α-Tricalcium phosphate: synthesis, properties and biomedical applications.

Nowadays, α-tricalcium phosphate (α-TCP, α-Ca(3)(PO(4))(2)) is receiving growing attention as a raw material for several injectable hydraulic bone cements, biodegradable bioceramics and composites for bone repair. In the phase equilibrium diagram of the CaO-P(2)O(5) system, three polymorphs corresponding to the composition Ca(3)(PO(4))(2) are recognized: ß-TCP, α-TCP and α'-TCP. α-TCP is formed by heating the low-temperature polymorph ß-TCP or by thermal crystallization of amorphous precursors with the proper composition above the transformation temperature. The α-TCP phase may be retained at room temperature in a metastable state, and its range of stability is strongly influenced by ionic substitutions. It is as biocompatible as ß-TCP, but more soluble, and hydrolyses rapidly to calcium-deficient hydroxyapatite, which makes α-TCP a useful component for preparing self-setting osteotransductive bone cements and biodegradable bioceramics and composites for bone repairing. The literature published on the synthesis and properties of α-TCP is sometimes contradictory, and therefore this article focuses on reviewing and critically discussing the synthetic methods and physicochemical and biological properties of α-TCP-based biomaterials (excluding α-TCP-based bone cements).

In vitro study of the proliferation and growth of human bone marrow cells on apatite-wollastonite-2M glass ceramics.

This study concerns the preparation and in vitro characterization of an apatite-wollastonite-2M bioactive glass ceramic which is intended to be used for the regeneration of hard tissue (i.e. in dental and craniomaxillofacial surgery). This bioglass ceramic has been obtained by appropriate thermal treatment through the devitrification (crystallization) of a glass with a stoichiometric eutectic composition within the Ca(3)(PO(4))(2)-CaSiO(3) binary system. Crack-free specimens of the bioglass ceramic were immersed in human bone marrow cell cultures for 3, 7, 14 and 21days, in order to study biocompatibility. Cell morphology, proliferation and colonization were assessed by scanning electron microscopy and confocal laser scanning microscopy. A total protein content assay was used to evaluate the viability and proliferation of cultured bone marrow cells. The results showed that the cells were able to adhere and proliferate on the designed material due to the essentiality of silicon and calcium as accessory factors for cell activity stimulation.

Devitrification studies of wollastonite-tricalcium phosphate eutectic glass.

The present paper describes and discusses the devitrification and crystallization process of wollastonite-tricalcium phosphate (W-TCP) eutectic glass. This process was studied in situ from room temperature up to 1375 degrees C, by neutron diffractometry in vacuum. The data obtained were combined and compared with those performed in ambient atmosphere by differential thermal analysis and with those of samples fired in air at selected temperatures, and then cooled down and subsequently studied by laboratory XRD and field emission scanning electron microscopy fitted with energy X-ray dispersive spectroscopy. The experimental evidence indicates that the devitrification of W-TCP eutectic glass begins at approximately 870 degrees C with the crystallization of a Ca-deficient apatite phase, followed by wollastonite-2M (CaSiO(3)) crystallization at approximately 1006 degrees C. At 1375 degrees C, the bio-glass-ceramic is composed of quasi-rounded colonies formed by a homogeneous mixture of pseudowollastonite (CaSiO(3)) and alpha-tricalcium phosphate (Ca(3)(PO(4))(2)). This microstructure corresponds to irregular eutectic structures. It was also found that it is possible to obtain from the eutectic composition of the wollastonite-tricalcium phosphate binary system a wide range of bio-glass-ceramics, with different crystalline phases present, through appropriate design of thermal treatments.

Synthesis, characterization, bioactivity and biocompatibility of nanostructured materials based on the wollastonite-poly(ethylmethacrylate-co-vinylpyrrolidone) system.

Composite materials are very promising biomaterials for hard tissue augmentation. The approach assayed in this work involves the manufacturing of a composite made of a bioactive ceramic, natural wollastonite (W) and a nanostructured copolymer of ethylmethacrylate (EMA) and vinylpyrrolidone (VP) to yield a bioresorbable and biocompatible VP-EMA copolymer. A bulk polymerization was induced thermally at 50 degrees C, using 1 wt % azobis(isobutyronitrile) (AIBN) as free-radical initiator. Structural characterization, compressive strength, flexural strength (FS), degradation, bioactivity, and biocompatibility were evaluated in specimens with a 60/40 VP/EMA ratio and ceramic content in the range 0-60%. A good integration between phases was achieved. Greater compression and FS, in comparison with the pure copolymer specimens was obtained only when the ceramic load got up to 60% of the total weight. The soaking in NaCl solution resulted in the initial swelling of the specimens tested. The maximum swelling was reached after 2-3 h of immersion and it was significantly greater for lower ceramic loads. This result makes the polymer component the main responsible for the interactions with the media. After soaking in SBF, microdomains segregation can be observed in the polymer component that can be related with a dramatic difference in the reactivity of both monomers in free radical polymerization, whereas the formation of an apatite-like layer on the W surfaces can be observed. Biocompatibility in vitro studies showed the absence of cytotoxicity of all formulations. The cells were able to adhere on the polystyrene negative control and on specimens containing 60 wt % wollastonite forming a monolayer and showing a normal morphology. However, a low cellular growth was observed.

Assessment of natural and synthetic wollastonite as source for bioceramics preparation.

Pseudowollastonite ceramics (beta-CaSiO3) from synthetic and natural sources were assessed with regard to their properties relevant to biomedical applications. Synthetic and natural CaSiO3 powders, with average particle size of 1.6 and 13.2 microm, respectively, were first employed. Powders were pressed and sintered at 1400 degrees C for 2 h. Pseudowollastonite was the only crystalline phase in sintered materials. Glassy phase, eight times more abundant in sintered natural wollastonite (SNW) than in the synthetic one (SSW), was observed in grain boundaries and in triple points. Larger grains and bigger and more abundant pores were present in SNW, resulting in lower diametral tensile strength (26 MPa), than in SSW (42 MPa). However, by milling the natural wollastonite starting powder to a particle size of 2.0 microm and sintering (SNW-M), the microstructure became finer and less porous, and diametral tensile strength increased (48 MPa). Weibull modulus of SNW and SNW-M samples was twice that of the SSW. All the samples released Si and Ca ions, and removed phosphate ions from simulated body fluid in similar amounts and were completely coated by apatite-like spherules after soaking in simulated body fluid for 3 wk. The aqueous extracts from all samples studied were not cytotoxic in a culture of human fibroblastic cells. No differences in fibroblast-like human cells adhesion and proliferation were observed between samples. According to the obtained results, properly processed pseudowollastonite bioceramics, obtained from the natural source, exhibit the same in vitro behavior and better performance in terms of strength and reliability than do the more expensive synthetic materials.

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.

Indirect cytotoxicity evaluation of pseudowollastonite.

This study aimed to evaluate the cytotoxicity of substances leached by pseudowollastonite (CaSiO(3)). It has been previously shown that calcium (Ca(2+)) and silicate (SiO(3)(-)) ions are released from pseudowollastonite into biological solutions. Both of these ions are known to influence the biological metabolism of osteoblastic cells essential in the mineralization process and bone-bonding mechanism. The indirect toxicity evaluation was performed by extraction method, according to International Standard Organization (ISO). Pseudowollastonite pellets obtained by solid-state reaction were incubated, in culture medium, during 24, 48, 72 or 168 h at different concentrations (5, 10, 15, 50, 100, 200 mg/ml). The cytotoxicity of each extract in presence of human osteoblastic cell line (SaOS-2) was quantitatively assessed by measuring the viability (succinate dehydrogenase activity, MTT), the membrane integrity (the uptake of the neutral red by viable cells, NR) as well as the cell necrosis by measuring the lactate dehydrogenase (LDH) released in the culture medium. No significant alteration of membrane integrity or cell suffering was detectable. However, increased cell metabolism was observed for cells exposed to pseudowollastonite extract with longest extraction time (168 h). In conclusion, mineral elements leached by pseudowollastonite do not significantly affect the metabolism of osteoblastic cells.

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.

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.

Analytical control of wollastonite for biomedical applications by use of atomic absorption spectrometry and inductively coupled plasma atomic emission spectrometry.

Preliminary in vitro experiments revealed that wollastonite (CaSiO3) is a potentially highly bioactive material that forms a hyroxyapatite (HA) surface layer on exposure to simulated body fluid with an ion concentration, pH and temperature virtually identical with those of human blood plasma. The formation of the HA layer is an essential requirement for an artificial material to be used as bioactive bone substitute. This finding opens up a wide field for biomedical applications of wollastonite. Biomaterials used as implants in the human body require strict control of trace elements and of the toxic species specified in American Society for Testing and Materials F-1185-88 (As, Cd, Hg and Pb) in ceramic hydroxyapatite for surgical implantation. In this work, two types of pseudowollastonite, the high temperature form of wollastonite, were analysed by using cold vapour atomic absorption spectrometry and hydride generation atomic absorption spectrometry, in order to determine the elements stated in the above-mentioned norm, and inductively coupled plasma atomic emission spectrometry to establish the SiO2/CaO ratio of the two materials and analyse for all other impurities introduced by the raw materials and by the processes of synthesis, sintering and grinding. Barium and Mg were especially prominent in raw materials, and Zr, Y, Mg, W, Co and Ni come mainly from the processing.

Bioeutectic: a new ceramic material for human bone replacement.

In the present work, a new way of obtaining bioactive ceramic materials with eutectic morphology is presented. To this purpose the binary system wollastonite-tricalcium phosphate was selected, taking into account the different bioactivity behaviour of both phases. The material is formed by quasi-spherical colonies composed of alternating radial lamellae of wollastonite and tricalcium phosphate. In in vitro experiments the material presents a high reactivity, with the formation of two well-differentiated zones of hydroxyapatite, one formed by alteration of the eutectic material with solution of the wollastonite into the simulated body fluid and subsequent pseudomorphic transformation of the tricalcium phosphate into hydroxyapatite, and the other, in the last stages of the experiments, by deposition of hydroxyapatite onto the surface of the material. The hydroxyapatite morphology, formed at the beginning of the reaction, is similar to that of porous bone. The method used opens the opportunity to develop a new family of bioactive materials with different constituents, binary or ternary, for which the authors propose the general name of bioeutectics.

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.