HA-based Materials Research Articles

Surface zwitterionization: Effective method for preventing oral bacterial biofilm formation on hydroxyapatite surfaces

In this study, we conducted surface zwitterionization of hydroxyapatite (HA) surfaces by immersing them in the zwitterionic polymer solutions to provide anti-bacterial properties to the HA surface. Three different monomers containing various zwitterionic groups, i.e., phosphorylcholine (PC), sulfobetaine (SB), and carboxybetaine (CB), were copolymerized with the methacrylic monomer containing a Ca2+-binding moiety, using the free radical polymerization method. As a control, functionalization of the copolymer containing the Ca2+-binding moiety was synthesized using a hydroxy group. The stable immobilization of the zwitterionic functional groups was confirmed by water contact angle analysis and X-ray photoelectron spectroscopy (XPS) measurement conducted after the sonication process. The zwitterionized HA surface showed significantly decreased protein adsorption, whereas the hydroxyl group-coated HA surface showed limited efficacy. The anti-bacterial adhesion property was confirmed by conducting Streptococcus mutans (S. mutans) adhesion tests for 6h and 24h. When furanone C-30, a representative anti-quorum sensing molecule for S. mutans, was used, only a small amount of bacteria adhered after 6h and the population did not increase after 24h. In contrast, zwitterionized HA surfaces showed almost no bacterial adhesion after 6h and the effect was retained for 24h, resulting in the lowest level of oral bacterial adhesion. These results confirm that surface zwitterionization is a promising method to effectively prevent oral bacterial adhesion on HA-based materials.

Injectable carboxymethylcellulose hydrogels for soft tissue filler applications

Disease, trauma and aging all lead to deficits in soft tissue. As a result, there is a need to develop materials that safely and effectively restore areas of deficiency. While autogenous fat is the current gold standard, hyaluronic acid (HA) fillers are commonly used. However, the animal and bacterial origin of HA-based materials can induce adverse reactions in patients. With the aim of developing a safer and more affordable alternative, this study characterized the properties of a plant-derived, injectable carboxymethylcellulose (CMC) soft tissue filler. Specifically, methacrylated CMC was synthesized and crosslinked to form stable hydrogels at varying macromer concentrations (2–4% w/v) using an ammonium persulfate and ascorbic acid redox initiation system. The equilibrium Young’s modulus was shown to vary with macromer concentration (ranging from ∼2 to 9.25kPa), comparable to values of native soft tissue and current surgical fillers. The swelling properties were similarly affected by macromer concentration, with 4% gels exhibiting the lowest swelling ratio and mesh size, and highest crosslinking density. Rheological analysis was performed to determine gelation onset and completion, and was measured to be within the ISO standard for injectable materials. In addition, hydrolytic degradation of these gels was sensitive to macromer concentration, while selective removal using enzymatic treatment was also demonstrated. Moreover, favorable cytocompatibility of the CMC hydrogels was exhibited by co-culture with human dermal fibroblasts. Taken together, these findings demonstrate the tunability of redox-crosslinked CMC hydrogels by varying fabrication parameters, making them a versatile platform for soft tissue filler applications.

Implantation of silicon dioxide-based nanocrystalline hydroxyapatite and pure phase beta-tricalciumphosphate bone substitute granules in caprine muscle tissue does not induce new bone formation

BackgroundOsteoinductive bone substitutes are defined by their ability to induce new bone formation even at heterotopic implantation sites. The present study was designed to analyze the potential osteoinductivity of two different bone substitute materials in caprine muscle tissue.Materials and methodsOne gram each of either a porous beta-tricalcium phosphate (β-TCP) or an hydroxyapatite/silicon dioxide (HA/SiO2)-based nanocrystalline bone substitute material was implanted in several muscle pouches of goats. The biomaterials were explanted at 29, 91 and 181 days after implantation. Conventional histology and special histochemical stains were performed to detect osteoblast precursor cells as well as mineralized and unmineralized bone matrix.ResultsBoth materials underwent cellular degradation in which tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells and TRAP-negative multinucleated giant cells were involved. The ß-TCP was completely resorbed within the observation period, whereas some granules of the HA-groups were still detectable after 180 days. Neither osteoblasts, osteoblast precursor cells nor extracellular bone matrix were found within the implantation bed of any of the analyzed biomaterials at any of the observed time points.ConclusionsThis study showed that ß-TCP underwent a faster degradation than the HA-based material. The lack of osteoinductivity for both materials might be due to their granular shape, as osteoinductivity in goat muscle has been mainly attributed to cylindrical or disc-shaped bone substitute materials. This hypothesis however requires further investigation to systematically analyze various materials with comparable characteristics in the same experimental setting.

Effect of highly dispersed yttria addition on thermal stability of hydroxyapatite

The capability of the colloidal method to produce yttria (Y2O3) dispersed hydroxyapatite (HA) has been investigated as an alternative method to the conventional method of mechanical mixing and sintering for developing HA-based materials that could exhibit controllable and enhanced functional properties. A water based colloidal route to produce HA materials with highly dispersed Y2O3 has been applied, and the effect of 10wt.% Y2O3 addition to HA investigated by thermal analysis, X-ray diffraction and Fourier transform infrared spectroscopy. These measurements evidence a remarkable effect of this Y2O3 addition on decomposition mechanisms of synthetic HA. Results show that incorporation of Y2O3 as dispersed second phase is beneficial because it hinders the decomposition mechanisms of HA into calcium phosphates. This retardation will allow the control of the sintering conditions for developing HA implants with improved properties. Besides, substitution of Ca2+ with Y3+ ions appears to promote the formation of OH− vacancies, which could improve the conductive properties of HA favorable to osseointegration.

Release of Ciprofloxacin-HCl and Dexamethasone Phosphate by Hyaluronic Acid Containing Silicone Polymers.

The purpose of this study was to determine the effect of the covalent incorporation of hyaluronic acid (HA) into conventional hydrogel and hydrogels containing silicone as models for contact lens materials on the uptake and release of the fluoroquinolone antibiotic ciprofloxacin and the anti-inflammatory steroid dexamethasone phosphate. A 3 mg/mL ciprofloxacin solution (0.3% w/v) and a 1 mg/mL dexamethasone phosphate solution (0.1%) was prepared in borate buffered saline. Three hydrogel material samples (pHEMA; pHEMA TRIS; DMAA TRIS) were prepared with and without the covalent incorporation of HA of molecular weight (MW) 35 or 132 kDa. Hydrogel discs were punched from a sheet of material with a uniform diameter of 5 mm. Uptake kinetics were evaluated at room temperature by soaking the discs for 24 h. Release kinetics were evaluated by placing the drug-loaded discs in saline at 34 °C in a shaking water bath. At various time points over 6–7 days, aliquots of the release medium were assayed for drug amounts. The majority of the materials tested released sufficient drug to be clinically relevant in an ophthalmic application, reaching desired concentrations for antibiotic or anti-inflammatory activity in solution. Overall, the silicone-based hydrogels (pHEMA TRIS and DMAA TRIS), released lower amounts of drug than the conventional pHEMA material (p < 0.001). Materials with HA MW132 released more ciprofloxacin compared to materials with HA MW35 and lenses without HA (p < 0.02). Some HA-based materials were still releasing the drug after 6 days.

Functionalization of Hyaluronic Acid with Chemoselective Groups via a Disulfide-Based Protection Strategy for In Situ Formation of Mechanically Stable Hydrogels

Functionalization of hyaluronic acid (HA) with chemoselective groups enables in situ (in vivo) formation of HA-based materials in minimally invasive injectable manner. Current methods of HA modification with such groups primarily rely on the use of a large excess of a reagent to introduce a unique reactive handle into HA and, therefore, are difficult to control. We have developed the new protective group strategy based on initial mild cleavage of a disulfide bond followed by elimination of the generated 2-thioethoxycarbonyl moiety ultimately affording free amine-type functionality, such as hydrazide, aminooxy, and carbazate. Specifically, new modifying homobifunctional reagents have been synthesized that contain a new divalent disulfide-based protecting group. Amidation of HA with these reagents gives rise to either one-end coupling product or to intra/intermolecular cross-linking of the HA chains. However, after subsequent treatment of the amidation reaction mixture with dithiothreitol (DTT), these cross-linkages are cleaved, ultimately exposing free amine-type groups. The same methodology was applied to graft serine residues to the HA backbone, which were subsequently oxidized into aldehyde groups. The strategy therefore encompasses a new approach for mild and highly controlled functionalization of HA with both nucleophilic and electrophilic chemoselective functionalities with the emphasis for the subsequent conjugation and in situ cross-linking. A series of new hydrogel materials were prepared by mixing the new HA-aldehyde derivative with different HA-nucleophile counterparts. Rheological properties of the formed hydrogels were determined and related to the structural characteristics of the gel networks. Human dermal fibroblasts remained viable while cultured with the hydrogels for 3 days, with no sign of cytotoxicity, suggesting that the gels described in this study are candidates for use as growth factors delivery vehicles for tissue engineering applications.

Role, Metabolism, Chemical Modifications and Applications of Hyaluronan

Hyaluronan (hyaluronic acid, HA) is a linear naturally occurring polysaccharide formed from repeating disaccharide units of N-acetyl-D-glucosamine and D-glucuronate. Despite its relatively simple structure, HA is an extraordinarily versatile glycosaminoglycan currently receiving attention across a wide front of research areas. It has a very high molar mass, usually in the order of millions of Daltons, and possesses interesting visco-elastic properties based on its polymeric and polyelectrolyte characteristics. HA is omnipresent in the human body and in other vertebrates, occurring in almost all biological fluids and tissues, although the highest amounts of HA are found in the extracellular matrix of soft connective tissues. HA is involved in several key processes, including cell signaling, wound repair and regeneration, morphogenesis, matrix organization and pathobiology. Clinically, it is used as a diagnostic marker for many disease states including cancer, rheumatoid arthritis, liver pathologies, and as an early marker for impending rejection following organ transplantation. It is also used for supplementation of impaired synovial fluid in arthritic patients, following cataract surgery, as a filler in cosmetic and soft tissue surgery, as a device in several surgical procedures, particularly as an anti-adhesive following abdominal procedures, and also in tissue engineering. This review will provide an overview of the structure and physiological role of HA, as well as of its biomedical and industrial applications. Recent advances in biotechnological approaches for the preparation of HA-based materials, and as a component of tissue scaffolding for artificial organs will also be presented.

361 EVALUATION OF THE PERSISTENCE OF DERIVATIZED HYALURONIC ACID FOLLOWING INJECTION INTO THE GUINEA PIG FEMOROPATELLAR FAT PAD TISSUES

Purpose: Hyaluronan (HA) based viscosupplements are used to reduce pain in patients with mild to moderate osteoarthritis. For the patient to receive the full benefit, these viscosupplements must be injected directly into the joint space. Extra-articular needle placement and injection of viscosupplements into the subsynovial tissues may be linked to a higher incidence of adverse reactions. In this study a guinea pig model was used to investigate the cellular response to two viscosupplements, non-animal stabilized hyaluronic acid (NASHA) and hylastan, following injection into the fat pad to simulate misinjection of the viscosupplement. These two crosslinked HA-based gels have distinctly different rheological and physical properties. NASHA is an epoxy crosslinked gel while hylastan is a non-animal HA gel lightly crosslinked with divinyl sulfone. Methods: Eighteen guinea pigs were randomized into two treatment groups of 6 animals each and 1 HA control group (1% w/v solution of unmodified HA) of 6 animals. Test or control material was injected into the fat pad of the assigned animal. Fat pads in the contra lateral joints were injected with lactated Ringer’s solution. Animals were sacrificed at 7 or 30 days following injection and the fat pads fixed in 4% paraformaldehyde for sectioning and staining with hematoxylin and eosin (H&E). Some slides were treated with hyaluronidase prior to staining with H&E to confirm the presence of HA-based material. The slides were examined by a board certified veterinary pathologist for the presence of residual HA-based material and cellular activity associated with this material. Rheological properties were characterized by a controlled stress rheometer and dynamic mechanical analysis. Results: On day 7, residual HA-based material was observed in tissues taken from both hylastan and NASHA-treated animals. Test material was noted as a basophilic substance that formed discrete clusters within the adipose tissue. The cellular response to these two materials was characterized by the presence of a thin layer of macrophages at the host-test material interface. Macrophages appeared to easily infiltrate and degrade the material in the hylastan-treated animals but not in the NASHA-treated animals. No residual control material was present in tissues from animals injected with the HA or lactated Ringer’s solution controls. At day 30, residual hylastan was not observed in any of the fat pad tissues. In contrast, residual NASHA was observed as discrete aggregates of basophilic material associated with a single layer of macrophages at the host material interface surrounded by layers of fibrous connective tissue that completely encapsulated the material. Rheological measurements demonstrate that hylastan is a softer, more cohesive gel than the NASHA material. Conclusions: We have evaluated the tissue distribution and cellular activity of two HA-based test articles following injection into the fat pads of guinea pigs. Microscopic analysis of the cellular activity associated with the dissolution of hylastan was quite different from that observed in tissue sections taken from the NASHA-treated animals. Hylastan injected tissue demonstrated a typical biomaterial response with macrophages that are facilitating the resorption of implanted material at early timepoints. This inflammatory response resolves at later times once the hylastan is completely resorbed. The NASHA injected tissue demonstrated a similar early time response, but at the later times showed a cellular and fibrous encapsulation of the injected material. Matrix Biochemistry

Hyaluronic acid: a natural biopolymer with a broad range of biomedical and industrial applications

Hyaluronic acid (hyaluronan, HA) is a linear polysaccharide formed from disaccharide units containing N-acetyl-D-glucosamine and glucuronic acid. It has a high molecular mass, usually in the order of millions of Daltons, and interesting viscoelastic properties influenced by its polymeric and polyelectrolyte characteristics. HA is present in almost all biological fluids and tissues. In clinical medicine, it is used as a diagnostic marker for many diseases including cancer, rheumatoid arthritis and liver pathologies, as well as for supplementation of impaired synovial fluid in arthritic patients by means of intra-articular injections. It is also used in certain ophthalmological and otological surgeries and cosmetic regeneration and reconstruction of soft tissue. Herein we present an overview of the occurrence and physiological properties of HA, as well as of the recent advances in production biotechnology and preparation of the HA-based materials for medical application.