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Influence of the cells on the pericellular environment. The effect of hyaluronic acid on proteoglycan synthesis and secretion by chondrocytes of adult cartilage.

The chondrocyte is a specialized cell that synthesizes proteoglycans of a type found only in cartilage and nucleus pulposus. These proteoglycans are distinct in forming multiple aggregates of unique structure in which hyaluronic acid provides a central chain to which many proteoglycan molecules are bound at one end only. Chondrocytes were isolated from adult cartilage and used in suspension culture to test the effect of compounds in the medium on the synthesis of proteoglycans. Hyaluronic acid alone, among a number of compounds extracted from or analogous to those in cartilage, reduced the incorporation of [35S] sulphate into macromolecular material. Oligosaccharides of hyaluronic acid of the size of decasaccharides and above also had this effect but hyaluronic acid already bound to proteoglycan did not. The proportion of total labelled material associated with the cells increased at the expense of that in the medium. Treatment of the cells with trypsin abolished the effect of hyaluronic acid but treatment with chondroitinase did not. It is suggested that hyaluronic acid interacts with proteoglycans at the cell surface by a specific mechanism similar to that involved in proteoglycan aggregation, as a result of which the secretion and synthesis of proteoglycans is reduced.

Animals↗

[Therapeutic effect of hyaluronic acid injection for knee and shoulder joint].

It is known that hyaluronic acid can protect against damage to articular cartilage and helps to lessen friction between joint surfaces. Furthermore, it is reported that hyaluronic acid is effective for pain relief and for the risk of adhesion, and contractures of joints. The injection of hyaluronic acid is useful for the treatment of frozen shoulder, osteoarthritis, and rheumatoid arthritis. The risk of infection is a rare, though important, possibility, however this method is relatively safe.

English Abstract↗

Platelet-derived growth factor stimulates the secretion of hyaluronic acid by proliferating human vascular smooth muscle cells.

Total glycans from the cell layer and the culture medium of human vascular smooth muscle cells (VSMC) that had been cultivated in the presence of platelet-derived growth factor (PDGF) were isolated and purified by gel filtration after Pronase and DNase digestion and alkaliborohydride treatment. Measurements of the content of neutral hexoses and uronic acids revealed that PDGF stimulates total glycan synthesis by proliferating VSMC in a linear fashion from 24 h to 72 h of incubation. In contrast, total glycan synthesis by human fibroblasts, epithelial cells, or endothelial cells was not affected by PDGF, indicating cell-type specificity. Chemical, biochemical, and enzymological characterization of the total glycans synthesized by VSMC showed that PDGF stimulates the secretion of a 340-kDa glycan molecule in a time-dependent manner from 24 h to 72 h. This molecule is highly acidic, shares a common structure with hyaluronic acid, and exhibits a potent antiproliferative activity on VSMC. These results suggest that VSMC in response to PDGF are capable of controlling their own growth and migration by the synthesis of a specific form of hyaluronic acid with antiproliferative potency, which may be involved in the regulation of the local inflammatory responses associated with atherosclerosis.

Amino Acids↗

Rheological effects of the presence of hyaluronic acid in the extracellular media of differentiated 3T3-L1 preadipocyte cultures.

The viscoelastic properties of culture medium obtained from confluent 3T3-L1 preadipocytes, after differentiation with isobutyl-methylxanthine and dexamethasone, were studied with a rotational Couette viscometer. In close association with adipocyte differentiation, the culture medium showed gel-like properties, in concert with an increase in viscosity. This behavior vanishes after digestion by Streptomyces hyaluronidase or chondroitinase ABC, but not after application of collagenase, pronase, trypsin, DNase, or neuraminidase, or by treatment with EDTA or mercaptoethanol, indicating that the primary substance responsible for this behavior is hyaluronic acid. The material revealed a non-Newtonian behavior with an irreversible disruption of the network by shear force at high speeds. The viscosity of the medium, containing about 1 microgram/ml of hyaluronic acid, was calculated to be similar to that of a solution containing 1.7 mg high molecular weight hyaluronic acid per milliliter of stock culture medium. The comparison of rheological properties between the culture medium and solutions of hyaluronic acid indicated the possibility of a highly organized network in the culture medium that is more complicated than a simple interaction between homologous hyaluronic acid molecules. The non-Newtonian behavior depends on the hyaluronic acid concentration in the medium as well as on the length of exposure of the 3T3-L1 cells to the isobutyl-methylxanthine/dexamethasone mixture. The results point toward the possibility of interaction between hyaluronic acid and binding proteins.

1-Methyl-3-isobutylxanthine↗

Hydroxyapatite-collagen-hyaluronic acid composite.

The new composite biomaterial made from hydroxyapatite and collagen conjugated with hyaluronic acid has been studied. The structure evaluation of the composite showed more dense arrangement due to the formation of collagen hyaluronic acid conjugate, and particles of inorganic component are closely anchored in the structure. Mechanical properties and model swelling of this composite are compared with properties of hydroxyapatite-collagen composite of similar material without hyaluronic acid. Higher cohesivity of the new biomaterial can give new possibilities for application as bone implant material. The test of contact cytotoxicity showed a very good biocompatibility of the biomaterial.

Biocompatible Materials↗

Synthesis and degradation of hyaluronic acid in the cultured fibroblasts of Marfan's disease.

Increased amounts of hyaluronic acid accumulate in fibroblasts cultured from patients with Marfan's disease, an autosomal dominant disorder. In the recessive Hurler's disease, the storage of glycosaminoglycan (GAG) is due to impaired degradation. This study examines the kinetics of GAG accumulation in Marfan's disease in order to determine whether the mechanism of accumulation differs from that in Hurler's disease.Marfan-derived fibroblasts incorporated [(14)C]acetate or [(14)C]glucosamine into GAG to a level 4-6 times greater than control fibroblasts. Sugar analyses, electrophoretic mobility, and enzyme susceptibility studies showed that the isolated material was hyaluronic acid. There were no differences in activity of a variety of glycosidases between Marfan and control fibroblasts, nor were there differences in the ability to degrade prelabeled hyaluronate by cell-free extracts. Finally, chase experiments showed parallel rates of loss of labeled GAG from control fibroblasts and fibroblasts from Marfan patients. It appears that hyaluronic acid was accumulating in greater amounts in the fibroblasts from patients with Marfan's disease because of a greater rate of synthesis as opposed to a decreased rate of breakdown.

Acetates↗

Hyaluronic acid in incision wound fluid: a clinical study with the Cellstick device in children.

When inserted into a human incision wound, the Cellstick device harvests inflammatory cells and collects wound fluid, reflecting time-related changes in cell populations and in wound fluid composition. Hyaluronic acid has been postulated to be an important factor in scar reduction in wound healing and in scarless fetal wound healing. The aim of this work was to determine the concentration and variation of hyaluronic acid and proportions of wound cells in closed surgical wounds in children at two time points. The Cellstick device was inserted subcutaneously into the wound at the end of an elective inguinal hernia operation on 37 healthy boys, and the devices were removed 3+/-1 or 24+/-3 hours after surgery. Haluronic acid concentration was measured from the wound fluid and a differential count of the wound cells was performed. There was a significant decrease in hyaluronic acid concentration from 3+/-1 to 24+/-3 hours after surgery (p<0.001, Kruskal-Wallis anova). The variance of hyaluronic acid concentration in wound fluid differed between the wounds at the two time points (p<0.01, Levene test for homogeneity of variance). A positive correlation between hyaluronic acid concentration and patient age (r=0.91, p<0.05, Spearman) at 3+/-1 hours post surgery and between HA and wound lymphocytes (r=0.38, p<0.05, Spearman) was also found. We conclude that the hyaluronic acid concentration in wound fluid peaks early in children and decreases significantly by 3 to 24 hours after surgery, and the concentrations in the wound fluid of healthy boys are more variable 3 hours than at 24 hours after surgery.

Analysis of Variance↗

Growth-related production of proteoglycans and hyaluronic acid in synchronous arterial smooth muscle cells.

1. The growth-stimulating effect of serum on the proteoglycan and hyaluronic acid production in arterial smooth muscle cells was investigated, using cells synchronized by serum deprivation. 2. After stimulation, synthesis of [35S]sulfated proteoglycans and [14C]hyaluronic acid increased during G1 and G2 phases (about 2- and 5-fold, respectively, in the culture medium), in comparison with quiescent cells. 3. Neither the size, nor the charge, nor the relative proportions of [35S]glycosaminoglycans of the proteoglycans were modified. 4. However, when the cells were stimulated to divide, increased synthesis of large [14C]hyaluronic acid was observed concomitantly with the production of higher hydrodynamic size [35S]proteoglycans, which aggregated with hyaluronic acid (20%).

Animals↗

Hyaluronic acid and proteoglycan accumulation in the cumulus oophorus matrix.

A factor produced by the oocytes induces in vitro FSH-treated cumulus and mural granulosa cells to synthesize hyaluronic acid. In the present study we examined hyaluronic acid synthesis by the two cell populations in vivo. After injection of hCG into PMSG-primed immature mice, cumulus and mural granulosa cells adjacent to the antrum synthesized a large amount of such glycosaminoglycan, while the outermost layers layers of mural granulosa cells did not. The results suggest that in vivo differences in hyaluronic acid synthesis between follicle cell subpopulations depend on a diffusion gradient of the oocyte factor. We also identified a proteoglycan species synthesized by cumulus cells which may contribute to hyaluronic acid organization in the intercellular spaces.

Animals↗

The kinetics of hydroxyl-radical-induced strand breakage of hyaluronic acid. A pulse radiolysis study using conductometry and laser-light-scattering.

Hydroxyl radicals were generated radiolytically in N2O- and N2O/O2(4:1)-saturated aqueous solutions of hyaluronic acid. The hydroxyl radicals react rapidly with hyaluronic acid mainly by abstracting carbon-bound H atoms. As a consequence of subsequent free-radical reactions, chain breakage occurs the kinetics of which has been followed using the pulse radiolysis technique. In the absence of oxygen, strand breakage was followed by the change in conductivity induced by the release of cationic counterions condensed at the surface of hyaluronic acid which is a polyanion consisting of subunits of glucuronic acid alternating with N-acetyl-glucosamine. It appears that strand breakage is not due to one single first-order process, however, the contributions of the different components cannot be adequately resolved. At pH 7 the overall half-life is 1.4 ms, in both acid and basic solutions the rate of free-radical induced strand breakage is accelerated (at pH 4.8, t1/2 = 0.6 ms; at pH 10, t1/2 = 0.18 ms). In the absence of oxygen there is no effect of dose rate on the kinetics of strand breakage. In the presence of oxygen in addition to conductometric detection, strand breakage was also followed by changes in low-angle laser light-scattering. These two techniques are complementary in that in this system the conductometry requires high doses per pulse while the light-scattering technique is best operated in the low-dose range. In the presence of oxygen a pronounced dose-rate effect is observed, e.g. at pH 9.7 after a dose of 9.4 Gy the overall half-time is approx. 0.5 s, while after a dose of 6.6 Gy the half-time is approx. 0.23 s. Both the yield and the rate of strand breakage increase with increasing pH, e.g. at pH 7 G(strand breaks) = 0.7 x 10(-7) mol J-1 and at pH 10.4, 4.8 x 10(-7) mol J-7. The radiolytic yields of CO2, H2O2, organic hydroperoxides, O2.- and oxygen consumption have been determined in gamma-irradiated N2O/O2(4:1)-saturated solutions of both hyaluronic acid and beta-cyclodextrin.

Carbohydrate Sequence↗

Identification and disruption of two discrete loci encoding hyaluronic acid capsule biosynthesis genes hasA, hasB, and hasC in Streptococcus uberis.

The hyaluronic acid capsule of Streptococcus uberis has been implicated in conferring resistance to phagocytosis by bovine neutrophils. Construction of a bank of random insertion mutants of S. uberis (strain 0140J) was achieved using the pGh9::ISS1 mutagenesis system (22). Phenotypic screening of approximately 5,000 clones enabled the isolation of 11 acapsular mutants. Southern hybridization indicated that two mutants carried a lesion within a group of genes similar to those involved in the assembly of the hyaluronic acid capsule found in the group A Streptococcus (GAS) has operon. The DNA sequence flanking the points of insertion confirmed the presence of homologues of GAS hasA and hasB in S. uberis. The DNA sequence flanking the ISS1 insertion in another mutant identified a homologue of hasC in S. uberis. The GAS hasABC operon structure was not conserved in S. uberis, and two discrete loci comprising homologues of either hasAB or hasC were identified. Disruption of S. uberis hasA or hasC resulted in the complete cessation of hyaluronic acid capsule production. Correspondingly, these mutants were found to have lost their resistance to phagocytosis by bovine neutrophils. The bactericidal action of bovine neutrophils on S. uberis 0140J was shown unequivocally to depend upon the capsule status of the bacterium.

Amino Acid Sequence↗

The repair of brain lesion by implantation of hyaluronic acid hydrogels modified with laminin.

The hyaluronic acid (HA) hydrogels modified with laminin were used for implantation in rat brain in present study, in order to investigate its effects in reparation of injury in the CNS. Cross-linked HA hydrogels were synthesized and their characteristics were analyzed. Laminin, an extracellular matrix protein, which participates in neuronal development and survival, was immobilized on the backbone of the hydrogels. Hydrogels unmodified and modified with laminin were implanted into cortical defects mechanically created in rats and their ability to improve tissue reconstruction was then evaluated. After 6 and 12 weeks of implantation, sections of brains were processed with Nissl and Glees staining for revealing neural cell bodies and fibers, with DAB histochemistry for detecting the blood vessels, as well as with immunocytochemistry for recognizing GFAP. The sections were also taken to SEM and TEM for ultrastructral examination. The results showed that the HA hydrogels synthesized had mechanical properties and rheological behavior similar to the brain tissue. After being implanted into the lesion of the cortex, the porous hydrogels created a scaffold, which could support cell infiltration and angiogenesis, and simultaneously inhibit the formation of glial scar. In addition, HA hydrogels modified with laminin could promote neurite extension. It seems possible that the tissue engineering technique may pave the way to repair injury in the CNS as suggested by the results in present study.

Animals↗

[Spin-lattice-relaxationtime-T1 measurements of hyaluronic acid (author's transl)].

Reductions with [3H5NaBH4 proof that the decrease in viscosity of hyaluronic acid solutions caused by lowering the pH does not depend on a depolymerisation of hyaluronic acid. At the same time investigations at different pH-values, show a sigmoide increase of the Spin-Lattice-Relaxationtime T1. This increase depends on a progressive aggregation of the hyaluronic acid molecule. The effect seems to be induced by the decrease of ionization of the carboxylgroups, by acidification of the solution.

Carbohydrate Conformation↗

Reticulated hyaluronic acid implant in nonperforating trabecular surgery.

PURPOSE: To evaluate experimentally and clinically the tolerance and efficacy of a reticulated hyaluronic acid implant in nonperforating trabecular surgery (NPTS). SETTING: Bascom Palmer Eye Institute, Miami, Florida, USA, and Clinique Sourdille and Clinique Ophtalmologique Universitaire, Nantes, France. METHODS: In experimental surgery, NPTS was performed with and without a hyaluronic acid implant in 25 rabbit eyes. In a pilot study, the results of NPTS with a hyaluronic acid implant in 72 human eyes were retrospectively analyzed in terms of visual acuity, intraocular pressure (IOP), external filtration, postoperative inflammation, and gonioscopy. Mean follow-up was 13.8 months (range 6 to 24 months). RESULTS: In the experimental surgery, the rabbit eyes with the implant showed a different healing process than the eyes without the implant. The implant was slowly bioabsorbed and remnants were seen at the operative site (where the tissue was removed) up to day 56 postoperatively. This site was detectable at all histology study periods. Intraocular pressure reduction was longer in the implant group: greater than 5 months versus 3 weeks (P < .05). In the pilot study, visual acuity remained stable, IOP decreased from a mean preoperative level of 26.3 mm Hg +/- 5.22 (SD) to a mean postoperative level without treatment of 15.4 +/- 3.1 mm Hg (P < .0001). No external filtration was detected in 60 eyes, a slightly elevated conjunctiva was noted in 12 eyes. Postoperative inflammation (laser flare and cell measurements) was low. Gonioscopy consistently demonstrated the persistence of a decompression space behind the trabeculum. CONCLUSION: Comparative experimental surgery results showed excellent tolerance and efficacy in the rabbit eyes with a hyaluronic acid implant. Clinical results, to be confirmed by a randomized comparative study, also showed excellent biocompatibility and encouraging efficacy.

Aged↗

Intra-articular injection of high-molecular hyaluronic acid. An experimental study on normal adult rabbit knee joints.

High-molecular hyaluronic acid was injected repeatedly into normal knee joints of adult rabbits. Histologically the articular cartilage was not affected by this treatment. The content of chondroitin sulphate, assessed by a histochemical method, was not altered and neither was the water content of the hyaline articular cartilage. On the other hand, in the synovial membrane of joints treated with hyaluronic acid a diffuse infiltration of plasma cells and lymphocytes was observed, indicating that the hyaluronic acid administered exerts its action via the synovial membrane.

Animals↗

Hyaluronic acid modulates cell proliferation unequally in intrasynovial and extrasynovial rabbit tendons in vitro.

As tendons differ in biochemical composition and cellular capacities, we have compared dose response effects of hyaluronic acid on cell proliferation and synthesis of matrix components in intermediate and proximal segments of intrasynovial deep flexor tendons and extrasynovial peroneus rabbit tendons in vitro. Compared with matched control tendons, hyaluronic acid inhibited cell proliferation in intermediate and proximal intrasynovial flexor tendon segments at the concentrations of 0.1-2.0 mg/ml and 0.5-2.0 mg/ml respectively, but in extrasynovial tendon segments only at the concentration of 0.5 mg/ml. Hyaluronic acid did not affect synthesis of proteoglycan, collagen and non-collagen protein in either type of tendon. These results show that hyaluronic acid modulates cell proliferation unequally in intra- and extrasynovial tendons without affecting the synthesis of matrix components in the two types of tendons, indicating differential hyaluronic acid sensitivity and a possible mechanism of action.

Animals↗

Biochemical characteristics of dissociatively isolated aortic proteoglycans and their binding capacity to hyaluronic acid.

Proteoglycans were extracted from pigeon aortas with 4.0 M guanidine hydrochloride containing protease inhibitors to characterize the proteoglycan (PG) types, to examine and define their association with hyaluronic acid, and to compare pigeon aortic PG with human aortic PG isolated and characterized under similar conditions. Following chromatography of guanidine hydrochloride extracts on Sepharose CL-4B under dissociative conditions, two populations of PG were isolated. PG I, eluting at the column void volume, contained predominantly (83.2%) chondroitin sulfate whereas PG II, eluting at a Kav of 0.40, contained predominantly dermatan sulfate (54.7%). Following centrifugation, 76 and 63% of PG I and PG II, respectively, were located in the bottom two-fifths (p greater than 1.43 g/ml) of a CsCl gradient. Only PG I, predominantly chondroitin sulfate, was effective in associating with hyaluronic acid to form high molecular weight aggregates. The specific association was illustrated by removing endogenous arterial hyaluronic acid remaining in the preparation and demonstrating increasing formation of aggregate with increasing addition of human umbilical cord high molecular weight hyaluronic acid. The results of the study illustrate that artery PG of the pigeon are similar in distribution to those described for the human aorta and contain at least three types of PG, a chondroitin sulfate PG, a dermatan sulfate PG, and a polydisperse heparan sulfate PG. The chondroitin sulfate PG but not the dermatan sulfate PG are capable of forming high molecular weight aggregates with hyaluronic acid.

Animals↗