density and on the N-sulfated glucosamine present in heparin. Surprisingly, PK binding to cell- or ECM-bound-HK and PK activation was not modified by heparin. However, the hydrolysis of HK by huPK, releasing BK in the fluid phase, was augmented by this glycosaminoglycan in the presence of Zn(2+). Thus, a functional dichotomy exists in which soluble glycosaminoglycans may possibly either increase or decrease the formation of BK. In conclusion, glycosaminoglycans that accumulated in inflammatory fluids or used as a therapeutic drug (e.g., heparin) could act as pro- or anti-inflammatory mediators depending on different factors within the cell environment.

Effect of amniotic membrane transplantation on corneal healing and proteoglycan expression in an experimental model of limbal deficiency in rabbits.

PURPOSE: Amniotic membrane transplantation (AMT) has been used as a graft or as a dressing in ocular surface reconstruction, facilitating epithelization, maintaining normal epithelial phenotype, and reducing inflammation, vascularization, and scarring. The corneal transparency is due, at least in part, to the arrangement in orthogonal lamellae of collagen fibrils, surrounded by proteoglycans (PGs). These PGs regulate fibrilogenesis, the matrix assembly, and ultimately the corneal transparency. The purpose of the present study was to investigate the effects of AMT upon the corneal PGs after severe limbal injury. METHODS: Experiments were performed on the right corneas of 22 New Zealand female albino rabbits, and their left corneas were used as matched controls. These animals were divided into 3 groups: G1 (n=10): total peritomy and keratolimbectomy, followed by application of 0.5 M NaOH; G2 (n=10): submitted to the same trauma as G1, and treated by AMT; G3: no trauma, only AMT (n=2). The right corneas of G2 and G3 were covered by DMSO4 cryopreserved human amniotic membrane, fixed by interrupted 9-0 mononylon sutures, with its stromal face toward the ocular surface. After 7 or 30 days, the corneas were removed and PGs were extracted. RESULTS: Normal corneas contained approximately 9 mg of PGs per gram of dry tissue. AMT on intact cornea (G3) did not cause any changes in the concentration of PGs. In contrast, injured corneas contained much less PGs, both on the seventh and on the 30th day posttrauma. The PG concentration was even lower in injured corneas treated by AMT. This decrease was due almost exclusively to dermatan sulfate PGs, and the structure of dermatan sulfate was also modified, indicating changes in the biosynthesis patterns. CONCLUSIONS: Although beneficial effects have been observed on clinical observation and concentration of soluble proteins after AMT, the normal PG composition of cornea was not attained, even 30 days postinjury, indicating that the normal ocular surface reconstruction, if possible, is a long-term process.

Proteoglycan synthesis by human corneal explants submitted to laser in situ keratomileusis (LASIK).

PURPOSE: To evaluate the acute effects of laser in situ keratomileusis (LASIK) upon the synthesis of proteoglycans (PGs) and collagen fibril organization in human corneal explants. METHODS: Human corneas that had been rejected for transplants were obtained at Banco de Olhos of Hospital São Paulo. For each eye pair, one cornea was submitted to refractive surgery, and the other was used as its matched control. After surgery, the corneas were excised from the eyes and immediately placed in a Ham F-12 nutrient mixture containing (35)S-sulfate for the metabolic labeling of PGs. After 24 h incubation, PGs were extracted and identified by a combination of agarose gel electrophoresis and enzymatic degradation with protease and specific glycosaminoglycan lyases. Histopathological and birefringence analysis were performed in fixed tissue slices. RESULTS: A marked decrease in (35)S-sulfate incorporation in PGs was observed in corneal explants that received LASIK, especially concerning dermatan sulfate-PGs, with keratan sulfate- and heparan sulfate-PG synthesis reduced to a lower degree. Only low molecular weight PGs were present in the corneas, both before and 24 h after LASIK. No sign of wound healing processes were observed, but a marked change in corneal birefringence was seen following LASIK treatment. CONCLUSIONS: Laser application led to decreased PG biosynthesis in human corneal explants, with marked changes in the collagen fibril organization, as revealed by changes in the tissue birefringence.

Reliability of 1,9-dimethylmethylene blue tests in comparison to agarose gel electrophoresis for quantification of urinary glycosaminoglycans.

BACKGROUND: The relevance of glycosaminoglycan determination in biological fluids is gradually gaining importance in the literature. Nevertheless, the results obtained by different methods vary widely. We evaluated 1,9-dimethylmethylene blue (DMB) dye-binding assays for quantification of urinary glycosaminoglycans, in comparison to densitometry after agarose gel electrophoresis. METHODS: Urinary glycosaminoglycans from different mammalian species were quantified by 3 different DMB dye-binding assays. The results were compared to those obtained by densitometry after agarose gel electrophoresis of glycosaminoglycans isolated from urine samples by ion exchange chromatography. RESULTS: Densitometry after agarose gel electrophoresis showed glycosaminoglycan urinary concentrations of 1-20 mg/l, and glycosaminoglycan/creatinine ratios of 2-25x10(-3), for all the mammalian species here studied. A decrease with age was observed for humans, cats and horses. In comparison, DMB assays gave much higher results - up to 200 mg/l and 500x10(-3) glycosaminoglycan/creatinine ratios. These values were greatly reduced after 4-h dialysis, suggesting that low molecular weight compounds do interfere. Furthermore, urinary anions such as sulfate, phosphate and citrate, react with metachromatic dyes, such as Toluidine Blue and DMB. CONCLUSION: DMB assays, although rapid and simple, are not appropriate to quantify urinary glycosaminoglycans in normal mammalians, since other urinary components interfere with the reactions.

Optical anisotropy of alcian blue-stained acid glycosaminoglycans.

Optical anisotropy as dispersion of birefringence (DB) (birefringence studied for light of different wavelengths) and linear dichroism (LD) (selective absorption of polarized light) in stained substrates reflects their macromolecular orientation states. Birefringence interference colors of alcian blue (AB)-stained glycosaminoglycans (GAG) and glycoconjugates observed with polarization microscopy have been found to vary, although their staining characteristics under unpolarized light are practically the same. We investigated the optical anisotropy of GAG-AB and some glycoconjugate-AB complexes used as standards, to provide a basis for interpreting results for AB-stained materials in situ. Filamentous preparations of hyaluronic acid (HA), chondroitinsulfates, proteoglycans, and a mucus sulfoglycoconjugate were studied. Anomalous DB (birefringence sign changing with the wavelength of the incident light) was generally observed, but LD was seen only in the AB-HA complex. LD simultaneous to anomalous DB characteristics on the AB-HA complex were assumed to be caused by a maximally oriented helical conformation of the HA. For the other AB-GAG studied, the optical anisotropic characteristics were suggestive of some degree of folding of their chains into a tertiary structure. The profiles of the anomalous DB curves for the AB-stained sulfoglycoconjugate differed from those of the other materials, probably due to different organization of its dye-binding sites.

Characterization of glycosaminoglycans in tubular epithelial cells: calcium oxalate and oxalate ions effects.

BACKGROUND: The interaction between tubular epithelial cells and calcium oxalate crystals or oxalate ions is a very precarious event in the lithogenesis. Urine contains ions, glycoproteins and glycosaminoglycans that inhibit the crystallization process and may protect the kidney against lithogenesis. We examined the effect of oxalate ions and calcium oxalate crystals upon the synthesis of glycosaminoglycans in distal [Madin-Darby canine kidney (MDCK)] and proximal (LLC-PK1) tubular cell lines. METHODS: Glycosaminoglycan synthesis was analyzed by metabolic labeling with (35)S-sulfate and enzymatic digestion with specific mucopolysaccharidases. Cell death was assessed by fluorescent dyes and crystal endocytosis was analised by flow cytometry. RESULTS: The main glycosaminoglycans synthesized by both cells were chondroitin sulfate and heparan sulfate most of them secreted to the culture medium or present at cellular surface. Exposition of MDCK cells to oxalate ions increased apoptosis rate and the incorporation of (35)S-sulfate in chondroitin sulfate and heparan sulfate, while calcium oxalate crystals were endocyted by LLC-PK1, induced necrotic cell death, and increased (35)S-sulfate incorporation in glycosaminoglycans. These effects seem to be specific and due to increased biosynthesis, since hydroxyapatite and other carboxylic acid did not induced cellular death or glycosaminoglycan synthesis and no changes in sulfation degree or molecular weight of glycosaminoglycans could be detected. Thapsigargin inhibited the glycosaminoglycan synthesis induced by calcium oxalate in LLC-PK1, suggesting that this effect was sensitive to the increase in cytosolic calcium. CONCLUSION: Tubular cells may increase the synthesis of glycosaminoglycans to protect from the toxic insult of calcium oxalate crystals and oxalate ions, what could partially limit the lithogenesis.