Chondroitin Sulfate Content Research Articles

Cytotoxicity of Viscoelastics on Cultured Corneal Epithelial Cells Measured by Plasminogen Activator Release

Plasminogen activator has been shown to be released by epithelial cells following corneal injury. The demonstration of the release of plasminogen activator from cultured corneal epithelial cells has been used for developing a cytotoxicity test, the Corneal Epithelial Plasminogen Activator test, which compares changes in the level of plasminogen activator in tissue culture media following chemical exposure as an index of chemical injury. Cultured rabbit corneal epithelial cells were exposed to varying concentrations of several viscoelastics for 1 hour. Release of plasminogen activator into the tissue culture media following exposure to the viscoelastic agent was studied as an index of chemical injury. The least cytotoxicity to cultured rabbit epithelium was associated with those viscoelastic agents containing methylcellulose. A 1-hour exposure to most concentrations of methylcellulose and chondroitin sulfate (Phacote) and methylcellulose (Occucoat) demonstrated release of greater amounts of plasminogen activator than was seen following a similar exposure to balanced salt solution, suggesting the greatest protective effect of these two viscoelastics. In contrast, sodium hyaluronate and chondroitin sulfate (Viscoat) showed decreased amounts of plasminogen activator release after a 1-hour exposure to cultured corneal epithelial cells demonstrating cytotoxicity. Polyacrylamide (Orcolon) and most diluted preparations of sodium hyaluronate (Healon and Healon Yellow) showed only mild reductions in the release of plasminogen activator, whereas undiluted sodium hyaluronate preparations were nearly as cytotoxic as Viscoat. This study suggests that viscoelastic agents containing methylcellulose (Phacote and Occucoat) may be most protective of the corneal epithelium during ophthalmic surgery. The clinical success of several dilute viscoelastic solutions as tear substitutes was corroborated by the lack of cytotoxicity seen in this study. Viscoat and undiluted sodium hyaluronate preparations showed the greatest cytotoxicity to cultured rabbit corneal epithelium.

Changes in the extracellular matrix of the autogenous patellar tendon graft after posterior cruciate ligament reconstruction: a biochemical study in sheep

The left knee joints of female German sheep were operated, replacing the posterior cruciate ligament by the central third of the patellar tendon. After 2, 6, 16, 26 and 52 weeks the graft of the operated leg as well as the contralateral central third of the patellar tendon and the posterior cruciate ligament were dissected and used for biochemical analysis. The total glycosaminoglycan content in grafts increases within the first year up to 5 fold and is higher than that of the patellar tendon, but is lower than that of the cruciate ligament. The distribution pattern of glycosaminoglycans differ in the posterior cruciate ligament and the patellar tendon. In cruciate ligament the main components are chondroitin sulfate (76%) and hyaluronan (15%). In the patellar tendon a higher portion of dermatan sulfate (approx. 16%) was found, next to 52% chondroitin sulfate and 22% hyaluronan. During the examination time the grafts show changes in the concentration and the distribution pattern of glycosaminoglycans. The chondroitin sulfate content increases during the experimental period from 1.4 +/- 1.2 mumol/g dry weight (d.w.) to 8.7 +/- 2.9 mumol/g d.w. After 1 year the chondroitin sulfate content in the graft does not differ significantly from that of the cruciate ligament. In the grafts the concentration of chondroitin (non sulfated) increases after 6 weeks up to 1.3 +/- 0.6 mumol/g d.w. in comparison to the value after 2 weeks (0.2 +/- 0.1 mumol/g d.w.) and also in the other groups (16, 26 and 52 weeks) it remains significantly increased. After 1 year the dermatan sulfate content in the graft has increased up to the fifth-fold compared to the value after 2 weeks and is higher than in the patellar tendon and in the cruciate ligament. In graft the hyaluronan content (1.0 +/- 0.4 mumol/g d.w. does not differ significantly in the groups 2, 6, 16, 26 and 52 weeks after operation, but in all five groups it is lower than in the cruciate ligament (2.4 +/- 1.0 mumol/g d.w.). Dermatan and heparan sulfate are not or only little detected in all three tissues. The distribution pattern of glycosaminoglycans in graft shows chondroitin sulfate and dermatan sulfate being the major parts of glycosaminoglycans with a slight increase of these components in the different groups during the experimental period. Hyaluronan makes up to 24 +/- 5% of the whole content of glycosaminoglycans after 2 weeks and decrease to 8 +/- 1% after 52 weeks.(ABSTRACT TRUNCATED AT 400 WORDS)

Corneal Cryopreservation with Chondroitin Surfate

In an experimental study using porcine corneas we investigated the influence of several variables of freeze-thaw trauma on survival of corneal endothelial cells after corneal cryopreservation employing chondroitin sulfate as a cryoprotectant. We examined the influence of: (1) the concentration of chondroitin sulfate in the cryopreservation medium, (2) the concentration of fetal calf serum in the cryopreservation medium, (3) the cooling rate, and (4) the preincubation period in the cryopreservation medium before freezing. Controls consisted of corneas cryopreserved in culture medium without cryoprotectants and corneas frozen in dimethyl sulfoxide (Me 2SO) by the method of Capella et al. (Preservation of viable corneal tissue, Cryobiology , 2, 116-121, 1965). Morphological evaluation was performed by determining endothelial cell density after staining with alizarin red S and trypan blue. Morphological evaluation was not performed directly after thawing but after a subsequent storage period in organ culture at 31°C in order to detect latent cell damage after freeze-thaw trauma. The group that yielded the best endothelial cell density was evaluated in perfusion chamber experiments in order to measure the functional integrity of the tissue. Controls included corneoscleral rims from freshly slaughtered pigs, corneas stored in organ culture for 1 day, and fresh corneas mechanically denuded of endothelium. It was demonstrated that corneas that had been cryopreseved in MEM medium containing 2% chondroitin sulfate and 20% fetal calf serum with a cooling rate of 1°C/min displayed the highest endothelial cell density (2430 cells/mm 2, SO = 383, n = 15) compared with freshly dissected corneas (3395 cells/mm 2, SD = 200, n = 48). Control corneas frozen by the method of Capella et al. demonstrated only a poor outcome. We conclude that in our experimental environment, corneal cryopreservation with chondroitin sulfate provides higher corneal endothelial cell densities than corneas preserved by conventional methods. Moreover, even slight changes in variables that affect the tissue in the freeze-thaw cycle have a major impact on corneal endothelial cell survival after cryopreservatiorn.

Optimization of the USP assay for hyaluronidase

The current USP XXII assay for hyaluronidase (EC 3.2.1.35, HAse) determines activity indirectly by measuring the amount of undegraded hyaluronic acid (HA) substrate remaining after the enzyme is allowed to react with the HA for 30 min at 37°C. To be acceptable as a substrate, the HA must pass a USP suitability test. In this study, seven HA samples, which differed in their anatomical origin, their commercial supplier, and their chondroitin sulphate content, were tested as substrates. One of these did not pass the USP suitability test and therefore would not be an officially acceptable substrate; however, it was carried through the investigation along with the others in order to demonstrate its effect on the analysis. All seven HAs were used as substrates to assay testicular hyaluronidases from three different suppliers. The standard by which the other hyaluronidase activities were measured was USP hyaluronidase reference standard. The activity values calculated for a particular hyaluronidase differed significantly depending on which HA was used as substrate in its assay. Optimal results, as judged on the bases of initial purity, suitability for the assay, linearity of the standard curve, and per cent relative standard deviation of the measured activity, were obtained with a HA substrate derived from vitreous humour.

Temporal matrix synthesis and histologic features of a chondrocyte-laden porous collagen cartilage analogue

SUMMARY Cartilage resurfacing by chondrocyte transplantation, using porous collagen matrices as a vehicle to secure the cells in cartilage defects, has been used experimentally in animals. This in vitro study evaluated the temporal morphologic features and proteoglycan synthesis of chondrocyte-laden collagen matrices. Forty-two porous collagen disks were implanted with a minimum of 6 × 106 viable chondrocytes, covered by a polymerized collagen gel layer, and 6 disks were harvested after 0, 3, 7, 10, 14, 18, or 22 days of incubation in supplemented Ham's F12 medium at 37 C and 5% CO2. Histologic and histo-chemical evaluation of formalin-fixed segments of the cultured disks indicated that the chondrocytes proliferated in the implant, producing small groups and linear segments of cells by day 14. The collagen framework remained intact over the course of the study with thick areas attributable to depositions of matrix material after day 10. Alcian blue-stained matrix was evident in the pericellular region of chondrocytes in sections of disks harvested on days 14, 18, and 22. Glycosaminoglycan (GAG) assay by dimethyl-methylene blue dye binding after papain digestion of the disk segments revealed negligible amounts of GAG at day 0. Significant (P ≤ 0.0001) increase in total GAG content was observed by day 3 (0.329 M-μg of disk) and further increases were observed until a plateau in GAG quantity was seen on day 14. Mean peak GAG content was 0.553 ± 0.062 μg/mg. Secondary treatment of the papain-digested implants with keratanase and chondroitinase ABC yielded similar trends in chondroitin sulfate (CS) and keratan sulfate (KS) concentrations. The CS content significantly (P = 0.0002) increased for the first 14 days of incubation, then a plateau was observed for the remainder of the study. Peak CS content was 0354 ± 0.037 μg/mg. Concentration of KS reached a plateau earlier than did CS content, with peak amount of 0.193 ± 0.027 μg/mg on day 10. Fluctuations in K5 content were not significant until an increase on day 22. Chondrocytes actively populated the collagen implants, increasing in number and synthesizing matrix GAG epitopes over the 22 days of incubation. These results indicate that chondrocyte-laden porous collagen matrices may be suitable cartilage analogue materials and the optimal metabolic time for transfer to cartilage defects is 10 to 14 days.

Possible role for chondroitin sulfate in urolithiasis: In vivo studies in an experimental model

The effect of chondroitin sulfate upon the growth of calcium oxalate crystals was measured in vivo by using an experimental model in rats. Adult male Wistar rats were treated by cronical i.p. injections of chondroitin sulfate solutions (1, 5 or 10 mg in 0.3 ml of saline, every 2 days). This treatment led to a dose-dependent increase in the urinary chondroitin sulfate concentration. Urolithiasis was induced by the introduction of a calcium oxalate seed into the bladder of the animals. Urine samples were collected and the calculi formed were removed after 42 days. The chondroitin sulfate concentration have decreased in the lithiasic urines, as compared to controls and higher chondroitin sulfate doses correlated with larger calculi. The presence of chondroitin sulfate in the matrices of stones obtained from chondroitin sulfatetreated animals suggested that there was some adsorption of chondroitin sulfate on to the growth sites of the calcium oxalate crystals. In contrast to the chondroitin sulfate effect observed in vitro, which inhibits the growth of calcium oxalate crystals, our results suggest that in vivo chondroitin sulfate promotes the growth of stones in the urinary tract.

The effects of epidermal growth factor and chondroitin sulfate on the animal corneal endothelial cell culture.

In order to investigate the effects of different culture media containing various concentrations of epidermal growth factor (EGF) and chondroitin sulfate (CDS) on the growth of cultured corneal endothelial cell, pig corneal endothelial cells were used for this study. The cells were divided into 7 groups and each group was cultured with a medium containing: 1. Eagle's minimal essential medium with Earle's salt (EMEM) only; 2. EMEM + EGF (10 ng/ml); 3. EMEM + EGF (100 ng/ml); 4. EMEM + CDS (1 mg/ml); 5. EMEM + CDS (25 mg/ml); 6. EMEM + EGF (10 ng/ml) + CDS (1 mg/ml); 7. EMEM + EGF (100 ng/ml) + CDS (25 mg/ml), respectively. The results are shown below: (1) High concentration of EGF (100 ng/ml) stimulated the growth of pig corneal endothelial cells, shortened doubling time and the cells reached confluence earliest in group 3. But a low concentration of EGF (10 ng/ml) showed no effects on the growth of pig corneal endothelial cells. (2) A high concentration of CDS (25 mg/ml) might retard the growth of pig corneal endothelial cell, but a low concentration of CDS (1 mg/ml) has no retarding effects on the growth of pig corneal endothelial cells. (3) A high concentration of EGF could "antagonize" the CDS induced growth retarding effects of endothelial cells. (4) Those cells cultured with a medium containing high concentrations of EGF had more mitotic activity, including many prominent binuclear and polynucleolar cells. (5) The cells cultured with a medium containing high concentrations of CDS showed a more flat-shaped morphology under observation with a phase contrast microscope.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of intratracheal endotoxin administration on hamster lung glycosaminoglycans

Incorporation of [3H]glucosamine and 35S into glycosaminoglycan (GAG) was measured in hamster lung explant cultures at 0, 1, 4, and 24 h after a single endotracheal instillation of Escherichia coli endotoxin. Lung content of GAG was measured in a second group of treated animals over an 8-day period. Albumin was detected after endotoxin treatment in bronchoalveolar lavage fluid at 24 h but was not found in lavage fluid 7 days later or in lavage fluid of saline-treated animals. Over the initial 24 h, increasing amounts of radiolabeled precursor molecules were incorporated into all classes of GAG. Proportionally more radiolabel was incorporated into hyaluronic acid and chondroitin sulfate, and less was incorporated into heparan sulfate. The proportion of radiolabel incorporated into dermatan sulfate did not change. Total lung content of hyaluronate and chondroitin sulfate was elevated at 24 h but was returning to baseline by 8 days. The lung content of dermatan sulfate was increased at 8 days; lung content of heparan sulfate did not change over the 8-day study period. Elevations in the amount of explant heparan sulfate that bound to antithrombin III (AT III) were found at 1 h after both saline and endotoxin treatment. Radiosulfated heparan sulfates were found in blood from hamsters treated with endotoxin 1 h previously; these heparan sulfates did not bind to AT III. However, blood contained heparin-like activity. We conclude that endotoxin differentially alters the metabolism of each class of hamster lung glycosaminoglycans and that metabolic changes begin very rapidly after endotoxin exposure. The relation of pulmonary endothelial injury to the presence of heparin-like activity in blood is not yet clear.

Effects of chondroitin sulphate, human serum albumin and Tamm-Horsfall mucoprotein on calcium oxalate crystallization in undiluted human urine

The effects of physiological concentrations of chondroitin sulphate, human serum albumin and Tamm-Horsfall mucoprotein on the crystallization of calcium oxalate in undiluted, ultrafiltered human urine were investigated using particle size analysis and scanning electron microscopy. Neither the amount of oxalate required to induce detectable calcium oxalate crystal nucleation nor crystal morphology was affected by the presence of any of these macromolecules. Chondroitin sulphate had no effect on the amount of crystalline material deposited or on the size of the particles precipitated in response to a standard oxalate load. Human serum albumin slightly reduced the size of the crystal aggregates and caused a small increase in the amount of crystal matter precipitated. By contrast, Tamm-Horsfall mucoprotein significantly inhibited crystal aggregation and markedly increased the volume of matter deposited, although this could not be attributed to a promotion of solute precipitation. It was concluded that chondroitin sulphate, human serum albumin and Tamm-Horsfall mucoprotein cannot account for the inhibitory effects of macromolecules with a relative mass greater than 10 kDa in spun and filtered urine. Nonetheless, Tamm-Horsfall mucoprotein is likely to inhibit crystal aggregation in whole urine in vivo and may therefore be instrumental in preventing calcium oxalate stone formation.

Endothelial Cells of Rabbit Cornea in Different Storage Conditions

The metabolic functions of endothelial cells of the rabbit cornea in different storage conditions were studied using quantitative cytochemistry. The corneas were stored for 2 h, and 1, 3, 7, 14 and 21 days in dexol at 4 degrees C and in culture medium at 37 degrees C. It was shown that glycolysis as expressed by the activity of the cytosolic enzymes glyceraldehyde 3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase and lactic dehydrogenase is well preserved for 1 week in dexol and for 3 days only in the culture medium. Mitochondrial enzymes as shown by succinate dehydrogenase and fatty acid oxidation activity show a similar pattern. Uridine diphosphoglucose dehydrogenase, which plays an important part in proteoglycan synthesis, is markedly decreased in both media, but retains its activity in dexol for a slightly longer time. Keratan and chondroitin sulfate content show a sharp drop in the culture medium at 37 degrees C as compared to dexol. This study demonstrates the superiority of dexol at 4 degrees C over the culture medium at 37 degrees C. Quantitative cytochemistry is a useful tool for studying the metabolism of endothelial cells.

Improved Storage Methods for Epikeratoplasty

A major variable in calculating the necessary curvatures and dimensions of the refractive lenticules for epikeratoplasty is the degree of swelling of the donor cornea during storage prior to lathing. The purpose of this study was to determine whether it is possible to restore donor corneas to their in vivo thickness using osmotic agents rather than physical means. The osmotic agents chosen were dextran and chondroitin sulfate. Donor porcine corneas stored in McCarey-Kaufman (MK) medium for seven days increased in thickness by almost 50%. Increasing concentrations of dextran and chondroitin sulfate, and increasing concentrations of dextran with 1.35% chondroitin sulfate added, were examined for their ability to reduce this thickness to normal levels. It was found that 15.00% dextran alone, and 10.00% dextran with 1.35% chondroitin sulfate, reduced corneal thickness to that of fresh tissue. Light and electron microscopy showed that the osmotic agents restored stromal ultrastructure to that of fresh tissue. The use of osmotic agents to reduce corneal thickness prior to lathing reduces physical deformation of the donor tissue associated with the use of a corneal press and may enhance the predictability of epikeratoplasty.

Glycosaminoglycans in Neurofibromas

Glycosaminoglycan (GAG) contents in neurofibromas (NFs) were examined to clarify how well they corresponded to their histological features. Eight cutaneous NFs and three plexiform NFs from five patients with Recklinghausen's (R) disease, two senile NFs and control dermis were subjected to the isolation of GAGs. The GAGs were then fractionated and quantitated with two-dimensional electrophoresis on cellulose acetate membranes. Dry weight/wet weight ratios of the NFs were lower than those of the controls; the plexiform NFs were the lowest. In these plexiform NFs, hyaluronic acid (HA) content was most increased and dermatan sulfate (DS) content most decreased, resulting in a marked decrease in the DS/HA ratio. Both cutaneous and senile NFs showed moderate decreases in DS content, increases in chondroitin sulfate and heparan sulfate contents, and slight increases in HA content, resulting in moderate decreases in the DS/HA ratio. Considering that cutaneous or senile NFs are relatively more fibrotic tumors than plexiform NFs, these differences in the GAGs between plexiform NFs and cutaneous or senile NFs appear to be consistent with the changes in GAGs previously described in physiological and pathological conditions such as fibrotic diseases. Thus these results suggest that studies of hereditary disorders like R disease might be useful for understanding the pathogenesis of so-called acquired diseases with unknown etiology.

Glycosaminoglycans of the hemodialysis-associated carpal synovial amyloid and of amyloid-rich tissues and fibrils of heart, liver, and spleen

Significant amounts of glycosaminoglycans (GAGs) were found in amyloid fibril preparations. Using two-dimensional electrophoresis to fractionate GAG mixtures, we quantified and identified for the first time the GAGs of the fibrils from carpal synovium of patients with amyloid associated with chronic hemodialysis. The total GAG content was small, but the GAG distribution (high relative content of chondroitin sulfate and hyaluronic acid and lack of the other GAGs) was unique, unlike that for the other amyloid fibril preparations. The amyloid-rich heart, liver, and spleen tissues, as well as the fibrils isolated from these tissues of patients with systemic forms (primary amyloid and secondary amyloid) of amyloid disease, were also analyzed for GAGs. Fibrils from heart tissue of a patient with primary amyloidosis, now examined for the first time, contained four major GAGs (chondroitin sulfate, dermatan sulfate, hyaluronic acid, and heparan sulfate).

Turnover of proteoglycans by chick lens epithelial cells in cell culture and intact lens.

Chick lens epithelial cells were cultured on plastic and type IV collagen substrata, and the confluent cultures were labeled continuously with [35S]sulfate for 20 h. Intact lenses were also labeled in the same way. 35S-Proteoglycans isolated from those cultures were compared for their molecular sizes and glycosaminoglycan compositions. The results have shown that: 1) Proteoglycans synthesized by cells on type IV collagen were significantly smaller than those by cells on plastic. 2) Proteoglycans of intact lens showed a broad distribution of molecular size and contained a high proportion of chondroitin sulfate in the medium fraction compared to those of the two cell cultures. In order to explain such differences between proteoglycans from cultures, label-chase experiments with [35S]sulfate were done for proteoglycans synthesized. 35S-Proteoglycans isolated at each chase time 0, 2.5, and 17 h) were compared and the following results were found: 1) The cell layers of both "plastic" and "type IV collagen" cultures contained glycosaminoglycan species predominantly at each chase time rather than proteoglycans. 2) Changes in the glycosaminoglycan compositions of medium fractions of cell cultures were observed during the chase period; in medium of the "plastic" culture, proteoheparan sulfate increased with chase time, whereas in medium of the "type IV collagen" culture, chondroitin sulfate glycosaminoglycan (not proteoglycan) increased with chase time. 3) In intact lens culture, lens capsule fraction at every chase time contained a proteoglycan unique in molecular size, which was not found in cell culture fractions. 4) All fractions from intact lens cultures contained a higher content of chondroitin sulfate at every chase time than the respective fractions from cell cultures. These results suggest that adhesion of the cells to type IV collagen or lens capsule influences the degradation and secretion of proteoglycans. In addition, they can account partially for the above-described differences in molecular sizes and glycosaminoglycan compositions between 35S-proteoglycans from various cultures continuously labeled with [35S]sulfate.

Effects of vanadate on tyrosine phosphorylation and the pattern of glycosaminoglycan synthesis in rabbit chondrocytes in culture

The present study examined the effects of high doses of vanadate on glycosaminoglycan (GAG) synthesis and tyrosine phosphorylation in rabbit chondrocytes in confluent cultures. Although 6 microM vanadate increased the incorporation of [3H]glucosamine into chondroitin sulfate proteoglycans twofold, 40-60 microM vanadate suppressed this incorporation fourfold. Although 6 microM vanadate had little effect on [3H]glucosamine incorporation into hyaluronate, 40-60 microM vanadate increased this incorporation threefold. Chemical analyses confirmed that the increase in [3H]glucosamine incorporation into hyaluronate and the decrease in the incorporation into chondroitin sulfate proteoglycan correlated with increased hyaluronate content and decreased chondroitin sulfate content in the cell layers of vanadate-transformed cells. Chondrocytes exposed to 40-60 microM vanadate became typically transformed spindlelike cells. Furthermore, vanadate, at 6 and 60 microM, increased the overall level of phosphotyrosine by 8- and 31-fold, respectively, and 60 microM vanadate enhanced phosphorylation of many phosphotyrosine-containing proteins. These observations suggest that vanadate induces transformation-associated changes in the pattern of GAG synthesis when it induces excess phosphorylation on tyrosine in chondrocyte proteins.

Soluble complex formation between low-density lipoprotein and glycosaminoglycans. A 2H and 31P-NMR, and quasi-elastic light scattering study

Soluble complex formation between LDL and heparin (HEP) and chondroitin sulfate (CS) has been studied by 2H- and 31P-NMR and light scattering. The 2H-NMR linewidths of [ 2H]HEP and [ 2H]C4S increase substantially upon binding to LDL, with the [ 2H]HEP linewidths broader at low glycosaminoglycan (GAG)/low density lipoprotein (LDL) ratios. Preliminary analysis of the bound C 2H 3 group correlation times suggests that the observed linewidths are determined by the complex size, and that both [ 2H]GAGs have similar motions when bound to LDL. The 31P-NMR data demonstrate that large LDL-HEP complexes (diameter approx. 50 nm) are formed only over a narrow range of HEP concentrations, whereas the size of LDL-CS complexes increases continuously over the range of CS concentrations studied, reaching values of 32–35 nm for both C4S and C6S. At the lower protein concentrations studied by light scattering (⩽1 mg/ ml), the same trends are observed, although the mean diameters are less than those estimated by 31P-NMR. Soluble complex formation was unaffected by the presence of 2 mM Ca 2+. Dilution studies demonstrate that complex size varies with protein concentration. The binding of GAGs to LDL was also examined by HEP-CS competition studies. HEP has the higher affinity while no differences in binding could be detected between C4S and C6S.

Pulsed electromagnetic fields alter phenotypic expression in chondroblasts in tissue culture.

We hypothesize that pulsed electromagnetic fields (PEMF) alter phenotypic expression of chondroblasts by promoting the production of alkaline phosphatase (AP) and altering the structure of proteoglycans. Chondroblasts from the hypertrophic zone of tibial epiphyses (HC), sternum (SC), and skin fibroblasts (F) were cultured from 16 day chick embryos. Cultures were randomly designated control (C) or experimental (E). E received PEMF for 24 h in a 6 h on, 6 h rest sequence. The controls were in the same incubator shielded by Mu metal. Assays for AP activity were performed and normalized to protein content. Proteoglycan synthesis assay involved labeling with 35S fractionating in a 5% to 20% surcrose gradient determining total protein and chondroitin sulfate content. PEMF showed no change of AP activity on F. A high AP basal activity was found in HC, but was not increased above the control. PEMF increased AP in the SC samples (E/C ratio). The sucrose gradient data showed a shift in peaks for SC only altering the ratio of carbohydrate to protein for the SC. Analysis of carbohydrate and protein indicated that the effect was decreased synthesis or degradation of protein. We conclude that PEMF alters the phenotypic expression of sternal chondroblasts in our in vitro system.

Sulfated glycosaminoglycans modify growth factor-induced neurite outgrowth in PC12 cells.

Glycosaminoglycans (GAGs), localized on the surfaces of cells and in the basement membrane, modulate the growth and differentiation of many cell types. Recent studies have shown that heparin, a GAG found in mast cells, potentiates the ability of acidic fibroblast growth factor (aFGF) to induce neurite outgrowth in pheochromocytoma (PC12) cells. We examined the effect of a variety of GAGs on aFGF, basic fibroblast growth factor (bFGF), and nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. The effects observed were dependent upon the specific GAG, the concentration of the GAG, and the growth factor. Heparin potentiated aFGF-induced neurite outgrowth in a concentration-dependent fashion; potentiation increased with increasing heparin concentrations of 0.01-100 micrograms/ml. At concentrations greater than 100 micrograms/ml, heparin potentiation decreased. The maximally active concentration of heparin (100 micrograms/ml) increased the potency of aFGF 102-fold. Increasing concentrations of heparan sulfate, dermatan sulfate, and chondroitin sulfate correlated with increasing aFGF potentiation. The maximally active concentrations of heparan sulfate (100 micrograms/ml), dermatan sulfate (10 mg/ml), and chondroitin sulfate (1 mg/ml) increased the activity of aFGF 11-, 110-, and 11-fold, respectively. Hyaluronic acid did not affect the neurite outgrowth-promoting activity of aFGF. Heparin also altered the activity of bFGF; increasing concentrations of heparin (0.01-1 micrograms/ml) correlated with increased potentiation. At concentrations greater than 1 microgram/ml, heparin concentration was inversely correlated with potentiation. Chondroitin sulfate only increased the percentage of neurite-bearing cells at concentrations greater than 10 micrograms/ml. Maximally active concentrations of heparin (1 microgram/ml) and chondroitin sulfate (1 mg/ml) increased the potency of bFGF 5-fold. The highest concentration of heparan sulfate studied (1 mg/ml) inhibited the activity of bFGF. Dermatan sulfate and hyaluronic acid (0.01-1000 micrograms/ml) had no effect on bFGF activity. Heparan sulfate and chondroitin sulfate showed concentration-dependent potentiation of NGF; maximally active concentrations of heparan sulfate (100 micrograms/ml) and chondroitin sulfate (1 mg/ml) increased the potency of NGF 3-fold, whereas heparin, dermatan sulfate and hyaluronic acid had no effect. None of the GAGs had any effect on PC12 neurite outgrowth when added alone. The specificity of the activity of the GAGs was verified by selective enzyme degradation.(ABSTRACT TRUNCATED AT 400 WORDS)

Keratan sulfate content in the superficial and deep layers of osteophytic and nonfibrillated human articular cartilage in osteoarthritis.

Very thin slices of the superficial and deep layers of osteophytic and apparently normal articular cartilage from six human osteoarthritic femoral heads were digested with papain. The digests were analyzed for keratin sulfate content using an enzyme-linked immunosorbent assay (ELISA) with inhibition step, and for chondroitin sulfate and collagen contents using biochemical assays. Although there were marked differences in Safranin-O staining of the superficial and deep layers of osteophytic cartilage, these two layers had identical high ratios of chondroitin sulfate/collagen. Keratan sulfate was present only in small amounts in osteophytic cartilage. However, the deeper layer contained significantly more of this glycosaminoglycan. The deeper layer of articular cartilage contained approximately twice as much chondroitin sulfate and six times more keratan sulfate relative to collagen than the superficial layer. The results of this study showed that this new sensitive approach, which requires as little as 200 micrograms wet cartilage as starting material, provides important qualitative and quantitative information about the major constituents of the matrix.

Running exercise as a modulatory of proteoglycan matrix in the articular cartilage of young rabbits.

Proteoglycans (PGs) and collagen were quantified in the knee articular cartilages of femoral and tibial medial condyles following 1-8 weeks of moderate running exercise of 4- to 6-month-old rabbits. The total content of PGs extractable with 4 mol/l guanidium chloride was elevated in the weight-bearing cartilage of the tibial medial condyle, while their concentration, expressed as uronic acid per wet weight, and collagen remained unchanged. The content of glucosamine (GlcN) and its ratio to galactosamine (GalN) was elevated in femoral cartilage PGs purified by centrifugation in dissociative CsCl gradients, indicating an increase in keratan sulfate. After 8 weeks of running, the chondroitin sulfate chains of PGs from tibial medial condyle contained less unsulfated disaccharide units. The content of chondroitin sulfate was elevated in the nonextractable residue of the tibial medial condyle as indicated by uronic acid and GaN assays. The content of nonextractable GlcN was increased even more, both in tibial medial and femoral cartilages. Moderate running thus increased (1) keratan sulfate-rich PGs, (2) the degree of sulfation of the chondroitin sulfate chains, and (3) nonextractable PGs; these modulatory alterations probably enhance the stability and elastic stiffness of the PG matrix.