Native Hyaluronic Acid Research Articles

Single step synthesis and characterization of thermoresponsive hyaluronan hydrogels

An efficient and scale-up ready single-step synthesis for the conjugation of thermoresponsive polymers to hyaluronic acid (HA) was established. Jeffamines® (JFM) and poly(N-isopropylacrylamide) (PNIPAM) were grafted to HA via direct amidation mediated by 1,1′-carbonyldiimidazole activation. The temperature-induced gelation of the semi-synthetic co-polymers was characterized by rheology as a function of the temperature and by differential scanning calorimetry (DSC). A HA–JFM conjugate with sol–gel transition in a physiologically relevant temperature range was identified. The grafting of PNIPAM resulted in the drastic change of the main rheological properties of native HA, revealing the hydrophobic non-covalent nature of the interactions between the thermoresponsive brushes in the gel state. Owing to the reversibility of these interactions and the sharpness of the transition, the HA–PNIPAM conjugates are suitable candidates for the incorporation of drugs, cells or ceramic materials for different biomedical applications.

Preparation of low-molecular-weight hyaluronic acid by ozone treatment

Recently, low-molecular-weight hyaluronic acid has been reported to have novel features, such as free radical scavenging activities, antioxidant activities, promotion of excisional wound healing, etc. In the present work, degradation of native hyaluronic acid by ozone treatment was performed for preparation of low-molecular-weight hyaluronic acid. The molecular weight of native hyaluronic acid was reduced from 1535 to 87kDa for 120min at 40°C. The rate of reduction of molecular weight was 94.33%. The FT-IR, 13C NMR, and UV–vis spectra suggested that there was no obvious modification of chemical structure of low-molecular-weight hyaluronic acid. The use of degradation of native hyaluronic acid by ozone treatment can be a useful alternative for production of low-molecular-weight hyaluronic acid.

Adipose-Derived Stem Cells on Hyaluronic Acid–Derived Scaffold

To evaluate the ability of human adipose-derived stem cells (h-ASCs) to survive and differentiate in corneal stroma. Our experiment consisted of 2 phases. First, we cultured h-ASCs in different types of hyaluronic acid (HA)-derived synthetic extracellular matrixes (sECMs) to determine the capability of proliferation and survival of the cells in hydrogels. Second, h-ASCs were grown in plastic flasks, labeled with an intracytoplasmic membrane fluorescent molecule, transferred onto different types of sECMs or the native HA product, and then inserted into the corneal stroma of the rabbits. After 10 weeks, we assessed the viability of the stem cells and the expression of cornea-specific proteins. The in vitro study showed that the HyStem-HP hydrogel had the highest yield of cells (1.1 × 10(6)/mL) compared with other types of HA-derived sECMs culture media, and the cells grown in the HyStem-HP hydrogel appeared more elongated and fibroblastlike. The in vivo study demonstrated that the labeled h-ASCs could be identified in the stroma with any type of sECM. The HA-derived sECMs, particularly the HyStem-HP hydrogel, showed better survival and cell morphologic features compared with pure HA. Immunostaining of keratocan, aldehyde dehydrogenase, and type I collagen revealed that the stem cells had expressed human cornea-specific proteins. Human adipose-derived stem cells can be successfully grown on HA-derived sECMs in vivo and can express human cornea-specific proteins. Human ASCs on an HA-derived scaffold may be used as a source of keratocytes to regenerate extracellular matrix-like material in situations where the cornea stroma has been compromised.

Improvement of hyaluronic acid enzymatic stability by the grafting of amino-acids

Injection of hyaluronic acid (HA)-based hydrogels has proven to provide many therapeutic benefits. To increase the stability of HA-based products against enzymatic digestion, we modified hyaluronic acid by grafting various amino acids on its carboxylic group and then evaluated the enzymatic stability of the various conjugates in presence of a hyaluronidase. Our results showed that all amino acid-modified HA polymers were more resistant to degradation compared to the native HA albeit with variation according to the amino acids. Amino acids with carboxylate groups such as aspartic acid or with hydroxyl functions (threonine, serine or tyrosine) conferred a particularly strong resistance to HA towards enzymatic digestion. The HA-amino acid products were then cross-linked with butanediol diglycidyl ether (BDDE). The swelling properties of the formed hydrogels appeared close to native HA whereas the increased resistance towards hyaluronidase digestion remained. These results suggest that amino acid-modified HA derivatives can become promising material for viscosupplementation or drug delivery.

Bioactivity of immobilized hyaluronic acid derivatives regarding protein adsorption and cell adhesion

Hyaluronic acid (HA) was chemically modified either by oxidation to obtain aldehyde-HA (aHA) or 3,3'-dithiobis(propanoic hydrazide) to obtain thiol-HA (tHA) that was covalently immobilized on model substrata such as amino-terminated surfaces or gold. Knowledge about the effect of modification with HA on physicochemical surface properties of these substrata and estimates of the quantities of immobilized HA were obtained by different physical methods such as contact angle measurements, ellipsometry, and atomic force microscopy. The bioactivity of aHA and tHA toward their natural binding partner aggrecan was studied by comparing surface plasmon resonance to native HA; this shows that binding of aggrecan was achieved in a similar way. Dermal human fibroblasts were used as a model cell to study how chemical modification and immobilization of HA impact adhesion and spreading of cells, which also affects cell growth and differentiation. A lower number and spreading of cells were observed on HA-modified surfaces compared to amino- and vinyl-terminated glass and silicon surfaces. Immunofluorescence microscopy also revealed that adhesion of fibroblast plated on HA-modified surfaces was mediated primarily by HA receptor CD44, indicating that bioactivity of HA was not significantly reduced by chemical modification.

High molecular weight hyaluronic acid limits astrocyte activation and scar formation after spinal cord injury

A major hurdle for regeneration after spinal cord injury (SCI) is the ability of axons to penetrate and grow through the scar tissue. After SCI, inflammatory cells, astrocytes and meningeal cells all play a role in developing the glial scar. In addition, degradation of native high molecular weight (MW) hyaluronic acid (HA), a component of the extracellular matrix, has been shown to induce activation and proliferation of astrocytes. However, it is not known if the degradation of native HA actually enhances glial scar formation. We hypothesize that the presence of high MW HA (HA with limited degradation) after SCI will decrease glial scarring. Here, we demonstrate that high MW HA decreases cell proliferation and reduces chondroitin sulfate proteoglycan (CSPG) production in cultured neonatal and adult astrocytes. In addition, stiffness-matched high MW HA hydrogels crosslinked to resist degradation were implanted in a rat model of spinal dorsal hemisection injury. The numbers of immune cells (macrophages and microglia) detected at the lesion site in animals with HA hydrogel implants were significantly reduced at acute time points (one, three and ten days post-injury). Lesioned animals with HA implants also exhibited significantly lower CSPG expression at ten days post-injury. At nine weeks post-injury, animals with HA hydrogel implants exhibited a significantly decreased astrocytic response, but did not have significantly altered CSPG expression. Combined, these data suggest that high MW HA, when stabilized against degradation, mitigates astrocyte activation in vitro and in vivo. The presence of HA implants was also associated with a significant decrease in CSPG deposition at ten days after SCI. Therefore, HA-based hydrogel systems hold great potential for minimizing undesired scarring as part of future repair strategies after SCI.

Network connectivity, mechanical properties and cell adhesion for hyaluronic acid/PEG hydrogels

The study aimed to explore the influence of the network architecture on the mechanical properties and degradability of HA/PEG gels, and to highlight the relationship between Young’s modulus and cell colonization with a selected architecture. Three different families of hyaluronic acid (HA)-based photopolymerized PEG diacrylate (PEGDA) hydrogels were compared, using different concentrations and molecular weights (64 and 234 kDa) of HA: semi-IPNs containing native HA in a PEG network (type I gels); co-networks obtained using thiolated HA as chain transfer agent during PEGDA polymerization (type II gels); co-networks obtained from the in situ preparation of a macromonomer derived from the Michael-type addition of thiolated HA on PEGDA (type III gels). From a comparative study of rheological properties and enzymatic degradability, type II gels were selected for a further study aiming to link their mechanical properties to cell spreading. Employing RGD-functionalized materials, Young’s moduli were measured via AFM nanoindentation while the cell spreading behavior was quantitatively evaluated by monitoring morphology and metabolic activity (MTS assay) of L929 fibroblasts. By revealing a clear relation between increasing modulus and increasing cell spreading/proliferation, the study showed the possibility to fine tune the cell/material interactions with appropriate reactive processing techniques.

Intra-articular Duration of Durolane™ after Single Injection into the Rabbit Knee

Objective:The aim of the present study was to investigate the intra-articular duration of Durolane™ in a rabbit model to allow comparison between Durolane™ residence time and data reported for other hyaluronic acid products as well as native hyaluronic acid.Design:14C-labeled Durolane™ was manufactured by labeling the cross-linker used for stabilization. A single injection of approximately 0.3 mL 14C-labeled Durolane™ was administered intra-articularly in both knee joints of male New Zealand White rabbits. At days 1, 2, 3, 7, 28, 60, 96, and 120 after injection, the knee joints of 4 animals were collected, and the radioactivity of the remaining gel was measured. The obtained data were fitted by exponential models to calculate the half-life of the gel. Two additional rabbits were used for histology of the joint 127 days after the injection.Results:The elimination of 14C-labeled Durolane™ followed first-order kinetics with an apparent half-life of approximately 32 days. Histology showed no morphological changes in the knee joints.Conclusions:This study shows that Durolane™ has a half-life of 32 days in the rabbit knee joint, which is much longer compared to literature data on hyaluronic acid and other modified hyaluronic acid products.

Comparative Histology of Intradermal Implantation of Mono and Biphasic Hyaluronic Acid Fillers

Hyaluronic acid (HA) gels have been used as filler material in the aesthetic field. Although the native HA molecule is without specificity of species and organs, synthetic cross-linked gels have differences in chemical composition and three-dimensional structure. Different technologies are employed in cross-linking, and the products have varying rheological properties. To determine whether the gels with differing chemical composition have differing histologic behavior when injected into human skin to determine if the histology changes after 14 days of implantation. Human volunteers consented to having controlled placement of HA intradermally into forearm or buttock skin. The trials were conducted in a single clinic in association with the Hôpitaux Universitaires de Genève, Geneva, Switzerland. The biopsies were taken immediately after implantation of the product and at day 14. Standard paraffin sections were prepared and stained with hematoxylin and eosin and Alcian blue and examined by an independent pathologist. Results show that each type of HA has a predictable histologic behavior in the skin. Biphasic gel has demonstrated deposition in big pools, often deep in the reticular dermis. The pools compress the collagen fibers. The papillary dermis and superficial reticular dermis are free of HA. Monophasic monodensified gels show large pools of hyaluronans throughout all the thickness of the reticular dermis. This material breaks up the collagen fibers of most of the dermal plane. The papillary dermis is free of exogenous hyaluronans. Monophasic polydensified cohesive gel penetrates into the dermis in a diffuse, evenly distributed manner, except in the papillary dermis, which remains free of exogenous material. The different types of cross-linked HA have different behaviors in the dermis immediately after their injection. The patterns are consistent between patients and are predictable. These histologic patterns do not change when biopsies are examined at 2 weeks.

Surgical correction of HIV-associated facial lipoatrophy

Surgical correction of HIV-associated facial lipoatrophy

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions. To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis(1). Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread(2-3). HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization (4-5). Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces(6-7). In these studies cell adhesion preferentially occurs on non-HA patterned regions. To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used (8). Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

Análise macroscópica dos efeitos dos hialuronatos e do corticosteroide no tratamento da osteoartrose induzida em joelhos de coelho

OBJECTIVE: The aim of this study was to evaluate the effects of intraarticular injections of corticosteroids, native hyaluronic acid and branched-chain hyaluronic acid in experimentally-induced osteoarthrosis. METHODS: 44 rabbits underwent anterior cruciate ligament resection and were subsequently divided into four groups, composed of eleven subjects each. Group 1: rabbits received weekly intraarticular injections of saline over a period of four weeks; Group 2: the animals received three weekly intraarticular injections of native hyaluronic acid; Group 3: rabbits in this group received three weekly intraarticular injections of branched-chain hyaluronic acid; Group 4: two injections of betamethasone over a period of three weeks. A macroscopic study of the cartilage of the tibial plateaus was performed twelve weeks after surgery and changes in the joint surface were graded accordingly: Grade 0: smooth joint surface without relief changes; Grade 1: rough surface without any depression; Grade 2: similar to grade 1, but with depressions on the joint surface and Grade 3: subchondral bone exposure. Statistical analysis was performed with the use of Student's T test, chi-square test and analysis of variance (ANOVA), with a significance level of 5%. RESULTS: A statistical difference was found between the control group and the three study groups (2, 3 and 4) in both the development and severity of arthrosis. However, there was no difference in outcome among the three different drugs. CONCLUSION: A similar degree of attenuation of the osteoarthrosis process in rabbits' knees was found in all three study groups (low-molecular-weight glycosaminoglycans, high-molecular-weight glycosaminoglycans and betamethasone) when compared to placebo.

MACROSCOPIC ANALYSES OF THE EFFECTS OF HYALURONATES AND CORTICOSTEROIDS ON INDUCED OSTEOARTHRITIS IN RABBITS’ KNEES

Objective: To evaluate the effects of intra-articular injections of corticosteroids, native hyaluronic acid and branched-chain hyaluronic acid in experimentally-induced osteoarthrosis. Methods: 44 rabbits underwent anterior cruciate ligament resection and were then divided into four groups of eleven. Group 1: one intra-articular injection of saline solution per week, for three weeks; Group 2: three injections (one per week) of native hyaluronic acid; Group 3: three injections (one per week) of branched-chain hyaluronic acid; Group 4: two injections of betamethasone with an interval of three weeks. The cartilage of the tibial plateaus was evaluated macroscopically twelve weeks after surgery. Changes to the joint surface were graded as follows: Grade 0: smooth joint surface without relief changes; Grade 1: rough surface without any depressions; Grade 2: similar to grade 1, but with depressions on the joint surface; and Grade 3: subchondral bone exposure. The statistical analysis consisted of the use of Student's t test, chi-square test and analysis of variance (ANOVA). The significance level used was 5%. Results: A statistical difference was found between the control group and the three study groups 2, 3, 4 in relation to the development and severity of arthrosis. However, there was no difference between the groups regarding the drugs studied. Conclusion: A similar degree of attenuation of the osteoarthrosis process in the rabbits’ knees was found with the use of intra-articular injections of low-molecular-weight and high-molecular-weight glycosaminoglycans, and the corticosteroid betamethasone, compared with placebo.

Gel‐Like Structure of a Hexadecyl Derivative of Hyaluronic Acid for the Treatment of Osteoarthritis

Hyaluronic acid is a polysaccharide with viscoelastic and mechanical properties that are crucial for the normal functioning of osteoarticular junctions. It is demonstrated that introduction of a hexadecyl side chain into HA yields an injectable polysaccharide capable of forming physical hydrogels, which are stable at very low polymer concentrations, whereas native hyaluronic acid forms viscous solutions at concentrations that are ten times higher. Characterization of this system showed that the driving force for its gel-like behavior is the occurrence of hydrophobic interactions involving aliphatic side chains, despite the low degree of substitution, as confirmed by molecular dynamics simulations of HYADD4 and HA hydrogels.

Effect of hyaluronic acid amide derivative on equine synovial fluid viscoelasticity

An amphiphilic hyaluronic acid (HA) derivative has been obtained by the amidation of the carboxylic group of the glucuronic acid. This derivative, HYADD4-G (HY4), is the hexadecylamide of 500-730 kDa hyaluronic acid, derived from Streptococcus equi at about 2% degree of substitution (2 mol hexadecylamine per 100 mol hexuronic acid). Its viscoelastic properties, at a concentration of 5 mg/mL in phosphate buffer saline, have been compared with those solutions of native HA, having the same molecular weight. Changes in the viscoelastic properties of equine synovial fluid (SF) when mixed with HY4 over a series of volume ratios-viz 1:2, 1:1, 3:1, and 7:1-have been evaluated. HY4 is able to associate into aqueous solution, and its rheological behavior is typical of a weak gel. Throughout the frequency range investigated (0.1-10 Hz), the elastic modulus G' is higher than the viscous modulus G'', and both moduli are frequency independent, and G' value is about two orders of magnitude higher than that of a comparable solution of native HA. The addition of HY4 to equine synovial fluid (SF) increased its viscoelasticity at all the SF:HY4 ratios tested. These results demonstrate that HY4 is able to integrate with SF, increasing the synovial fluid rheology, and could be an interesting new option in viscosupplement therapy of osteoarthritis, particularly considering its low degree of chemical modification from native HA.

Native hyaluronic acid in dermatology – results of an expert meeting

The glycosaminoglycan hyaluronic acid (HA) is a major component of the extracellular matrix of the skin and plays an important role in the metabolism of the dermis.HA is responsible for hydration,nutrient exchange and protects against free radical damage;via signaling pathways it is also involved in biologic processes like cell differentiation and motility. Native HA has been employed for several years to help the skin to regain elasticity,turgor and moisture. In a clinical study an increase in elasticity and turgor following repeated injections with HA could be demonstrated, but this treatment approach is discussed controversially. An expert conference took place to find a consensus regarding use, aims of treatment, indications and limitations of this therapy. The decisions of the expert meeting are presented in this report.

Einsatz biophysikalischer Messverfahren zur Untersuchung der hautphysiologischen Wirkung injizierbarer Hyaluronsäure

To evaluate the effects of intradermal injection of native hyaluronic acid on physiology of aged skin using bioengineering methods. 10 female patients aged 44-69 were included. Study duration was 24 weeks. Each patient received 3 treatments (at week 0, 2 and 4) with native hyaluronic acid (Hyalsystem Merz Pharmaceuticals, Frankfurt/Main, Germany) and 4 examinations (week 0, 4, 12 and 24). Biophysical properties assessed were skin elasticity and surface roughness, as well as skin thickness and density. Skin elasticity increased while skin surface roughness was reduced. High frequency ultrasound revealed an increase of skin thickness, whereas skin density decreased during treatment and slightly increased above baseline at week 24. The evaluation of the effects of an intradermal injection of hyaluronic acid on aged skin showed an improvement of skin elasticity and skin surface roughness.

Preparation and characterization of new hydrogels based on hyaluronic acid and α,β-polyaspartylhydrazide

Hyaluronic acid (HA) has been crosslinked with α,β-polyaspartylhydrazide (PAHy). The crosslinking reaction has been performed in acidic medium in the presence of various amounts of N-ethyl- N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC). All obtained samples have been characterized by FT-IR analysis and swelling measurements in double distilled water that have confirmed the occurrence of a chemical linkage between two polymers and the affinity towards aqueous medium of HA–PAHy networks, respectively. In vitro degradation assays have been performed in simulated physiological conditions as well as in the presence of hyaluronidase. Experimental data evidenced that HA–PAHy samples undergo a poor chemical and a reduced enzymatic degradation unlike native HA.

ACP Gel: A New Hyaluronic Acid???Based Injectable for Facial Rejuvenation. Preclinical Data in a Rabbit Model

Facial aging results from reduced biosynthetic activity of dermal fibroblasts and decreased deposition of extracellular matrix components, such as hyaluronic acid, a glycosaminoglycan responsible for skin hydration and turgidity. Exogenous hyaluronic acid injections provide a safe though short-term solution for facial rejuvenation. Using a rabbit model, the authors investigated residence time and tolerability of ACP gel, a new hyaluronic acid cross-linked derivative, compared with high-molecular-weight native hyaluronic acid currently used for facial rejuvenation (Ial System). ACP gel 1% and 2%, Ial System, and saline were intradermally injected into the backs of 12 New Zealand rabbits: six animals were used to follow volume maintenance and redness up to 10 days and the other six animals were euthanized at days 2, 6, 8, 10, 14, and 21 (one animal per time point) to histologically assess biocompatibility. ACP gel 2% had the longest residence time, showing a significantly better volume maintenance than the other samples, especially in the initial study period (71 percent of original volume versus 23 percent and 21 percent of ACP gel 1% and Ial System, respectively, at day 2). Macroscopically, no adverse events were observed in the treated animals. Histologic examination confirmed the absence of adverse events, persistent inflammation, tissue degeneration, or necrosis. ACP gel macrophage-mediated phagocytosis was more persistent with respect to the Ial System, consistent with its longer residence time. ACP gel 2% is a promising dermal biorevitalizer, characterized by a high safety profile and prolonged residence time in relation to native high-molecular-weight hyaluronic acid.