Synthesis and characterization of a novel hyaluronic acid hydrogel

Hyaluronic acid (hyaluronan, HA) has many medical applications as a biomaterial. To enhance its biostability, a novel hydrogel of cross-linked hyaluronic acid was prepared using a double cross-linking process, which involves building cross-linkages between hydroxyl group pairs and carboxyl group pairs. The present study explored a number of cross-linking processes in order to obtain different degrees of cross-linking, which were evaluated by the measurement of water absorption capacity as an index of the gel network density. To gain a better understanding of the stability of the gel, the chemical structure and particularly the rheological behaviour of the cross-linked HA, which included the influences of factors, such as degree of cross-linking, HA concentration and gel particle size, were investigated. The in vitro biostability against hyaluronidase and free radical degradation was tested to show that the cross-linked hydrogel had improved resistance to in vitro hyaluronidase and free radical degradation.     

Synthesis and characterization of a novel hyaluronic acid hydrogel

Hyaluronic acid (hyaluronan, HA) has many medical applications as a biomaterial. To enhance its biostability, a novel hydrogel of cross-linked hyaluronic acid was prepared using a double cross-linking process, which involves building cross-linkages between hydroxyl group pairs and carboxyl group pairs. The present study explored a number of cross-linking processes in order to obtain different degrees of cross-linking, which were evaluated by the measurement of water absorption capacity as an index of the gel network density. To gain a better understanding of the stability of the gel, the chemical structure and particularly the rheological behaviour of the cross-linked HA, which included the influences of factors, such as degree of cross-linking, HA concentration and gel particle size, were investigated. The in vitro biostability against hyaluronidase and free radical degradation was tested to show that the cross-linked hydrogel had improved resistance to in vitro hyaluronidase and free radical degradation.     

Synthesis,chemical and rheological characterization of new hyaluronic acid-based hydrogels

New hyaluronic acid-based hydrogels have been synthesized. The carboxylate groups of hyaluronan were activated in order to bind the amino terminal groups of the di-amine cross-linking reagent. Different hydrogels were obtained according to the different di-amine cross-linking agents (1,3-diaminepropane, 1,6-diaminohexane, PEG500 di-amine, and PEG800 di-amine). The crosslinked polymer (C.L.Hyal) was then sulphated (C.L.HyalS) by a heterogeneous reaction using sulphur trioxide pyridine complex (SO₃-Py). The thermo-mechanical properties and swelling degree were evaluated and are discussed in relation to the chemical structure and the hydrophilic character of the gels. The different behaviours of C.L.Hyal and C.L.HyalS indicate the important role of sulphated groups.     

Synthesis, chemical and rheological characterization of new hyaluronic acid-based hydrogels

New hyaluronic acid-based hydrogels have been synthesized. The carboxylate groups of hyaluronan were activated in order to bind the amino terminal groups of the di-amine cross-linking reagent. Different hydrogels were obtained according to the different di-amine cross-linking agents (1,3-diaminepropane, 1.6-diaminohexane, PEG500 di-amine. and PEG800 di-amine). The cross-linked polymer (C.L.Hyal) was then sulphated (C.L.HyalS) by a heterogeneous reaction using sulphur trioxide pyridine complex (SO3-Py). The thermo-mechanical properties and swelling degree were evaluated and are discussed in relation to the chemical structure and the hydrophilic character of the gels. The different behaviours of C.L.Hyal and C.L.HyalS indicate the important role of sulphated groups.     

Characterization of chemisorbed hyaluronic acid directly immobilized on solid substrates

Hyaluronic acid (HA) has a number of potential biomedical applications in drug delivery and tissue engineering. For these applications, a prerequisite is to understand the characteristic of HA films directly immobilized to solid substrates. Here, we demonstrate that high molecular weight HA can be directly immobilized onto hydrophilic substrates without any chemical manipulation, allowing for the formation of an ultrathin chemisorbed layer. Hyaluronic acid is stabilized on these surfaces through hydrogen bonding between the hydrophilic moieties in HA [such as carboxylic acid (-COOH) or hydroxyl (-OH) groups] with silanol (-SiOH), carboxylic acid or hydroxyl groups on the hydrophilic substrates. Despite the water solubility, the chemisorbed HA layer remained stable on glass or silicon oxide substrates for at least 7 days in phosphate-buffered saline. Furthermore, HA immobilized on silicon and other dioxide surfaces in much higher quantities than other polysaccharides including dextran sulfate, heparin, heparin sulfate, chondroitin sulfate, dermatan sulfate, and alginic acid. This behavior is related to the molecular entanglement and intrinsic stiffness of HA as a result of strong internal and external hydrogen bonding as well as high molecular weight. These results demonstrate that HA can be used to coat surfaces through direct immobilization.     

PEGylation of hyaluronic acid nanoparticles improves tumor targetability in vivo

A major drawback of hyaluronic acid (HA)-based drug conjugates or nanoparticles for cancer therapy is their preferential accumulation in the liver after systemic administration. In an attempt to investigate the physicochemical characteristics and in vivo fates of poly(ethylene glycol) (PEG)-conjugated HA nanoparticles (HA-NPs), amphiphilic HA derivatives were prepared by varying the degree of PEGylation. The PEGylated HA-NPs formed self-assembled nanoparticles (217-269 nm in diameter) with the negatively charged surfaces in the physiological condition. Although PEGylation of HA-NPs reduced their cellular uptake in vitro, larger amounts of nanoparticles were taken up by cancer cells over-expressing CD44, an HA receptor, than by normal fibroblast cells. The ex vivo images of the organs using an optical imaging technique after the intravenous injection of Cy5.5-labeled nanoparticles into normal mice demonstrated that PEGylation could effectively reduce the liver uptake of HA-NPs and increase their circulation time in the blood. When the nanoparticles were systemically administered into tumor-bearing mice for in vivo real-time imaging, the strongest fluorescence signals were detected at the tumor site of the mice for the whole period of time studied, indicating their high tumor targetability. Interestingly, PEGylated HA-NPs were more effectively accumulated into the tumor tissue up to 1.6-fold higher than bare HA-NPs. The high tumor targetability of PEGylated HA-NPs was further supported by the intravital tumor imaging, in which their rapid extravasation into the tumor tissue was clearly observed. These results suggest that PEGylated HA-NPs can be useful as a means for cancer therapy and diagnosis.     

Synthesis and characterization of polypyrrole-hyaluronic acid composite biomaterials for tissue engineering applications

New tissue engineering technologies will rely on biomaterials that physically support tissue growth and stimulate specific cell functions. The goal of this study was to create a biomaterial that combines inherent biological properties which can specifically trigger desired cellular responses (e.g., angiogenesis) with electrical properties which have been shown to improve the regeneration of several tissues including bone and nerve. To this end, composites of the biologically active polysaccharide hyaluronic acid (HA) and the electrically conducting polymer polypyrrole (PP) were synthesized and characterized. Electrical conductivity of the composite biomaterial (PP/HA) was measured by a four-point probe technique, scanning electron microscopy was used to characterize surface topography, X-ray photoelectron spectroscopy and reflectance infrared spectroscopy were used to evaluate surface and bulk chemistry, and an assay with biotinylated hyaluronic acid binding protein was used to determine surface HA content. PP/HA materials were also evaluated for in vitro cell compatibility and tissue response in rats. Smooth, conductive, HA-containing PP films were produced; these films retained HA on their surfaces for several days in vitro and promoted vascularization in vivo. PP/HA composite biomaterials are promising candidates for tissue engineering and wound-healing applications that may benefit from both electrical stimulation and enhanced vascularization.     

Development, characterization, and diagnostic applications of monoclonal antibodies against bovine rotavirus

Hybridomas secreting monoclonal antibodies (MAbs) against the Nebraska calf diarrhea strain of bovine rotavirus (BRV) were characterized. Indirect fluorescent-antibody assay, immunodot assay, and immunoprecipitation were used to select hybridomas that produced anti-BRV MAbs. Seven of the MAbs were shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot assay to be reactive with the BRV outer capsid protein, VP7, which has a molecular mass of 37.5 kDa. None of the seven MAbs were reactive with canine rotavirus, bovine coronavirus, or uninfected Madin-Darby bovine kidney cells. Two clones, 8B4 (immunoglobulin G2a [IgG2a]) and 2B11 (IgG1), were found suitable for use in an antigen capture enzyme-linked immunosorbent assay for detecting BRV in bovine fecal samples. Both were subtype A specific (G6 subtype) but did not react with all isolates of BRV group A.     

Preparation and characterization of gelatin/hyaluronic acid cryogels for adipose tissue engineering: In vitro and in vivo studies

Macroporous elastic scaffolds containing gelatin (4% or 10%) and 0.25% hyaluronic acid (HA) were fabricated by cryogelation for application in adipose tissue engineering. These cryogels have interconnected pores (～200 μm), high porosity (>90%) and a high degree of cross-linking (>99%). The higher gelatin concentration reduced the pore size, porosity and swelling ratio of the cryogel but improved its swelling kinetics. Compressive mechanical testing of cryogel samples demonstrated non-linear stress–strain behavior and hysteresis loops during loading–unloading cycles, but total recovery from large strains. The presence of more gelatin increased the elastic modulus, toughness and storage modulus and yielded a cryogel that was highly elastic, with a loss tangent equal to 0.03. Porcine adipose-derived stem cells (ADSCs) were seeded in the cryogel scaffolds to assess their proliferation and differentiation. In vitro studies demonstrated a good proliferation rate and the adipogenic differentiation of the ADSCs in the cryogel scaffolds, as shown by their morphological change from a fibroblast-like shape to a spherical shape, decreased actin cytoskeleton content, growth arrest, secretion of the adipogenesis marker protein leptin, Oil Red O staining for triglycerides and expression of early (LPL and PPARγ) and late (aP2 and leptin) adipogenic marker genes. In vivo studies of ADSCs/cryogel constructs implanted in nude mice and pigs demonstrated adipose tissue and new capillary formation, the expression of PPARγ, leptin and CD31 in immunostained explants, and the continued expression of adipocyte-specific genes. Both the in vitro and in vivo studies indicated that the gelatin/HA cryogel provided a structural and chemical environment that enabled cell attachment and proliferation and supported the biological functions and adipogenesis of the ADSCs.     

Stimulation of in vivo angiogenesis by cytokine-loaded hyaluronic acid hydrogel implants

Crosslinked hyaluronic acid (HA) hydrogels were evaluated for their ability to elicit new microvessel growth in vivo when preloaded with one of two cytokines, vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF). HA film samples were surgically implanted in the ear pinnas of mice, and the ears retrieved 7 or 14 days post implantation. Histologic analysis showed that all groups receiving an implant demonstrated significantly more microvessel density than control ears undergoing surgery but receiving no implant (p < 0.01). Moreover, aqueous administration of either growth factor produced substantially more vessel growth than an HA implant with no cytokine. However, the most striking result obtained was a dramatic synergistic interaction between HA and VEGF. Presentation of VEGF in crosslinked HA generated vessel density of NI = 6.7 at day 14, where NI is a neovascularization index defined below, more than twice the effect of the sum of HA alone (NI = 1.8) plus VEGF alone (NI=1.3). This was twice the vessel density generated by co-addition of HA and bFGF (NI=3.4, p<0.001). New therapeutic approaches for numerous pathologies could be notably enhanced by the localized, synergistic angiogenic response produced by release of VEGF from crosslinked HA films.     

Hyaluronic acid nanofiber wound dressing--production, characterization, and in vivo behavior

Hyaluronic acid (HA) was electrospun. The effects of flow rate, spin length, and the applied voltage on the diameter of the HA nanofibers were analyzed. The average thickness of the webs was 0.041 cm. The air permeability of sterilized HA nanofiber wound dressing was much higher than that of gauze with Vaseline. The degree of crystallinity of HA nanofibers was characterized using powder X-ray technique and was found to be 20.6%. A preclinical study was conducted to compare healing of wounds covered by an adhesive bandage, a sterilized solid HA, gauze with Vaseline, an antibiotic dressing, and a sterilized HA nanofiber wound dressing. Results of the two methods of appraisal showed that the sterilized HA nanofiber wound dressing was the best type of dressing out of the five types of dressings compared.     

Metabolic and cytoprotective effects of in vivo peri-patellar hyaluronic acid injections in cultured tenocytes

Abstract

The purpose of this study was to investigate tenocyte mechanobiology after sudden-detraining and to examine the hypothesis that repeated peri-patellar injections of hyaluronic acid (HA) on detrained patellar tendon (PT) may reduce and limit detrained-associated damage in tenoctyes.

Twenty-four male Sprague–Dawley rats were divided into three groups: Untrained, Trained and Detrained. In the Detrained rats, the left tendon was untreated while the right tendon received repeated peri-patellar injections of either HA or saline (NaCl). Tenocyte morphology, metabolism and synthesis of C-terminal-propeptide of type I collagen, collagen-III, fibronectin, aggrecan, tenascin-c, interleukin-1β, matrix-metalloproteinase-1 and-3 were evaluated after 1, 3, 7 and 10 days of culture. Transmission-electronic-microscopy showed a significant increase in mitochondria and rough endoplasmic reticulum in cultured tenocytes from Detrained-HA with respect to those from Detrained-NaCl. Additionally, Detrained-HA cultures showed a significantly higher proliferation rate and viability, and increased synthesis of C-terminal-Propeptide of type I collagen, fibronectin, aggrecan, tenascin-c and matrix-metalloproteinase-3 with respect to Detrained-NaCl ones, whereas synthesis of matrix-metalloproteinase-1 and interleukin-1β was decreased. Our study demonstrates that discontinuing training activity in the short-term alters tenocyte synthetic and metabolic activity and that repeated peri-patellar infiltrations of HA during detraining allow the maintenance of tenocyte anabolic activity.     

Role of hyaluronic acid in the in vivo aggregation of cartilage proteoglycans.

From studies of isolated cartilage proteoglycans in solution it has been inferred that they occur in the tissue as aggregates of high molecular weight which consist of proteoglycan monomers, hyaluronic acid and specific link proteins. The present investigation provides direct evidence for the existence of hyaluronic acid-containing aggregates in vivo, as indicated by the following observations: Treatment of sections of coastal cartilage from newborn rabbits with Streptomyces hyaluronidase led to complete disappearance of the electron dense granules, which have been previously identified as chondroitin sulfate proteglycans, from the extracellular matrix. Similar results were obtained on digestion with leech hyaluronidase which, like the Streptomyces enzyme, specifically degrades hyaluronic acid. Proteoglycan aggregation occurs not only in the extracellular compartment but intracellularly as well, since a portion of the hyaluronidase-senstive, electron dense proteoglycan granules are found in intracellular vesicles. It is concluded that the ability of proteoglycan monomers to form aggregates is a true reflection of the in vivo organization of these molecules and that aggregate formation is an important factor in the maintenance of the normal physiological function of cartilage tissue.     

Synthesis and characterization of a novel N-vinylcaprolactam-containing acrylic acid terpolymer for applications in glass-ionomer dental cements

In this study a novel N-vinylcaprolactam (NVC)-containing copolymer of acrylic–itaconic acid was synthesized, characterized and incorporated into Fuji IX conventional glass-ionomer cement (GIC). Subsequently, the effects of incorporation of synthesized terpolymer on the mechanical properties of GIC were studied. The synthesized terpolymer was characterized using ¹H nuclear magnetic resonance, Fourier transform infrared and Raman spectroscopy. The viscosity and molecular weight of the terpolymer were also measured. The compressive strength (CS), diametral tensile strength (DTS) and biaxial flexural strength (BFS) of the modified GICs were evaluated after 24 h and 1 week of immersion in distilled water at 37 °C. The handling properties (working and setting times) of the resulting modified cements were also evaluated. One-way analysis of variance was used to study the statistical significance of the mechanical strengths and handling properties in comparison to the control group. The results showed that NVC-containing GIC samples exhibited significantly higher (P < 0.05) DTS (38.3 ± 10.9 MPa) and BFS (82.2 ± 12.8 MPa) in comparison to Fuji IX GIC (DTS = 19.6 ± 11.4 MPa; BFS = 41.3 ± 10.5 MPa). The experimental cement also showed higher but not statistically significant values for CS compared to the control material (CS for NVC-containing sample = 303 ± 32.8 MPa; CS for Fuji XI = 236 ± 41.5 MPa). Novel NVC-containing GIC has been developed in this study, with a 28% increase in CS. The presented GIC is capable of doubling the DTS and BFS in comparison to commercial Fuji IX GIC. The working properties of NVC-containing glass-ionomer formulations are comparable and are acceptable for water-based cements.     

Immobilization and characterization of gamma-aminobutyric acid on gold surface

gamma-Aminobutyric acid (GABA) is one of two main inhibitory neurotransmitters in the central nervous system that plays an important role in neuronal function and dysfunction. Immobilization of GABA molecules on a rigid surface in an ordered fashion will provide an opportunity to understand some of the fundamental properties related to its structure and function. In this study, we report a novel strategy for immobilization of bioactive GABA on gold substrate. GABA was immobilized in three consecutive steps, namely gold substrate amination, dextran covalent attachment, and GABA immobilization. Surface chemistry was verified at each step using XPS and FTIR. Bioactivity of GABA immobilized on the gold surface was studied using atomic force microscopy to reveal antigen-antibody binding. Nonspecific protein adsorption on the bioactive surface was analyzed quantitatively using anti-GABA antibody and an enzyme linked nonspecific anti-immunoglobulin-G antibody in an ELISA assay. GABA functionalized surface has high affinity for anti-GABA, while showing significantly low affinity for nonspecific anti-IgG antibody. All these data support the presence of a bio-functional immobilized GABA on the gold surface. In conclusion, we report a novel technique for immobilizing bioactive GABA molecules in an orderly fashion on gold substrates.     

Chemical-physical characterization and in vitro preliminary biological assessment of hyaluronic acid benzyl ester-hydroxyapatite composite

HYAFF11 is a biocompatible, biodegradable benzyl ester of hyaluronic acid. However, in order to use it for orthopedic application, its mechanical performance needs to be improved. In this study, a novel composite based on HYAFF11 polymer matrix reinforced with hydroxylapatite (HA) has been developed. Its advantage is having a similar component of the mineral phase of bone resulting in favorable osteoconductive properties. The present study has examined the compressive mechanical and surface chemical-physical properties of the novel HYAFF11-HA composite. Preliminary biological investigations, including pH and cytotoxicity studies of the material extracts, have also been performed using an in vitro primary human osteoblast-like cell model. Moreover, protein, especially fibronectin adsorption has been investigated following incubation in culture medium and human plasma. The results show a grainy surface topography composed mainly of C, P, and Ca, with a Ca/P atomic ratio indicating HA on the composite surface. Mechanical analysis shows an improvement of the compressive properties of HYAFF11 matrix, both in the dry and swollen states, with values in the range of that of spongy bone. No cytotoxic effects and no inhibition of cell proliferation have been observed in the presence of the material extracts with pH values within acceptable ranges for cell vitality. Protein studies reveal a similar pattern, but a higher amount of fibronectin following incubation in human plasma when compared with culture medium. The results show that the novel HYAFF11-HA composite shows a great potential for application in orthopedic fields, especially as vertebral trabecular bone substitute.     

The effects of covalently immobilized hyaluronic acid substrates on the adhesion, expansion, and differentiation of embryonic stem cells for in vitro tissue engineering

We investigated the in vitro effects of the molecular weight (MW) of hyaluronic acid (HA) on the maintenance of the pluripotency and proliferation of murine embryonic stem (ES) cells. High (1000 kDa) or low (4-8 kDa) MW HA was derivatized using an ultraviolet-reactive compound, 4-azidoaniline, and the derivative was immobilized onto cell culture cover slips. Murine ES cells were cultured on these HA surfaces for 5 days. High-MW HA interacted with murine ES cells via CD44, whereas low-MW HA interacted with these cells mostly via CD168. ES cells grown on both high- and low-MW HA appeared undifferentiated after 3 days. However, more cells adhered, proliferated, and exhibited greater amounts of phospho-p42/44 mitogen-activated-protein-kinase on low- compared with high-MW HA. Expression of Oct-3/4 and phosphorylation of STAT3 were enhanced by ES cells on low-MW HA, not on high-MW HA. After release from HA, cells cultured on low-MW HA in the presence of differentiating medium showed enhanced expression of α-SMA or CD31 compared with cells cultured on high-MW HA. It was concluded that low-MW HA substrates were effective in maintaining murine ES cells in a viable and undifferentiated state, which favors their use in the propagation of ES cells for tissue engineering.     

Design and characterization of microporous hyaluronic acid hydrogels for in vitro gene transfer to mMSCs

The effective and sustained delivery of DNA locally could increase the applicability of gene therapy in tissue regeneration and therapeutic angiogenesis. One promising approach is to use porous hydrogel scaffolds to encapsulate and deliver nucleotides in the form of nanoparticles to the affected sites. We have designed and characterized microporous (μ-pore) hyaluronic acid hydrogels which allow for effective cell seeding in vitro post-scaffold fabrication and allow for cell spreading and proliferation without requiring high levels of degradation. These factors, coupled with high loading efficiency of DNA polyplexes using a previously developed caged nanoparticle encapsulation (CnE) technique, then allowed for long-term sustained transfection and transgene expression of incorporated mMSCs. In this study, we examined the effect of pore size on gene transfer efficiency and the kinetics of transgene expression. For all investigated pore sizes (30, 60, and 100 μm), encapsulated DNA polyplexes were released steadily, starting by day 4 for up to 10 days. Likewise, transgene expression was sustained over this period, although significant differences between different pore sizes were not observed. Cell viability was also shown to remain high over time, even in the presence of high concentrations of DNA polyplexes. The knowledge acquired through this in vitro model can be utilized to design and better predict scaffold-mediated gene delivery for local gene therapy in an in vivo model where host cells infiltrate the scaffold over time.