Racquel Z. LeGeros

Biphasic calcium phosphate bioceramics: preparation, properties and applications

Biphasic calcium phosphate (BCP) bioceramics belong to a group of bone substitute biomaterials that consist of an intimate mixture of hydroxyapatite (HA), Ca10(PO4)6(OH)2, and beta-tricalcium phosphate (β-TCP), Ca3(PO4)2, of varying HA/β-TCP ratios. BCP is obtained when a synthetic or biologic calcium-deficient apatite is sintered at temperatures at and above 700 °C. Calcium deficiency depends on the method of preparation (precipitation, hydrolysis or mechanical mixture) including reaction pH and temperature. The HA/β-TCP ratio is determined by the calcium deficiency of the unsintered apatite (the higher the deficiency, the lower the ratio) and the sintering temperature. Properties of BCP bioceramics relating to their medical applications include: macroporosity, microporosity, compressive strength, bioreactivity (associated with formation of carbonate hydroxyapatite on ceramic surfaces in vitro and in vivo), dissolution, and osteoconductivity. Due to the preferential dissolution of the β-TCP component, the bioreactivity is inversely proportional to the HA/β-TCP ratio. Hence, the bioreactivity of BCP bioceramics can be controled by manipulating the composition (HA/β-TCP ratio) and/or the crystallinity of the BCP. Currently, BCP bioceramics is recommended for use as an alternative or additive to autogeneous bone for orthopedic and dental applications. It is available in the form of particulates, blocks, customized designs for specific applications and as an injectible biomaterial in a polymer carrier. BCP ceramic can be used also as grit-blasting abrasive for grit-blasting to modify implant substrate surfaces. Exploratory studies demonstrate the potential uses of BCP ceramic as scaffold for tissue engineering, drug delivery system and carrier of growth factors.

Biodegradation and bioresorption of calcium phosphate ceramics.

The use of several calcium phosphate (Ca-P) materials for bone repair, augmentation, substitution and as coatings on metal implants has gained clinical acceptance in many dental and medical applications. These Ca-P materials may be of synthetic or natural origin, available in different physical forms (dense or macroporous, particles or blocks) and are used in bulk as coatings for metallic and non-metallic substrates or as components in composites, cements and bioactive glasses. Biodegradation or bioresorption of calcium phosphate materials implies cell-mediated degradation in vitro or in vivo. Cellular activity during biodegradation or bioresorption occurs in acid media; thus the factors affecting the solubility or the extent of dissolution (which in turn depends on the physico-chemical properties) of the Ca-P materials are important. Enrichment of the microenvironment due to the release of calcium and phosphate ions from the dissolving Ca-P materials affects the proliferation and activities of the cells. The increase in the concentrations of the calcium and phosphate ions promotes the formation of carbonate apatite which are similar to the bone apatite. The purpose of this invited paper is to discuss the processes of biodegradation or bioresorption of Ca-P materials in terms of the physico-chemical properties of these materials and the phenomena involved including the formation of carbonate apatite on the surfaces and in the vicinity of these materials. This phenomenon appears to be related to the bioactivity of the material and the ability of such materials to directly attach to bone and to form a uniquely strong material-bone interface.

Formation of carbonate-apatite crystals after implantation of calcium phosphate ceramics.

SummaryThe aims of this study were (1) to determine at the crystal level, the nonspecific biological fate of different types of calcium phosphate (Ca−P) ceramics after implantation in various sites (osseous and nonosseous) in animals and (2) to investigate the crystallographic association of newly formed apatitic crystals with the Ca−P ceramics.Noncommercial Ca−P ceramics identified by X-ray diffraction as calcium hydroxylapatite (HA), beta-tricalcium phosphate (β-TCP), and biphasic calcium phosphates (BCP) (consisting of β-TCP/HA=40/60) were implanted under the skin in connective tissue, in femoral lamellar cortical bone, articular spine bone, and cortical mandibular and mastoidal bones of animals (mice, rabbits, beagle dogs) for 3 weeks to 11 months. In humans, HA or β-TCP granules were used to fill periodontal pockets, and biposies of the implanted materials were recovered after 2 and 12 months.Results of this study demonstrated the following: (1) the presence of needle-like microcrystals (new crystals) associated with the Ca−P ceraiic macrocrystals in the microporous regions of the implants regardless of the sites of implantation (osseous or nonosseous), type of Ca−P ceramics (HA, β-TCP, BCP), type of species used (mice, rabbits, dogs, humans), or duration of implantation; (2) decrease in the area occupied by the ceramic crystals and the subsequent filling of the spaces between the ceramic crystals by the new crystals; (3) these new crystals were identified as apatite by electron diffraction and as carbonate-apatite by infrared absorption spectroscopy; (4) high resolution transmission electron microscopy (Hr TEM) revealed one family of apatite lattice fringes in the new crystals in continuity with the lattice planes of the HA of β-TCP ceramic crystals; (5) Hr TEM also demonstrated the presence of linear dislocations at the junction of the new apatite crystals and ceramic crystals.It is suggested that the formation of the CO3 apatite crystals associated with the implanted Ca−P ceramic is due to dissolution/precipitation and secondary nucleation involving an epitatic growing process and not to an osteogenic property of the ceramic.

Apatite crystallites: effects of carbonate on morphology.

Carbonate is a substituent in the apatite structure; when present, it limits the size of the growing apatite crystals and so influences their shape that they grow more equiaxed than needle-like. The tendency for carbonate apatites to be equiaxed is related to the nature of the chemical bonds formed in the crystal. The interference of carbonate with the good crystallization of apatite, and its weakening effect on the bonds in the structure, increase the dissolution rate and the solubility, thereby presumably contributing to the susceptibility to caries of dental apatites containing carbonate.

Two types of carbonate substitution in the apatite structure.

Um die Art des Karbonateinbaues in die Apatitstruktur zu klären, wurden zwei Typen von synthetischen Karbonatapatiten untersucht: solche, die sich in wässrigen Medien bildeten, und andere, die bei hohen Temperaturen und unter Ausschluss von Wasser entstanden.

Phosphate Minerals in Human Tissues

The mineralized or calcified tissues in biological systems are composed of two phases: organic and inorganic or mineral phases. In the invertebrates (e.g., echinoderms, mollusks, arthropods, etc.), the inorganic phase is usually calcium carbonate, CaCO3, predominantly in the form of either calcite or aragonite or both. In the invertebrates, the inorganic phase consists of one or more types of phosphate minerals (predominantly calcium phosphates) depending on the nature of calcification, i.e., normal (e.g., bones and teeth) or abnormal or pathological (e.g., dental calculi, salivary and urinary stones, soft tissue calcifications, etc.). In several pathologically calcified tissues, the mineral is non-phosphatic, such as calcium oxalates (whewellite and weddellite), sodium urates, uric acid, cysteine.

ADAPTIVE CRYSTAL FORMATION IN NORMAL AND PATHOLOGICAL CALCIFICATIONS IN SYNTHETIC CALCIUM PHOSPHATE AND RELATED BIOMATERIALS

Mineralization and crystal deposition are natural phenomena widely distributed in biological systems from protozoa to mammals. In mammals, normal and pathological calcifications are observed in bones, teeth, and soft tissues or cartilage. We review studies on the adaptive apatite crystal formation in enamel compared with those in other calcified tissues (e.g., dentin, bone, and fish enameloids) and in pathological calcifications, demonstrating the adaptation of these crystals (in terms of crystallinity and orientation) to specific tissues that vary in functions or vary in normal or diseased conditions. The roles of minor elements, such as carbonate, magnesium, fluoride, hydrogen phosphate, pyrophosphate, and strontium ions, on the formation and transformation of biologically relevant calcium phosphates are summarized. Another adaptative process of crystals in biology concerns the recent development of calcium phosphate ceramics and other related biomaterials for bone graft. Bone graft materials are available as alternatives to autogeneous bone for repair, substitution, or augmentation. This paper discusses the adaptive crystal formation in mineralized tissues induced by calcium phosphate and related bone graft biomaterials during bone repair.

XRD, SEM-EDS, and FTIR studies of in vitro growth of an apatite-like layer on sol-gel glasses

A glass with a composition (in mole %) of: SiO2 (70), CaO (26), and P2O5 (4) was obtained using a sol-gel method. The in vitro bioactivity of the glass was assessed by determining the changes in surface morphology and composition after soaking in simulated body fluid (SBF) for periods of up to 14 days at 37 degrees C. X-ray diffraction, scanning electron microscopy, X-ray energy dispersive spectroscopy, and FTIR analyses of the glass surface after different soaking periods in SBF demonstrated the growth of an apatite-like layer on the glass surface. In the first stage, an amorphous calcium phosphate layer was formed; after 7 days this surface consisted of spheres, with diameters ranging between 2 and 15 microm, composed of needle-like apatite crystallites (250 x 100 nm) with a crystallinity similar to that of a biological apatite.

Crystal dissolution of biological and ceramic apatites

SummaryHigh resolution transmission electron microscopy (Hr TEM) studies on biological and synthetic calcium phosphate have provided information on the dissolution process at the crystal level. The purpose of this study was to investigate the dissolution of ceramic hydroxyapatite (HA) after implantation using Hr TEM. Recovered HA ceramic implanted in bony and nonbony sites in animals and in periodontal pockets in humans were used for the study. For comparison, sections of human fluorotic enamel with caries and sections of shark enameloid previously exposed to 0.1 HCl were similarly investigated. Hr TEM studies demonstrated that in both the biological and ceramic apatites, the lattice and atomic defects were the starting points in the dissolution process. However, significant differences in the process of dissolution were observed: (1) biological apatite crystals showed preferential core dissolution whereas ceramic apatite crystals showed nonspecific dissolution at the cores and at the surfaces; (2) the dissolution of biological apatites appeared to consistently extend along the crystals c-axis whereas dissolution of the ceramic HA did not appear to be correlated with the crystals c-axis. The observed differences in crystal dissolution between biological and ceramic apatites may be attributed to the following: (1) the unique crystal/protein interaction present with biological apatites but absent in ceramic HA; (2) differences in defect distribution between biological and ceramic apatites which are due to the differences in the original of these defects; and (3) the longer morphological c-axis of biological apatites compared with that of ceramic apatites. This study provided for the first time, information on the dissolution process of implanted ceramic HA crystals and suggests that the crystal defects resulting from the sintering processes during the preparation of ceramic HA affect itsin vivo degradation and performance.

Physicochemical Characterization of the Nucleational Core of Matrix Vesicles

While previous studies revealed that matrix vesicles (MV) contain a nucleational core (NC) that converts to apatite when incubated with synthetic cartilage lymph, the initial mineral phase present in MV is not well characterized. This study explored the physicochemical nature of this Ca2+ and Pi-rich NC. MV, isolated from growth plate cartilage, were analyzed directly by solid-state 31P NMR, or incubated with hydrazine or NaOCl to remove organic constituents. Other samples of MV were subjected to sequential treatments with enzymes, salt solutions, and detergents to expose the NC. We examined the NC using transmission electron microscopy, energy-dispersive analysis with x-rays, and electron and x-ray diffraction, Fourier transform-infrared spectroscopy, high performance thin-layer chromatographic analysis, and SDS-polyacrylamide gel electrophoresis. We found that most of the MV proteins and lipids could be removed without destroying the NC; however, NaOCl treatment annihilated its activity. SDS-polyacrylamide gel electrophoresis showed that annexin V, a phosphatidylserine (PS)-dependent Ca2+-binding protein, was the major protein in the NC; high performance thin-layer chromatographic analysis revealed that the detergents removed the majority of the polar lipids, but left significant free cholesterol and fatty acids, and small but critical amounts of PS. Transmission electron microscopy showed that the NC was composed of clusters of ∼1.0 nm subunits, which energy-dispersive analysis with x-rays revealed contained Ca and Pi with a Ca/P ratio of 1.06 ± 0.01. Electron diffraction, x-ray diffraction, and Fourier transform-infrared analysis all indicated that the NC was noncrystalline. 1H-Cross-polarization 31P NMR indicated that the solid phase of MV was an HPO42−-rich mixture of amorphous calcium phosphate and a complex of PS, Ca2+, and Pi. Taken together, our findings indicate that the NC of MV is composed of an acid-phosphate-rich amorphous calcium phosphate intermixed with PS-Ca2+-Pi, annexin V, and other proteins and lipids.