High Molecular Hyaluronic Acid Research Articles

Abstract NG09: Bioengineered tissue mimetic hydrogels to study brain tumor biology and screen therapeutics

Abstract NG09: Bioengineered tissue mimetic hydrogels to study brain tumor biology and screen therapeutics

Influence of high-molecular hyaluronic acid on MCF-7 breast cancer stem cells

In recent years, more and more data have been accumulating on the high radio- and chemo-resistance of tumor stem cells (CSCs), confirming the assumption that these cells play an important role in the recurrence of the tumor process in some cancer patients. Therefore it is of considerable interest to elucidate the patterns and mechanisms of the formation of the CSC pool under the influence of various factors, including one of the main components of the extracellular matrix, hyaluronic acid (HA). The purpose of the study was to elucidate the effects of HA with a molecular weight of (1-3) x 106 Dа on the amount of CSCs in MCF-7 breast cancer cell culture in vitro. Material and methods. Using the MTT test, the viability of MCF-7 cancer cells was determined after incubation with HA at a concentration of 0.3-5.0 mg/ml. Using flow cytometry, we evaluated the change in the relative and absolute amount of CSCs under the influence of HA (0.6 mg/ml). CSCs were detected by CD44+CD24-/low immunophenotype, as well as by their ability to efficiently efflux Hoechst 33342 fluorescent dye from cells and form so-called side population (SP). Results. Viability of cancer cells in the general population decreased with increasing concentration of HA (R=-0.94, p=0.006). At the same time the incubation of cells with HA led to an increase in both the relative and absolute amounts of CSCs (SP) compared with the control. Thus, the relative number of SP cells increased by 2.0 times (p = 0.002), CD44 + CD24- / low cells - by 6.1 times (p = 0.005). The absolute number of these cells changed to a lesser extent compared to the control: the increase was 1.6 (p = 0.004) and 2.9 times (p = 0.02), respectively. Conclusion. Thus, the effect of high-molecular weight HA on breast cancer cells of MCF-7 line leads to an increase in amount of CSCs, while the viability of non-stem cancer cells decreases under the same conditions.

Management tactics for patients with osteoarthritis: a joint view of a rheumatologist and an orthopedic traumatologist

Osteoarthritis is a common disease and occupies the leading place in the number of cases of disability in the world. The pathogenesis of this topical nosology is actively condemned, and researchers have offered a lot of evidence for the leading role of inflammation in the pathogenesis of osteoarthritis, which, by stimulating internal catabolic reactions of chondrocytes, leads to inflammation of the synovial membrane and, as a result, the occurrence of joint pain. Osteoarthritis is the disease with the highest incidence of comorbidities, which complicates the choice of medications for its treatment. And, despite the variety of recommendations, the issue of treating patients with OA remains complex. Based on the recommendations of ESCEO, which were updated in 2019, it is necessary to follow a step-by-step tactic, according to which the introduction of hyaluronic acid preparations intra-articularly is recommended at the 2nd stage of therapy as part of advanced pharmacological correction. Intra-articular hyaluronic acid injection is an alternative local treatment option that provides symptomatic benefit without systemic adverse events. The clinical example demonstrates the experience of joint decision-making on the tactics of managing a patient with persistent OA and the ineffectiveness of therapy at the previous stages. Preference was given to the preparation with high-molecular hyaluronic acid, in the production of which no cross-linking agents were used, due to its effectiveness and safety. A good analgesic and symptom modifying effect of the drug was demonstrated with the result remaining for 6 months. The authors believe that in patients with high comorbidity, drugs for intra-articular administration of hyaluronic acid can take a worthy place in the treatment of osteoarthritis.

Noncanonical effects of vasopressin in angiogenesis

The molecular action of vasopressin depends on the localization of hormonal receptors. The basic physiological effects of vasopressin are manifested in the blood vasculature, renal inner medulla and brain. To date, new information concerning the tissue-specific spreading of vasopressin receptors has been accumulated, and it needs to be summarized. Platelets and endotheliocytes expressing V1a and V2 receptor types, respectively, are related to less investigated targets of the hormone. Vasopressin induces the initial reversible stage of platelet activation, required for interaction with intercellular matrix proteins. Platelet adhesion on endothelium activates cellular secretion of growth factors and enzymes for intercellular matrix glucosamine metabolism. Platelet hyaluronidase HYAL2 hydrolyses high-molecular hyaluronic acid to shorter fragments. Unlike intact hyaluronic acid with a molecular weight of several megadaltons, generally showing distinctive antiangiogenic properties, intermediate fractions of hyaluronan hydrolysis in a range from 2.5 to 200 kilodaltons have a stimulating effect on angiogenesis. Intercellular contacts between platelets and endotheliocytes are stabilized due to adhesive transmembrane glycoprotein PECAM-1 interaction. Resulting PECAM-1 heterodimers acquire conformation with high affinity to integrins αvβ3. Integrin activation forms contact links between endothelium and fibrillar proteins. Activated endotheliocytes secrete von Willebrand factor and P-selectin. These proteins are accumulated in Weibel–Palade bodies. Vasopressin stimulates cAMP-dependent ACAP-regulated exocytosis of Weibel–Palade bodies. von Willebrand factor possesses adhesive properties and additionally accelerates interaction of cells with the intercellular matrix. Adhesion on fibrillar collagen and membrane glycoproteins in cooperation with effects of PECAM-1–αvβ3 integrin complexes fixes cell aggregates in the surrounding interstitium and promotes proliferating endotheliocyte migration in according to the direction of local growth factor gradients during angiogenesis. Neurohormonal regulation of platelet and endotheliocyte secretory activity functionally link proliferation and migration of endotheliocytes during angiogenesis and integrate it according to the adaptive capacity of the entire organism.

Efficacy of local injection therapy with hyaluronic acid of different molecular weight in patients with knee osteoarthritis

Intra-articular administration of hyaluronic acid (HA) is one of the methods of conservative treatment of knee osteoarthritis (OA). However, the data of its efficacy in the modern literature are contradictory.Objective: to assess the efficacy of intra-articular administration of HA preparations with different molecular weight in the treatment of patients with knee OA and to study the factors that can influence the effectiveness of such therapy.Subjects and methods. HA preparations were administered to 60 patients with idiopathic and posttraumatic knee OA. The ratio of women to men was 3:1. The average age was 59.5±13.7 years and body mass index – 30.3±5.8 kg/m2. The presence of enthesitis was evaluated prior to treatment by the characteristic clinical picture. The x-ray stage of OA was determined in accordance with the classification of Kellgren–Lawrence. Distribution of of knee joint lesions, relationship of femur and tibia anatomical axes, presence of varus and valgus deformities were also evaluated. The patients were divided into three groups: 18 patients received low-molecular (LM), 19 – medium-molecular (MM) and 23 – high-molecular (HM) HA. To assess the efficacy of the treatment the severity of pain on a visual analog scale (VAS) and the total score of the Knee injury and Osteoarthritis Outcome Score (KOOS) questionnaire before, 1, 3 and 6 months after treatment were evaluated.Results and discussion. Enthesitis was detected in 17 (28,3%) patients. Stage I of knee OA was diagnosed in 10 (16.6%), II – in 36 (60%) and III – in 14 (23.3%) patients. Varus deformity was detected in 43 (71.7%) patients, neutral axis of the lower limb - in 14 (23.3%), valgus deformity – in 3 (5%). The most significant decrease in the severity of pain was noted in patients with stage I of knee OA: from 54±17.1 to 10.0±10.0 mm. At stage II, the pain decreased from 59.4±12.6 to 45.5±20.3 mm, and at stage III – from 66.4±13.5 to 60.7±23.3 mm. By the 6th month, the severity of pain in patients with enthesitis was higher, and the values of KOOS total score were worse than in patients without it. At stages II and III of knee OA, the ratio of positive to unsatisfactory results for MM, LM and HM HA was as follows: 2:1, 3:1 and 3.6:1. In the group of patients with excellent results, the value of varus deformation averaged 8.5±1.5°, in patients with a good result – 9.8±2.8°, with a satisfactory result – 11.3±4.5°, and with an unsatisfactory – 11.5±3.2°.Conclusion. Intra-articular injection of HA is very effective at stage I of knee OA. The expression and duration of this effect are reducedwith decrease of cartilage thickness and increase of knee joint varus deformation (i.e., at the II and III stages of knee OA). The presence of varus deformation does not affect HA efficacy. Three factors are important: the appearance of varus deformation of the lower limb with initially neutral axis; the increase of varus deformation; the value of varus deformation >10°.

Preparation and Characterization of Sponge Using Different Ratios of High Molecular Hyaluronic Acid and Small Intestine Submucosa

Preparation and Characterization of Sponge Using Different Ratios of High Molecular Hyaluronic Acid and Small Intestine Submucosa

Hydroxyapatite combined with hyaluronic acid metronidazole gel increased the quantity of osteoblasts in the alveolar bone wistar rat

Background: Bone graft material have been used extensively in bone healing and periodontal treatment. Alloplast such as hydroxyapatite are frequently used to repair and reconstruct bone defects. By merely applying hydroxyapatite for the treatment of bone is not fully effective yet to produce new bone regeneration. Locally applied high molecular hyaluronic acid (HA) has been shown to stimulate differentiation and migration of mesenchymal cells. Recent studies on regenerative surgical procedures indicate that reduction of bacterial burden at the wound site may improve the clinical outcome of regenerative therapy. Metronidazole has the greatest bacteriostatic effect. A clinical application of HA metronidazole gels during the surgical therapy may reduce the bacterial contamination of surgical wound site. Purpose: The purpose of this study was to examine the effect of combination hydroxyapatite and HA metronidazole gel 1% on osteoblast cell number after wound healing process in the wistar rats incisor tooth extraction socket. Method: Twenty seven wistar rats were divided randomly into 3 groups. The first group consisted of wistar rats given hydroxyapatite were subjected to the mandibular incisor extraction socket. The second group were given hydroxyapatite combined with HA metronidazole gel 1%. The control group were filled with blood. Wistar rats were euthanized on day 14 and then preparation for histological examination was stained using hematoxylin-eosin and then the numbers of the osteoblasts were calculated. Result: The differences in each group were tested by one way Anova test ( α =0.05). The numbers of osteoblasts in each group had a significant difference (p<0.05): the highest numbers of osteoblasts were found in the group that was given hydroxyapatite combined with HA metronidazole, followed by the lower numbers of osteoblasts in the hydroxyapa ite group and the lowest numbers of osteoblasts were found in the control group. Conclusion: Adjunctive application of HA metronidazole to hydroxyapatite after tooth extraction increase the number of osteoblast in the treatment of bone defects on a wistar rat model.

COMPARATIVE EFFICACY OF INTRAARTICULAR THERAPY OF GONARTHROSIS WITH HYALURONIC ACID OF DIFFERENT MOLECULAR WEIGHT (A DOUBLE-BLIND RANDOMI7ED STUDY)

Objectives. To study the comparative efficacy and safety of intra-articular treatment of knee osteoarthritis (OA) with hyaluronic acid (HA) of different molecular weights (MW). Material and methods. Randomized double-blind controlled study included 50 patients with osteoarthritis of the knee stage II-III, weight-bearing pain more 40 mm on VAS and Lequesne index score &gt; 4 and &lt; 12. 25 patients received HA with MW 3500 kDa (Rusvisk group) and 25 patients were treated with HA with MW 1200-1400 kDa (Ostenil group) injected at 3-weekly intervals. During the 22-week observation period we assessed dynamics of pain on VAS (weightbearing pain, pain at rest), index WOMAC, and recorded the overall assessment of the effectiveness of therapy by patient and physician, OMERACT-OARSI criteria and the need for NSAIDs. Results. One week after the last injection, a significant decrease in weight-bearing pain (in the group Rusvisk by 69% in the group Ostenil 55%) and the total index WOMAC (group Rusvisk by 63% in the group Ostenil 60%), continuing until the end of observation. A significant decrease in pain at rest observed in the group Rusvisk at all visits and in the group Ostenil only at 6 and 10 weeks of therapy. The overall evaluation of effectiveness of therapy by patient and physician yielded the benefits observed in high molecular HA group. A high percentage of responses to therapy using OMERACT-OARSI criteria and reduced need for NSAIDs were observed in both groups. Tolerability was satisfactory and did not differ significantly between the groups. Most common adverse reactions were mild pain at the injection site. Conclusion. Intra-articular therapy with HA of different molecular weight Rusvisk and Ostenil resulted in pain reduction and improved joint function in patients with knee OA with satisfactory tolerability.

AB0066 Cyclic phosphatidic acid (cpa) stimulates hyaluronic acid production in human osteoarthritic articular chondrocytes, and intra-articular administration of cpa suppresses pain, swelling, and cartilage destruction in rabbit experimental osteoarthritis.

BackgroundCyclic phosphatidic acid (cPA) is one of lipid mediators, has been shown various biological effects1. On human skin fibroblasts, cPA stimulates high molecular hyaluronic acid (HA) production through up-regulating HA...

BMP-2 and BMP-7: differential regulation of chondrogenic differentiation

BMP-2 and BMP-7: differential regulation of chondrogenic differentiation

Establishment of novel three-dimensional culture system for human chondrocytes using micro-space plate

Purpose: It has been reported that monolayer culture of human articular chondrocytes results in dedifferentiation and loss of chondrogenic property. Therefore, the dedifferentiation is a big problem when the influence of cytokines and drugs were evaluated. In order to solve this problem, we performed a novel three-dimensional culture system for human chondrocytes using micro-space plates compared with monolayer culture. Methods: The micro-space plate has regularly arranged 200 mm×100 mm×50 mm square compartments on its surface. Human articular chondrocytes were cultured in flat plates or micro-space plates for 1 week. After culture, the level of collagen 1A1 (COL1A1) and aggrecan mRNA expressions were measured by real-time PCR, and cartilage matrix production was assessed by alcian blue staining. Furthermore, chondrocytes were cultured with IL-1β, IL-6+soluble IL-6 receptor (sIL-6R) in the presence or absence of high molecular hyaluronic acid (HA) for additional 24 h and then the production of MMP-3 in cell supernatant was measured by ELISA. Results: At the same passage of cells, human articular chondrocytes grown on micro-space plates in clusters after 5-7 days’ culture, whereas cells grown on flat plates in fibroblast-like morphology. In three-dimensional culture, the mRNA expression levels of COL1A1 and aggrecan were higher than those in monolayer culture. And the cartilage matrix production in three-dimensional culture was higher than that in monolayer culture. Moreover, the production of MMP-3 induced by IL-1β and IL-6+sIL-6R was higher in three-dimensional culture than in monolayer culture. Inhibitory activity of HA on MMP-3 production was similar between three-dimensional culture and monolayer culture. Over the subsequent passage, the expression levels of COL1A1 and aggrecan mRNA and MMP-3 production by IL-1β and IL-6+sIL-6R gradually decreased in monolayer culture, but they remained at higher levels in three-dimensional culture. Conclusions: In conclusion, the present study demonstrated that the novel three-dimensional culture using micro-space plates maintains chondrogenic properties. Three-dimensional culture using micro-space plates is a useful tool for chondrocyte culture.

The effect of high molecular hyaluronic acid on the induction of matrix degradation enzymes By IL-6, IL-1β and TNF-α

The effect of high molecular hyaluronic acid on the induction of matrix degradation enzymes By IL-6, IL-1β and TNF-α

Specific degradation of hyaluronic acid and its rheological properties

High molecular hyaluronic acid was degraded by ultrasonication to definite molecular weight varying from 0.4 to 2.0 × 10 6g mol −1. The weight average molecular weight (M w) of degraded hyaluronic acid in 0.2 M NaCl was determined by size exclusion chromatography combined with multi-angle-laser-light-scattering (SEC-MALLS). Comparison measurements by using an Ubbelohde viscosimeter and a low shear rate viscosimeter (Zimm-Crothers) were performed for all sonicated samples. The relation of M w of the hyaluronic acid to the intrinsic viscosity [η] was calculated according to the Mark-Houwink equation, [η] = K M w α. The coefficients K = 5.075 10 −2 and α = 0.716 determined by Ubbelohde and K = 3.023 10 −2 and α = 0.770 by Zimm-Crothers viscosimeter. Furthermore, the dependence on pH and the influence of bivalent cations were investigated. In the range of pH 5.0 to 11.0 the intrinsic viscosity [η] was constant. At pH less than 5.0, the intrinsic viscosity [η] and the molecule volume decreased probably due to the protonation of glucoronic acid groups. Most bivalent cations do not affect the rheological behaviour significantly, only Fe 2+ causes to decrease of the intrinsic viscosity [η] and the molecule volume by intramolecular chain linkage combined with displacement of hydrating water.

Stimulation of osteoinduction in bone wound healing by high-molecular hyaluronic acid

To study the osteoinductive action of hyaluronic acid (HA), we examined the effects of applying an elastoviscous high-molecular HA preparation on bone wound healing after bone marrow ablation. The middiaphyses of cortical bones from rat femurs were perforated with a round bar, and excavated marrow cavities were filled immediately with high-molecular HA. Bone marrow ablation without HA was used to prepare controls. On post-ablation days 1, 2, 4, 7, and 14, animals were perfusion-fixed with an aldehyde mixture, and dissected femurs were examined by means of light, transmission-, and scanning-electron microscopy. In controls, the wounded marrow cavities were first filled with blood and fibrin clots (days 1 and 2), then with granulated tissues containing macrophages, neutrophils, and fibroblastic cells (day 4). New bone formation by differentiated osteoblasts was observed at 1 week post-ablation; at 2 weeks, the perforated cortical bones and marrow cavities were filled mostly with newly formed trabecular bone. In bones to which HA had been applied, new bone formation already had been induced by day 4 on both the peri- and endosteal surfaces of the existing cortical bones. At 1 week post-ablation, marrow cavities were completely filled with newly formed trabecular bones, in which active bone remodeling by osteoblasts and osteoclasts had occurred. Granulated tissues were replaced rapidly by normal marrow cells. These results suggest that high-molecular HA is capable of accelerating new bone formation through mesenchymal cell differentiation in bone wounds.

Stimulation of osteoinduction in bone wound healing by high-molecular hyaluronic acid.

To study the osteoinductive action of hyaluronic acid (HA), we examined the effects of applying an elastoviscous high-molecular HA preparation on bone wound healing after bone marrow ablation. The middiaphyses of cortical bones from rat femurs were perforated with a round bar, and excavated marrow cavities were filled immediately with high-molecular HA. Bone marrow ablation without HA was used to prepare controls. On post-ablation days 1, 2, 4, 7, and 14, animals were perfusion-fixed with an aldehyde mixture, and dissected femurs were examined by means of light, transmission-, and scanning-electron microscopy. In controls, the wounded marrow cavities were first filled with blood and fibrin clots (days 1 and 2), then with granulated tissues containing macrophages, neutrophils, and fibroblastic cells (day 4). New bone formation by differentiated osteoblasts was observed at 1 week post-ablation; at 2 weeks, the perforated cortical bones and marrow cavities were filled mostly with newly formed trabecular bone. In bones to which HA had been applied, new bone formation already had been induced by day 4 on both the peri- and endosteal surfaces of the existing cortical bones. At 1 week post-ablation, marrow cavities were completely filled with newly formed trabecular bones, in which active bone remodeling by osteoblasts and osteoclasts had occurred. Granulated tissues were replaced rapidly by normal marrow cells. These results suggest that high-molecular HA is capable of accelerating new bone formation through mesenchymal cell differentiation in bone wounds.

The glycosaminoglycans in cultures of stimulated rat peritoneal macrophages. 2. Gel chromatographic studies and the behaviour of heparan sulfate.

The molecular weight distribution of pMP-derived glycosaminoglycans (GAG), i.e. non-sulfated GAG, chondroitin sulfate (CS), and heparin sulfate (HS)-like material was determined. The peritoneal macrophages (pMP) were harvested from rats normal or stimulated by i.p. injection of thioglycolate, carrageenan or BCG, and maintained in culture. The GAG of cell layer and medium were isolated separately after labeling with 35S-sulfate and 3H-acetate. Treatment with nitrous acid served to remove HS-like material. Labeling with 3H-acetate served to detect synthesis of the high m. w. hyaluronic acid (HA). Gel chromatic separation was done using Sephadex G-200 columns. The maximal size of 35S-labeled GAG, especially HS (36 kDa), was reduced in cultural medium and cell layer after stimulation in vivo. Reduction was most pronounced after application of carrageenan followed by thioglycolate and BCG/LPS stimulation. The extracellular GAG of BCG-stimulated pMP were smallest, probably due to degradation. Heparan sulfate-like material made up a larger proportion in monolayer and medium, comprising the total m.w. range up to 36 kDa. The GAG sensitive to nitrous acid were maximal in cultures of carrageenan-stimulated pMP and minimal in those of thioglycolate-stimulated pMP. This type of HS was sensitive to hyaluronidase, too. Any synthesis of high molecular hyaluronic acid was not found in normal or stimulated rat pMP. Therefore MP-associated HA must be adsorbed from other sources or synthesized by early forms of macrophages.

ラットにおける実験的関節とう痛モデルとヒアルロン酸ナトリウムの鎮痛作用

Using the model of knee pain reaction induced by intra-articular injection of endogenous pain substances, especially bradykinin (BK) in rats, the mechanism of the analgesic effect of sodium hyaluronate (SPH) was investigated. The simultaneous administration of prostaglandin E2 with BK or hyaluronidase digestion of endogenous hyaluronic acid (HA) in our experiments brought remarkable hyperalgesia on BK-induced knee pain. These results suggest that higher sensitivity to the pain reaction is induced in a diseased joint (higher prostaglandin content, lower concentration and molecular size of HA in synovial fluid) than in a normal one. SPH definitely decreased BK-induced pain, and its analgesic effect was observed for a longer period, depending on its dose in pre-treatment and the degree of its distribution in synovial tissues. As the analgesic effect of SPH was observed in the hyaluronidase-treated joint as well, it is suggested that the increasing viscosity of synovial fluid caused by increasing HA concentration can decrease the pain even without normalizing molecular size of HA in the joint. HA oligomer and other compounds with similar viscosity or with similar polyanionic character as SPH showed no analgesic effect. From these results, it seems that the characteristic steric configurations of higher molecular HA are needed for the manifestation of the analgesic effect. SPH seems to show its analgesic effect by covering pain receptors in synovial tissues and holding endogenous pain substances in its molecule.

The healing of cartilage injuries under the influence of joint immobilization and repeated hyaluronic acid injections. An experimental study.

In an experimental animal model the influence of intraarticularly injected high-molecular hyaluronic acid on the healing of superficial and deep lesions of the articular cartilage in freely mobile and immobilized joints was investigated. In the right knee joint in 42 adult rabbits two intracartilaginous lesions were produced in one of the femoral condyles and an osteochondral lesion in the other. In half of the animals the injured knee joint was immobilized in plaster of Paris, while the other half were allowed to move the joint freely. Half of the injured joints in each group were then injected with high-molecular hyaluronic acid once a week for 8 weeks. After 2 months the knee joints were examined histologically, histochemically and microangio-graphically and the water content of the articular cartilage was determined. The intracartilaginous lesions remained unchanged in all knee joints. The wound margins of the osteochondral lesions showed better closure in the immobilized animals. As a rule the osteoc...

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.

The effect of oestrogenic treatment on the acid mucopolysaccharide pattern in skin of mice.

Acid mucopolysaccharides obtained from the skin of the back of mice were determined after precipitation with cetylpyridinium chloride and cellulose‐column fractionation. The results are given as μmol uronic acid per gram dry fat free tissue. Following treatment with oestradiol monobenzoate the hyaluronic acid (HA) increased from 2.7 to 14.6, while the chondroitin sulphates were unaffected (3.7 to 4.2). The increase (from 1.3 to 10.8) of a fraction probably consisting of low molecular HA was considered as reflecting an intensified metabolic activity of HA in the connective tissue. A very close linear correlation (r = 0.924) was demonstrated between the increase in the high molecular HA and the increase in tissue water.