Umbilical Cord Hyaluronic Acid Research Articles

Production of a composite hyaluronic acid/gelatin blood plasma gel for hydrogel‐based adipose tissue engineering applications

Standard approaches to soft-tissue reconstruction include autologous adipose tissue transplantation, but most of the transferred adipose tissue is generally reabsorbed in a short time. To overcome this problem, long lasting implantable hydrogel materials that can support tissue regeneration must be produced. The purpose of this study was to evaluate the suitability of composite 3D natural origin scaffolds for reconstructive surgery applications through in vitro tests. The Young's modulus of the glutaraldehyde crosslinked hyaluronic acid/gelatin (HA/G) plasma gels, composed of human platelet-poor plasma, gelatin and human umbilical cord hyaluronic acid, was determined as 3.5 kPa, close to that of soft tissues. The composite HA/G plasma gels had higher porosity than plain plasma gels (72.5% vs. 63.86%). Human adipose tissue derived stem cells (AD-MSCs) were isolated from human lipoaspirates and characterized with flow cytometry, and osteogenic and adipogenic differentiation. Cell proliferation assay of AD-MSCs on the HA/G plasma gels revealed the nontoxic nature of these constructs. Adipogenic differentiation was distinctly better on HA/G plasma gels than on plain plasma gels. The results showed that the HA/G plasma gel with its suitable pore size, mechanical properties and excellent cell growth and adipogenesis supporting properties can serve as a useful scaffold for adipose tissue engineering applications.

Effect of Emblica officinalis (fruit) against UVB-induced photo-aging in human skin fibroblasts

Emblica officinalis fruit (EO), commonly known as Amla is a reputed traditional medicine and functional food used in Indian subcontinent. It has long been used in Indian folk medicine to treat liver diseases, stomach ulcers, inflammatory diseases, metabolic disorders, geriatric complaints, skin disorders and beauty care. Recently, it has been shown to promote pro-collagen content and inhibit matrix metalloproteinase levels in skin fibroblast. The aim of the present study was to investigate the efficacy of EO to inhibit UVB-induced photo-aging in human skin fibroblasts. Mitochondrial activity of human skin fibroblasts was measured by MTT-assay. Quantifications of pro-collagen 1 and matrix metalloproteinase 1 (MMP-1) release were performed by immunoassay techniques. Hyaluronidase inhibition assay was studied in vitro using bovine testicular hyaluronidase and human umbilical cord hyaluronic acid. Cell cycle analysis was performed by flowcytometry using propidium iodide. EO stimulated, the otherwise UVB inhibited cellular proliferation and protected pro-collagen 1 against UVB-induced depletion via inhibition of UVB-induced MMP-1 in skin fibroblasts (10-40 μg/mL, p>0.001). EO exhibited inhibitory activity of hyaluronidase (10-40 μg/mL, p>0.001). Treatment with EO also prevented UVB disturbed cell cycle to normal phase. The results of the present study suggests that EO effectively inhibits UVB-induced photo-aging in human skin fibroblast via its strong ROS scavenging ability and its therapeutic and cosmetic applications remain to be explored.

Electrical conduction in macroscopically oriented deoxyribonucleic and hyaluronic acid samples

Measurements of the quasistatic and frequency dependent electrical conductivity below 1 MHz were carried out on wet-spun, macroscopically oriented, calf thymus deoxyribonucleic (DNA) and umbilical cord hyaluronic acid (HA) bulk samples. The frequency dependence of the electrical conductivity in the frequency range of approximately 10(-3) - 10(6) Hz of both materials is surprisingly rather similar. Temperature dependence of the quasistatic electrical conductivity above the low temperature saturation plateau can be well described by the activated Arrhenius law with the activation energy of approximately 0.8 eV for both DNA and HA. We discuss the meaning of these findings for the possible conduction mechanism in these particular charged polyelectrolytes.

Plasmodium falciparum Cytoadherence to Human Placenta: Evaluation of Hyaluronic Acid and Chondroitin 4-Sulfate for Binding of Infected Erythrocytes

Valiyaveettil, M., Achur, R. N., Alkhalil, A., Ockenhouse, C. F., and Gowda, D. C. 2001. Plasmodium falciparum cytoadherence to human placenta: Evaluation of hyaluronic acid and chondroitin 4-sulfate for binding of infected erythrocytes. Experimental Parasitology99, 57–65. Chondroitin 4-sulfate (C4S) is known to mediate the adherence of Plasmodium falciparum infected red blood cells (IRBCs) to human placenta. Recently, hyaluronic acid (HA) has also been reported to bind IRBCs, and HA has been suggested as an additional receptor for the sequestration of IRBCs in the placenta. In this study, we assessed the adherence of 3D7 parasite strain, which has been reported to bind both C4S and HA, using highly purified clinical grade rooster comb HA, Streptococcus HA, several preparations of human umbilical cord HA (hucHA), and bovine vitreous humor HA (bvhHA). While all hucHA preparations and bvhHA bound with moderate to high density to IRBCs, the rooster comb and bacterial HAs did not bind IRBCs. IRBCs binding to the hucHA and bvhHA could be abolished by pretreatment with testicular hyaluronidase but not with Streptomyces hyalurolyticus hyaluronidase, suggesting that IRBC binding to hucHA and bvhHA was due to chondroitin sulfate (CS) contaminants in HAs. Compositional analysis confirmed the presence of CS in both hucHA and bvhHA. The CSs present in these commercial hucHA and bvhHA samples were isolated, characterized, and studied for their ability to bind IRBCs. The data suggested that IRBC adherence to hucHA and bvhHA was mediated by the CS present in these samples. However, our data did not exclude the possibility of a minor population of distinct parasite subtype adhering to HA and further studies using pure HA conjugated to proteins or lipids and placental parasite isolates should clarify whether HA is an in vivo receptor for IRBC adherence.

Assay of synovial fluid hyaluronic acid using high-performance liquid chromatography of hyaluronidase digests

A high-performance liquid chromatographic method for the determination of hyaluronic acid levels in synovial fluids has been developed. The hyaluronidase sample digests, containing an internal standard (benzoic acid), were separated on a reversed-phase octadecylsilyl column eluted with 0.01 M tetrabutylammonium phosphate-acetonitrile (83:17, v/v) at pH 7.35. The determination was made on 1:10 diluted samples, by using a calibration curve from 50 to 500 micrograms/ml of human umbilical cord hyaluronic acid. For validation, the synovial fluids were simultaneously analysed by this method and a radiometric method: a high correlation was found between the two (correlation coefficient 0.94). The proposed method can be used to determine specifically the high hyaluronic acid levels of synovial fluids without interferences from other glycosaminoglycans or non-steroidal anti-inflammatory drug treatment.

PH control of the third virial coefficient of hyaluronate solutions calculated from van der Waals forces by means of the Lifshitz-McLachlan susceptibility theory

Positive third virial coefficients and osmotic coefficients have been calculated for human umbilical cord hyaluronic acid solutions at pHs 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 and constant ionic strength 0.1. The calculations are based on experimental axial flow birefringence and radial linear dichroism data previously reported and the Lifshitz-McLachlan field theory of van der Waals forces. The second virial coefficients are negative, according to both this analysis and light scattering evidence, and reflect the tendency of hyaluronic acid to associate. This negativity denies the assumption of force additivity required by virial expansion theory.

Substrate-bonded hyaluronic acid exhibits a size-dependent stimulation of chondrogenic differentiation of stage 24 limb mesenchymal cells in culture

Extracellular matrix molecules including glycosaminoglycans have been implicated in several differentiative and morphogenetic processes including cell aggregation and migration. Previous reports have shown that plating of stage 24 limb mesenchyme cells onto hyaluronic acid (HA) bonded to the culture substrate causes an increase in the number of cells exhibiting chondrogenesis. This increased chondrogenesis is now shown to be dependent upon the source of the HA. When limb mesenchymal cells are plated onto HA from bovine vitreous humor, human umbilical cord, or large molecular weight HA (Healon), increased chondrogenesis is observed only on the bovine vitreous humor HA. Unsulfated chondroitin, which has a structure and charge density similar to those of HA, is capable of enhancing chondrogenesis, while cells plated onto sulfated glycosaminoglycan substrates are indistinguishable from controls. The evidence in this report suggests that the differentiation response is related to the molecular size of the HA bound to the culture substrate. Healon and human umbilical cord HA are ineffective because their molecular weight is too large, while smaller HA derived from these larger molecules or normally present in bovine vitreous humor preparations stimulates the chondrogenic differentiation of stage 24 limb mesenchymal cells in culture. The most active size class of HA elutes from a Sepharose CL-2B column with a K av between 0.6 and 0.7 and, thus, has a molecular weight of approximately 200,000–400,000. These observations reinforce the hypothesis that local cues have an informational effect on the differentiation of chick limb mesenchymal cells.

Depolymerisation products of hyaluronic acid after exposure to oxygen-derived free radicals.

Preparative chromatographic fractions of human umbilical cord hyaluronic acid (HA) of a molecular weight of 10(6) were subjected to graded oxygen-derived free radical (oxy radical) fluxes produced by: (a) the autoxidation of ferrous ions; (b) the action of xanthine oxidase (XO) on hypoxanthine (HX); and (c) by peripheral blood polymorphonuclear leucocytes that had been stimulated by phorbol myristate acetate (PMA). Analysis by gel chromatography of the products obtained with each of the oxy radical generating systems showed polydispersity in size. The smallest molecules detected had a molecular weight of 10(4). This limiting size was not reduced further by exposure to a second oxy radical flux. The relative proportions of large, medium, and small degradation products were established for various levels of oxy radical flux. Consistently a relatively rapid transition from large to small material was seen on Sepharose 2B chromatography, suggesting an ordered element to the breakdown process. Although the decrease in molecular weight after oxy radical exposure was confirmed by analytical ultracentrifugation, this procedure showed that those samples of lowest viscosity did not have the lowest sedimentation values, possibly reflecting oxy radical-induced repolymerisation. If the size and possibly the conformational characteristics of HA are altered, oxy radical exposure might be expected to alter its biological properties.

Inhibition of oxidative degradation of hyaluronic acid by uric acid.

It has been postulated that glycosaminoglycans in the trabeculum have an influence on aqueous humor drainage. Ascorbate reduces the viscosity of hyaluronic acid, and also increases outflow facility. Our recent observation of high urate concentrations in some glaucomatous eyes led us to study the influence of urate on oxidative degradation of hyaluronic acid by ascorbate. The viscosity of rooster comb hyaluronic acid was reduced slowly by ascorbate. Cupric sulfate accelerated ascorbate oxidation and also enhanced hyaluronic acid degradation. Urate inhibited ascorbate oxidation and prevented the copper catalyzed oxidative degradation of rooster comb hyaluronic acid. The range of urate concentrations used in this study was within the range of urate concentrations observed in glaucomatous eyes. The partially purified umbilical cord hyaluronic acid had lower viscosity than rooster comb hyaluronic acid, and rapidly degraded in the presence of ascorbate. The ascorbate effect on umbilical cord hyaluronic acid was partially prevented by urate.

Dielectric changes in hyaluronate solutions at microwave frequencies as a function of concentration, system pH, and temperature

AbstractThe dielectric response of human umbilical cord hyaluronic acid in various environments has been studied at microwave frquencies using a resonant microwave cavity as a probe. Both the real and imaginary parts of complex dielectric constant and the loss tangent for hyaluronate solutions are obtained by utilizing equations for perturbation of a resonant cavity. Dielectric changes at room temperature have been observed in aqueous solutions of hyaluronic acid as a function of concentration ranging from 0 to 350 mg/ml. The data indicate the existence of ordered phases in hyaluronate solutions at selective concentrations, that is, exhibiting lyotropic‐type transitions. Hyaluronate solutions at 1.5 and 3 mg/ml concentrations have been studied at various pH in the range of 6–8 and at constant ionic strength 0.1. A temperature‐dependent transition in hyaluronate solution of 120 mg/ml concentration has been observed at physiological temperature. It is shown that this temperature‐dependent behavior can be related to the orientational polarizability term in the Debye theory of polar molecules in liquids.

Cartilage proteoglycan aggregates. Electronmicroscopic studies of native and fragmented molecules

1. Proteoglycan aggregates from bovine nasal cartilage were studied by using electron microscopy of proteoglycan/cytochrome c monolayers. 2. The aggregates contained a variably long central filament of hyaluronic acid with an average length of 1037nm. The proteoglycan monomers attached to the hyaluronic acid appeared as side chain filaments varying in length (averaging 249nm). They were distributed along the central filament at an average distance of about 36nm. 3. Chondroitin sulphate side chains were removed from the proteoglycan monomers of the aggregates by partial chondroitinase digestion. The molecules obtained had the same general appearance as intact aggregates. 4. Proteoglycan aggregates were treated with trypsin and the largest fragment, which contains the hyaluronic acid, link protein and hyaluronic acid-binding region, was recovered and studied with electron microscopy. Filaments that lacked the side chain extensions and had the same length as the central filament in the intact aggregate were observed. 5. Hyaluronic acid isolated after papain digestion of cartilage extracts gave filaments with similar length and size distribution as observed for the central filament both in the intact aggregate and in the trypsin digests. 6. Umbilical-cord hyaluronic acid was also studied and gave electron micrographs similar to those described for hyaluronic acid from cartilage. However, the length of the filament was somewhat shorter. 7. The electron micrographs of both intact and selectively degraded proteoglycans corroborate the current model of cartilage proteoglycan structure.

Linear dichroism and radial flow birefringence of stream‐oriented hyaluronate solutions as a function of system pH

AbstractThe linear dischroism in the radial direction of solution flow over a velocity gradient of approximately 10–850 sec−1 has been obtained for human umbilical cord hyaluronic acid at concentrations of 0.1, 0.0666, and 0.0333%, and pHs 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5, at constant ionic strength 0.1. The redial flow briefingenece has also been obtained for these solutions by a Kroing‐Kramers transformation. The data indicate a change in linear dichroism and optical anisotrophy as a function of pH, with most of the transition across the physiological range: 7.0–7.5. Previously reported axial flow birefringence and extinction angles indicating a similar pH‐dependent structural change in hyaluronate solutions across the same pH range are thus confirmed with other instrumentation. The radial birefringence measurements reported here, together with the axial measurements previously reported, permit a complete description of the refractive index tensor for each of the six solutions. The interpretation of these tensors in terms of the Peterlin modification of the Rouse‐Zimm Gaussian subchain theory results in a description of entropy production induced by changes in system pH.

Purification and characterization of a hyaluronidase associated with a temperate bacteriophage of group A, type 49 streptococci.

Urea treatment of a temperate bacteriophage from a type 49 strain of group A streptococcus (Streptococcus pyogenes) followed by ammonium sulfate fractionation, ion exchange, and affinity chromatography of solubilized proteins provided for the recovery (12%) and purification (44-fold) of the phage-associated hyaluronidase. The molecular weight of the homogeneous, purified enzyme was estimated to be 71,000 by polyacrylamide gel electrophoresis (in the presence of sodium dodecyl sulfate) and 75,000 by gel filtration with Sephacryl S-200. The enzyme has a pH optimum of 5.5, a Vmax of 0.1 absorbance unit/min per microgram of protein, and a Km of 4.8 X 10(-2) mg/ml with umbilical cord hyaluronic acid as substrate. Of the cations tested, calcium and magnesium were the only effectors of the enzyme. The enzyme is a glycoprotein (7.25% carbohydrate) containing glucose, galactose, and glucosamine. Analysis of the amino acid composition revealed a predominance of acidic amino acids and a relatively high content of cysteine. The partial specific volume, estimated from the amino acid and sugar analyses, was 0.725 cm3/g.

Low velocity gradient flow birefringence and viscosity changes in hyaluronate solutions as a function of pH.

AbstractThe flow birefringence and extinction angle over a velocity gradient range of approximately 5–100 sec−1, and the zero shear‐viscosity have been obtained from human umbilical cord hyaluronic acid at concentrations of 0.25, 0.125 and 0.0625%, and pHs 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 and constant ionic strength 0.1. The data indicate a large change in optical anisotropy as a function of pH, with most of the transition in the pH range 7.0–7.5, i.e., across the physiological range. The sign of the anisotropy changes between pH 8.0 and 8.5. These results, together with changes in the extinction angle and intrinsic viscosity as a function of pH, suggest a pH‐dependent structural change in the system. Due to the abruptness of the transition, as evidenced by the intrinsic viscosity and flow birefringence, it is probable that the structural transition is cooperative. If the data are interpreted in terms of the Rouse‐Zimm Gaussian subchain theory, a modification of the model in terms of the Haller‐Cerf concept of internal viscosity is required. Thus, the demonstrated properties of hyaluronate solutions indicate a system with memory of stress. Due to the presence of large concentration effects discernible in the extinction angle measurements, hyaluronic acid probably exists as a network in solution. The results are discussed with respect to the mechanoelectrical transducing properties of hyaluronates and stress‐dependent changes in ORD already reported.

Simultaneous determination of neutral sugars and hexosamines in glycoproteins and acid mucopolysaccharides (glycosaminoglycans) by gas-liquid chromatography

A reliable and reproducible method for the simultaneous determination of neutral sugars and hexosamines in glycoproteins and acid mucopolysaccharides has been described. It involves the following steps: the release of neutral sugars and hexosamines from biopolymers by resin-catalysed hydrolysis, the nitrous acid deamination of resin-bound hexosamines in this hydrolysate to aldoses, and the determination of these newly formed anhydroaldoses together with the pre-existing neutral sugars as aldononitrile acetates by gas chromatography. Application of this method to analyses of glycoprotein from urina of schizophrenic patients, bovine thyroglobulin, human brain glycoprotein, chondroitin sulfates from bovine vitreous humor, and veal brain, and human umbilical cord hyaluronic acid are given.

Antigenicity of hyaluronic acid.

SummaryAntigens were prepared from human umbilical cord hyaluronic acid, streptococcal hyaluronic acid, group A hemolytic streptococci and Staphylococcus aureus and these same bacteria incubated with human umbilical cord hyaluronic acid. Rabbits were injected intravenously, intramuscularly and subcutaneously with these antigens. Neither precipitating nor non-precipitating, skin-sensitizing antibodies against hyaluronic acid developed. Anti C carbohydrate and anti M protein antibodies were produced in the animals which received streptococci, indicating that the rabbits had received enough of at least 2 antigens, one a carbohydrate the other a protein, to stimulate antibody production. The development of skin sensitivity to hyaluronic acid was suggested but by no means proved. These experiments furnish further evidence that hyaluronic acid is probably not antigenic by itself, nor does it act as a hapten capable of adsorption to group A hemolytic streptococci or Staphylococcus aureus.

ISOLATION OF THE ALDOBIONIC ACID OF UMBILICAL CORD HYALURONIC ACID

ISOLATION OF THE ALDOBIONIC ACID OF UMBILICAL CORD HYALURONIC ACID