Electrohydrodynamic atomization of protein (bovine serum albumin).

Bovine serum albumin (BSA) was chosen as a model protein. Three solutions of different concentrations of 5, 20 and 50 mg/ml were prepared, characterised and subjected to electrohydrodynamic atomization (EHDA). The 5 and 20 mg/ml solutions were atomized successfully and mode selection (M-S) maps were drawn for both concentrations to find out regions of stable cone-jet mode atomizaton. Droplet relics of these two solutions were investigated by electron microscopy. Samples were investigated by UV spectroscopy and circular dichroism (CD) spectroscopy before and after electrohydrodynamic atomization. We conclude that, particularly at the higher concentration of protein, EHDA does not result in significant structural change of BSA, and therefore is a processing route that can be considered for encapsulating drugs in proteins.

Development of high-viscosity, two-paste bioactive bone cements.

Self-curing two-paste bone cements have been developed using methacrylate monomers with a view to formulate cements with low polymerization exotherm, low shrinkage, better mechanical properties, and improved adhesion to bone and implant surfaces. The monomers include bis-phenol A glycidyl dimethacrylate (bis-GMA), urethane dimethacrylate (UDMA) and triethylene glycol dimethacrylate (TEGDMA) as a viscosity modifier. Two-paste systems were formulated containing 60% by weight of a bioactive ceramic, hydroxyapatite. A methacroyloxy silane (A174) was used as a coupling agent due to its higher water stability in comparison to other aminosilanes to silanate the hydroxyapatite particles prior to composite formulation. A comparison of the FT-infrared spectrum of hydroxyapatite and silanated hydroxyapatite showed the presence of the carbonyl groups ( approximately 1720 cm(-1)), -C=C-( approximately 1630 cm(-1)) and Si-O- (1300-1250 cm(-1)) which indicated the availability of silane groups on the filler surface. Two methods of mixing were effected to form the bone cement: firstly by mixing in an open bowl and secondly by extruding the two pastes by an auto-mixing tip using a gun to dispense the pastes. Both types of cements yielded low polymerization exotherms with good mechanical properties; however, the lower viscosity of UDMA allowed better extrusion and handling properties. A biologically active apatite layer formed on the bone cement surface within a short period after its immersion in simulated body fluid, demonstrating in vitro bioactivity of the composite. This preliminary data thus suggests that UDMA is a viable alternative to bis-GMA as a polymerizable matrix in the formation of bone cements.

In vivo response to HA-polyhydroxybutyrate/polyhydroxyvalerate composite.

This study examined the morphological and compositional structure of bone-implant interfaces after in vivo implantation into the tibias of rabbits. The implants were composed of biodegradable polyhydroxybutyrate/polyhydroxyvalerate copolymer reinforced with synthetic hydroxyapatite (HA) particles. Optical and scanning electron microscopy techniques were used, including energy-dispersive X-ray analysis. The interface was found to be morphologically, biologically and chemically active throughout the period of study. There was a strong tendency to rebuild the bone structure at the interface after implantation, independent of the composition of the implant, but direct bone bonding with the implant depended on the bioactive nature of the interface, as represented by the HA particles. At all implantation times, lamellar bone formed at the interface and replaced degrading polymer matrix, while engulfing HA filler particles. In regions about 50-100 from the interface, the bone region displayed an osteon organization. Osteoblasts and osteocytes were identified throughout the interface region. The thickness of the newly formed bone significantly increased over the period of the experiment from about 130 microm at 1 month to about 770 microm at 6 months. Materials that behave in this manner may be useful in some bone replacement therapies.

Mechanism of bone-like formation on a bioactive implant in vivo.

The physical and chemical nature of the remodelled interface between the porous A3 glass-ceramic, composed of (wt%): SiO(2) = 54.5; CaO = 15.0; Na(2)O = 12.0; MgO = 8.5; P(2)O(5) = 6.0 K(2)O = 4.0, and the surrounding bone was studied after implantation into rat tibias. The interfaces which developed new bone layer in direct contact with the implants were examined by analytical scanning and transmission electron microscopy after implantation for 6, 8 and 12 weeks. Degradation processes of the implants also encouraged osseous tissue ingrowths into the pores of the material, changing drastically the macro- and microstructure of the implants. The ionic exchange initiated at the implant interface with the physiological environment was essential in the integration process of the implant, through a dissolution-precipitation-transformation mechanism. The interfaces developed non-toxic biological and chemical activities and remained reactive over the 12-week implantation period. These findings were significant as indicative of morphological and chemical integration of the A3 glass-ceramic into the structure of living bone tissue. A3 glass-ceramic could be suitable for the repair or replacement of living bone.

Synthesis and characterization of nano-biomaterials with potential osteological applications.

The manufacture of high-surface area, un-agglomerated nano-sized (1-100 nm) bioceramic particles are of interest for many applications including injectable/controlled setting bone cements, high strength porous/non-porous synthetic bone grafts, and the reinforcing phase in nano-composites that attempt to mimic the complex structure and superior mechanical properties of bone. In the present study, we report on the manufacture of nano-particle hydroxyapatite powders by several wet chemical methods, which incorporate a freeze-drying step. In particular, it was found that the emulsion-based syntheses yielded powders with high surface areas and small primary particle sizes. Freeze drying rather than oven drying of powders prepared by conventional wet chemical synthesis yielded a nano-sized powder with a comparatively higher surface area of 113 m(2)/g. All powders were calcined in air in a furnace at 900 degrees C to investigate the effects of synthesis method on phase purity and surface area. The materials were characterized by a range of analytical methods including Fourier-transform infrared spectroscopy employing the photo acoustic (PAS-FTIR) sampling technique, BET surface area analysis, X-ray powder diffraction (XRD), and the particles were examined using a transmission electron microscope (TEM).

Effect of glass-ceramic microstructure on its in vitro bioactivity.

Two routes were used to obtain a glass-ceramic composed of 43.5 wt % SiO(2) - 43.5 wt % CaO - 13 wt % ZrO(2). Heat treatment of a glass monolith produced a glass-ceramic (WZ1) containing wollastonite-2M and tetragonal zirconia as crystalline phases. The WZ1 did not display bioactivity in vitro. Ceramizing the glass via powder technology routes formed a bioactive glass-ceramic (WZ2). The two glass-ceramics, WZ1 and WZ2, were composed of the same crystalline phases, but differed in microstructure. The in vitro studies carried out on WZ2 showed the formation of an apatite-like layer on its surface during exposure to a simulated body fluid. This paper examined the influence of both chemical and morphological factors on the in vitro bioactivitity. The interfacial reaction product was examined by scanning and transmission electron microscopy. Both instruments were fitted with energy-dispersive X-ray analyzers. Measurements of the pH made directly at the interface of the two glass-ceramics were important in understanding their different behavior during exposure to the same physiological environment.

Transmission electron microscopy of the interface between bone and pseudowollastonite implant.

This paper reports on the structural morphology of the interface in vivo between implants composed of bioactive synthetic pseudowollastonite ceramic and bone in rat tibias. Thin sections of the interfaces were examined after 6 and 8 weeks of implantation period in a high resolution transmission electron microscope up to the lattice plane resolution level. The interfaces developed normal biological and chemical activities and remained reactive over the 8-week period. The regions showing direct bone tissue bonding to the implant contained nanocrystals of hydroxyapatite-like phase growing epitaxially across the interface in the [002] direction. The nanocrystals were also identified in the bone tissue formed in the interfacial area. The reactivity of the implant caused in the first instance formation of an amorphous woven type of bone, which transformed into a crystalline lamellar type containing collagen fibres. The Ca/P ratio of the interfacial region was found to be between 1.67 in the mature bone tissue formed about 5 microm from the interface, and 2.06 in the regions right at the interface.

Hydroxyapatite as a filler for dental composite materials: mechanical properties and in vitro bioactivity of composites.

Hydroxyapatite (HAp) powder was treated with gamma-methacryloxypropyltrimethoxy-silane (gamma-MPS) using standard techniques in both non-polar and polar systems. Infrared spectra (DRIFT) and thermogravimetric analysis (TGA) confirmed the presence of gamma-MPS on the surface of the HAp filler particles. Series of experimental composites consisting of bisphenol-a-glycidyl methacrylate (BisGMA) based resin and untreated or treated HAp filler were produced to determine the mechanical properties and in vitro bioactivity. The incorporation of HAp filler into the BisGMA base resin had an enhancing effect on the flexural strength and Young's modulus of the base resin, the latter being increased by a factor of three. The mechanical properties of the filled resin were not affected by the surface treatment of the HAp, but filler loading was found to have a significant effect on Young's modulus. Higher proportions of silane-treated HAp of smaller particle size could be incorporated in the monomer phase giving rise to composites of higher stiffness. Examination of the fracture surfaces showed that the silanized HAp particles maintained better contact with the polymer matrix. In vitro study revealed that the composites incorporating silanized HAp formed a compact and continuous calcium phosphate layer on their surface after 4 weeks immersion in a simulated body fluid (SBF).

Reactivity of a wollastonite-tricalcium phosphate Bioeutectic ceramic in human parotid saliva.

In a previous study, a new ceramic material (Bioeutectic), prepared by slow solidification through the eutectic temperature region of the wollastonite-tricalcium phosphate system, was found to be reactive in a simulated body fluid. In the present study, the reactivity of the Bioeutectic was assessed in human parotid saliva. Samples of the material were soaked for one month in human parotid saliva at 37 degrees C. The experiments showed the formation of two separate zones of carbonate-hydroxyapatite-like phase on the periphery of the samples. The first zone was formed by reaction of the bioeutectic with the saliva and progressed inside the material. The other zone developed on the surface of the bioeutectic by precipitation from the media. The mechanism of carbonate-hydroxyapatite-like phase formation in human parotid saliva appeared to be similar to that of apatite-like phase found in a simulated body fluid.

Morphological and structural study of pseudowollastonite implants in bone.

In vitro experiments show that pseudowollastonite (alpha-CaSiO3) is a highly bioactive material that forms a hydroxyapatite surface layer on exposure to simulated body fluid and also to human parotid saliva. This finding is very significant, as it indicates that the pseudowollastonite can be physically and chemically integrated into the structure of living bone tissue, and therefore could be suitable for repair or replacement of living bone. The physical and chemical nature of the remodelled interface between the pseudowollastonite implants and the surrounding bone has been studied after in vivo implantation of 20 pseudowollastonite cylinders into rat tibias. The interfaces formed after 3, 6, 8 and 12 weeks of implantation were examined histologically using an optical microscope and also by analytical scanning electron microscopy. SEM and X-ray elemental analysis showed that the new bone was growing in direct contact with the implants. Other examinations found that the bone was fully mineralized. The ionic exchange taking place at the implant interface with the body fluids was essential in the process of the implant integration through a dissolution-precipitation-transformation mechanism. The study found the interface biologically and chemically active over the 12-week implantation period. The rate of new bone formation decreased after the first 3 weeks and reached constant value over the following 9 weeks. The osteoblastic cells migrated towards the interface and colonized the surface at the contact areas with the cortical regions and also bone marrow.

Bioactivity of pseudowollastonite in human saliva.

OBJECTIVES: Pseudowollastonite (CaO.SiO2) was found to be bioactive in a simulated body fluid environment. In the present study, 'in vitro' bioactivity of pseudowollastonite was further assessed in human parotid saliva. The main objective was to compare behaviour of the material in a natural medium of high protein content (human parotid saliva) with its behaviour in an acellular protein-free solution (simulated body fluid). METHODS: Samples of polycrystalline pseudowollastonite were immersed for one month in human parotid saliva at 37 degrees C. Changes in ionic concentrations in the human parotid saliva and the pH right at the interface of pseudowollastonite/human parotid saliva were determined. The products of the interfacial reactions were studied by thin-film X-ray diffraction, scanning and transmission electron microscopy. RESULTS: The results confirmed formation of a hydroxyapatite-like layer on the surface of the material, and also suggested that the mechanism of hydroxyapatite-like layer formation in saliva was similar to that showed in simulated body fluid. CONCLUSIONS: The hydroxyapatite-like layer formed at the interface was found to be compact, continuous and composed of many small crystallites with ultrastructure similar to that of natural cortical bone and dentine. The study also concluded that the high pH conditions (10.32) existing right at the pseudowollastonite/human parotid saliva interface promoted hydroxyapatite-like precipitation. At this stage of the study, similarities of the material behaviour in saliva and acellular simulated body fluid suggest that the pseudowollastonite could be of interest in specific periodontal applications for bone restorative purposes.

Morphology and ultrastructure of the interface between hydroxyapatite-polyhydroxybutyrate composite implant and bone.

A composite of polyhydroxybutyrate (PHB) polymer, reinforced with synthetic hydroxyapatite (HA) particles, with potential as a bone-analogue material, was examined microscopically using scanning electron microscopy and transmission electron microscopy. These imaging techniques provide the means of understanding and monitoring the morphological and structural behaviour of retrieved implants. Scanning electron microscopy was used to assess the overall mechanism of new bone formation at the implant interface after up to 6 months implantation. This procedure was followed by a detailed ultrastructural examination at lattice plane resolution level, using high resolution electron microscopy and selected area diffraction of the regions showing bone apposition. Fine hydroxyapatite crystallites were found to form at the interface after in vivo implantation into cortical bone.

Morphological studies of pseudowollastonite for biomedical application.

Pseudowollastonite ceramic (psW) composed of CaO.SiO2 was found to be bioactive in a simulated body fluid environment. The chemical reaction initiated at the material surface resulted in hydroxyapatite (HA) formation. These bone-bonding properties are essential for securing the necessary physico-chemical integration of the material with living bone. Materials behaving in this way can be considered for potential biomedical application as bone tissue substitute for a natural bone repair or replacement as implant. A mechanism of hydroxyapatite formation on pseudowollastonite ceramics surface was investigated during exposure to a stimulated body fluid (SBF) for a period of 3 weeks. Morphology and structure of the surface product and its original substrate was examined by thin-film X-ray diffraction, scanning electron microscopy and high-resolution transmission electron microscopy. HA crystals were found to form on an amorphous silica intermediate layer. (100) lattice planes of HA were resolved and identified. Concentration of ions in the SBF and pH of the SBF were monitored throughout the exposure. Additional pH measurements were made at the interface of psW with SBF. The HA formation occurred when there was a sudden increase of pH from 7.25 to 10.5 at the interface of psW with SBF as a result of ionic exchange between 2H+ and Ca2+ within the psW network. This ionic exchange transformed the psW crystals into an amorphous silica phase. The appropriate pH and the ion concentrations were essential for partial dissolution of the amorphous silica phase and subsequent precipitation of a Ca-P rich phase which then transformed to HA.