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PubMed2006

Nature of phosphate substrate as a major determinant of mineral type formed in matrix vesicle-mediated in vitro mineralization: An FTIR imaging study.

Membrane-bound extracellular matrix vesicles play an important role in the de novo initiation and propagation of calcium-phosphate mineral formation in calcifying cartilage, bone, dentin, and in pathologic calcification. Characterization of the phase, composition, crystal size, and perfection provides valuable insight into the mechanism of the mineral deposition. In the present study, Fourier transform infrared imaging spectroscopy (FT-IRIS) was used to characterize the mineral phase generated during MV-mediated in vitro mineralization. FT-IRIS studies revealed that the mineral phase associated with MVs calcified in the presence of AMP and beta-GP was always found to be crystalline hydroxyapatite while with ATP only a small amount of immature mineral, most likely an amorphous or poorly crystalline hydroxyapatite, was observed. Low concentrations of pyrophosphate (PPi) (< or = 0.01 mM) showed apatitic mineral while high concentrations showed immature calcium pyrophosphate dihydrate (CPPD). The implications of these findings are that (a) hydrolysis of AMP or beta-GP, monophosphoester substrates of MV-5' AMPase (substrate: AMP) and TNAP (substrates: AMP, beta-GP), yields orthophosphate (Pi) which leads to the formation of mature crystalline, apatite mineral, while the hydrolysis of ATP, substrate for MV-TNAP or ATPase or NPP1, inhibits the formation of mature hydroxyapatite, and (b) pyrophosphate (PPi) has a bimodal effect on mineralization, i.e., at low PPi concentrations, alkaline phosphatase activity of matrix vesicles is able to hydrolyze PPi to orthophosphate and thus facilitates the formation of basic calcium phosphate mineral which subsequently transforms into apatitic mineral. We hypothesize that, at high PPi concentrations, PPi by itself or Pi released by partial PPi hydrolysis could act as inhibitors of alkaline phosphatase activity, thereby preventing complete hydrolysis of PPi to Pi, and thus resulting in the accumulation of calcium pyrophosphate dihydrate. Therefore, in order for physiological mineralization to proceed, a balance is required between levels of Pi and PPi.

Animals↗

PubMed2006

Novel synthesis and structural characterization of fluorine and chlorine co-substituted hydroxyapatites.

Fluorine and chlorine co-substituted hydroxyapatites (HAP) were prepared through aqueous precipitation method. Characterization results from X-ray diffraction (XRD) revealed that co-substitution of these anions resulted in the formation of pure HAP phase except for the small observed difference in the lattice parameters of the resultant apatites. The elemental analysis and FT-IR results have also confirmed the incorporation of substituted anions in the apatite structure. The calculated crystallite size for all anionic substituted apatite fall well below the range of 50 nm size that tend to coincide with the value of bone mineral crystallite size.

Chemistry, Inorganic↗

PubMed2002

Sol-gel hydroxyapatite coatings on stainless steel substrates.

Thin film hydroxyapatite deposits onto sandblasted 316L stainless steel substrates were prepared using water-based sol-gel technique recently developed in our lab. The coatings were annealed in air at 375 degrees C, 400 degrees C, and 500 degrees C. Phase formation, surface morphology, interfacial microstructure, and interfacial bonding strength of the coatings were investigated. Apatitic structure developed within the coatings while annealing at temperatures > or = 400 degrees C, while those heat-treated at 375 degrees C showed poor crystallinity. The coatings were dense and firmly attached to the underlying substrates, reaching an average bonding strength (as determined through the pull-out test) of 44 MPa. Nano-porous structure was found for the coatings annealed at 500 degrees C, believed to result from grain growth, and causing a slight decrease in the bonding strength. Surface microcracking, although not extensive, occurred after annealing at temperatures > or = 400 degrees C, and was linked to non-uniform thickness of the coating due to roughness of the substrate. A contraction of the coatings as a result of sintering, and phase transition from amorphous (or poor crystalline) to reasonably good crystalline apatite, may be responsible for the loss of structural integrity of the thicker sections of the coatings. It seems quite promising that a dense and adhesive apatite coating can be achieved through water-based sol gel technology after short-term annealing at around 400 degrees C in air.

Bone Substitutes↗

PubMed2002

The ultrastructure of the plasma-sprayed hydroxyapatite-bone interface predisposing to bone bonding.

The deposition of biological apatite and subsequent formation of bone on hydroxyapatite implants depends on the partial dissolution of the implant surface and the reprecipitation of carbonated apatite from the biological milieu. Previous investigations in vitro have shown that the degree of dissolution and reprecipitation decreases as the coating crystallinity increases. These findings prompted the current study of the effects of coating crystallinity on the mechanism of bone bonding. The process of mineralization of bone associated with a hydroxyapatite coating was compared to the normal process of ossification. Plasma-sprayed hydroxyapatite (PSHA) coated titanium alloy (6% Al-4% V) rods as received and annealed for 0.7 h at 600 degrees C in air to increase the coating crystallinity were implanted in the proximal and distal femora and proximal tibiae of adult mongrel dogs for 3 h, 3 and 10 days. Bony sites containing the implant were prepared for ultramicrotomy and transmission electron microscopy using an anhydrous embedding procedure: fixation in ethylene glycol and embedment in Spurr's resin. The results demonstrated the precipitation of biological apatite crystallites on non-annealed PSHA coatings in vivo within 3 h of implantation. After 3 and 10 days there were differences in the ultrastructure of the mineral phase on the surfaces of non-annealed and annealed surfaces. Observations showed that there was little difference in the mechanism of mineralization of bone associated with HA-coated prostheses and the normal process of ossification.

Alloys↗

PubMed2002

The ultrastructure of anorganic bovine bone and selected synthetic hyroxyapatites used as bone graft substitute materials.

The objective of this study was to investigate the morphology and organization of apatite crystallites in mature mammalian bone. Anorganic bovine bone was studied in this investigation to allow for the examination of the mineral crystallites after removal of the organic phase. Field-emission low-voltage scanning electron microscopy (FE-LVSEM) was employed to obtain images at nanometer resolution without the application of a conductive coating. Transmission electron microscopy (TEM) of the samples was also performed to confirm the identification of features observed in the SEM and to allow for comparison with earlier studies of bone mineral architecture. For comparison, in order to demonstrate how the interaction of collagen and apatite results in the architecture and crystal structure of bone mineral, two synthetic hydroxyapatite materials were also analyzed: OsteoGen and OsteoGraf/LD300. FE-LVSEM revealed distinctive features of bone mineral: a fibrillar organization of crystallites, a periodic spacing of crystallites along the fibrils consistent with the banding pattern of collagen, inter-fibrillar bridging crystallites, and a plate-like habit of the crystallites. These findings supported the hypothesis, derived from the earlier TEM data of others, that the mineralization of collagen comprising osteoid proceeds by the formation of apatite crystallites within the fibers at selected periodic sites along their length. Moreover, the very presence in this anorganic material of distinct fibers comprised of the crystallites is demonstration of inter-crystallite bonding. The crystallites of the synthetic hydroxyapatite materials did not display any of these ultrastructural features.

Animals↗

PubMed2002

Study of implantable calcium phosphate systems for the slow release of methotrexate.

The authors prepared and studied systems implantable in bone, for the slow release of an antineoplasic agent, methotrexate (MTX). The systems were made by compaction of a powdered mixture of an apatitic deficient calcium phosphate, dextran and various amounts of MTX. Used as a matrix, this calcium phosphate has outstanding adsorption and compaction properties. It is an osteoconductor and biodegradable. The in vitro study carried out on these systems showed that the release of MTX with time is slow and prolonged due to the phenomena of adsorption/desorption of MTX onto deficient apatite. The composition of the implants changed with time towards that of stoichiometric apatite. The in vivo pilot study was performed by implantation in the external femoral condyle of rabbits. A pharmacokinetic study revealed that the circulating concentration of MTX in the blood was always below toxic levels. Twenty percent of the initial MTX remained in the implants after 7 days. A study of the biocompatibility and bioreactivity showed no local necrosis at any time, while implants degraded and new bone formed simultaneously. These implantable systems seem appear suitable for use immediately.

Animals↗

PubMed2003

In vivo dissolution behavior of various RF magnetron-sputtered Ca-P coatings on roughened titanium implants.

RF magnetron sputter deposition was used to produce 0.1, 1.0 and 4.0 microm thick Ca-P coatings on TiO(2)-blasted titanium discs. Half of the as-sputtered coated specimens were subjected to an additional infrared heat treatment for 30s at 425-475 degrees C. X-ray diffraction demonstrated that infrared radiation changed the amorphous 4 microm sputtered coatings into an amorphous-crystalline structure, while the amorphous 0.1 and 1 microm changed in a crystalline apatite structure with the presents of tetracalciumphosphate as a second phase. Scanning electron microscopically examination of the sputtered coatings revealed that annealing of the 4 microm thick coatings resulted in the appearance of small cracks. Subsequently, the discs were implanted subcutaneous into the back of rabbits. After 1, 4, 8 and 12 weeks of implantation, the implants were retrieved and prepared for histological and physicochemical evaluation. Histological evaluation revealed that the tissue response to all coated implants was very uniform. A very thin connective tissue capsule surrounded all implants. The capsule was usually free of inflammatory cells. At the interface, there was a close contact between the capsule and implant surface and no inflammatory cells were seen. Physicochemical evaluation showed that the 0.1 and 1 microm thick amorphous coatings had disappeared within 1 week of implantation. On the other hand, the 4 microm thick amorphous phase disappeared during the implantation periods, which was followed by the precipitation of a crystalline carbonate apatite. Further, at all implantation periods the heat-treated 1 and 4 microm thick coatings could be detected. Occasionally, a granular precipitate was deposited on the heat-treated 4 microm thick coating. Fourier transform infrared spectroscopy showed the formation of carbonate apatite (CO(3)-AP) on the 4 microm thick amorphous coating and on the heat-treated specimens. On basis of our findings, we conclude that 1 microm thick heat-treated Ca-P sputter coating on roughened titanium implants appear to be of sufficient thickness to show bioactive properties, under in vivo conditions.

Animals↗

PubMed2003

Action of FGMgCO3Ap-collagen composite in promoting bone formation.

To improve the biological properties of materials as bone substitutes, functionally graded CO3 apatite crystals containing magnesium, FGMgCO3Ap, were synthesized to be mixed with atelocollagen and made into a composite pellet. A radio-labeled cell adhesion experiment showed that the degree of adherence of mouse MC3T3E1 osteoblast-like cells to the FGMgCO3Ap-collagen composite was better than to CO3Ap-collagen and much better than to the Ti plate. When the composites were implanted beneath the periosteum cranii of rats, the FGMgCO3Ap-collagen composite was metabolized faster than the CO3Ap-collagen composite and better formation of new bone and osteoblast arrangement at the interface between the composite and the periosteum cranii was observed. When the composites were implanted into the femur of rabbits, clear bone formation with a higher degree of bone density was observed for the FGMgCO3Ap-collagen composite. These results suggest that the Mg2+ ions taken into the apatite crystals may contribute to the acceleration of osteoblast adhesion to apatites and promote bone formation, cross-talking with osteoblasts at the molecular level.

3-Hydroxysteroid Dehydrogenases↗

PubMed2004

In vitro Ca-P precipitation on biodegradable thermoplastic composite of poly(epsilon-caprolactone-co-DL-lactide) and bioactive glass (S53P4).

Bioactive properties of composites containing poly(epsilon-caprolactone-co-DL-lactide) with molar ratio 96/4 and bioactive glass (BAG), S53P4, were tested in vitro. The glass content in the tested materials was 40, 60 or 70 wt%, and two granule size ranges (<45 and 90-315 microm) were used. The composites were analysed for their apatite-forming ability. This was determined as a function of time by the dissolution pattern of Si and Ca ions and structural changes on the specimen surfaces. Composite specimens were immersed in simulated body fluid at 37 degrees C for up to 6 months. The changes in Si and Ca concentrations of the immersion medium were determined with UV-Vis and atomic absorption spectrophotometry. The calcium phosphate precipitation and apatite formation were evaluated by scanning electron microscopy (SEM) and infra-red spectroscopy (IR) using the attenuated total reflectance (ATR) system. The SEM and SEM-EDX analysis of the depositions formed on the composite surfaces was in line with the changes in ion concentrations. The clearest results with IR were seen in the material containing 60 wt% small glass particles. The results indicate that composites containing over 40 wt% BAG granules are bioactive, and that a higher BAG surface area/volume ratio favors the apatite formation in vitro.

Absorbable Implants↗

PubMed1998

Growth and differentiation of human bone marrow osteoprogenitors on novel calcium phosphate cements.

Materials that augment bone cell proliferation and osteogenic activity have important therapeutic implications for bone regeneration and for use in skeletal reconstruction and joint replacement. We have studied the growth and interactions of human bone marrow cells on a variety of new cement composites in vitro. These cement materials are composed of calcium-deficient hydroxyapatites, carbonated apatite and amorphous calcium phosphate. Cell proliferation was significantly reduced and cell differentiation increased in the presence of these cements compared with cells cultured on tissue culture plastic. Alkaline phosphatase, one of the markers of the osteoblast phenotype, was dramatically stimulated by 3 of the 4 cements examined between day 4 and day 10, above levels observed following culture of human osteoblasts on plastic alone. Photomicroscopic examination demonstrated growth and close integration of bone marrow cells and 3 of the composites. Longer term marrow cultures (15 day) on the cements confirmed the stimulation of cell differentiation over proliferation. From these studies, enhanced osteoblastic differentiation was observed on a 70% carbonated apatite, which has a composition similar to bone mineral, whereas, cell toxicity was observed on cells grown on amorphous calcium phosphate. This in vitro culture system demonstrates the use of human bone marrow cells for the potential evaluation of new biomaterials and the development of a novel carbonated apatite that may be of potential use in orthopaedic implants.

Adult↗

PubMed1997

Chemical and structural characterization of the mineral phase from cortical and trabecular bone.

X-ray diffraction, infrared spectroscopy and chemical investigations have been carried out on the inorganic phases from rat cortical and trabecular bone. Although both inorganic phases consist of poorly crystalline B carbonated apatite, several significant differences have been observed. In particular, trabecular bone apatite displays reduced crystallite sizes, Ca/P molar ratio, and carbonate content, and exhibits a greater extent of thermal conversion into beta-tricalcium phosphate than cortical bone apatite. These differences can be related to the different extents of collagen posttranslational modifications exhibited by the two types of bone, in agreement with their different biological functions.

Animals↗

PubMed1997

Behavior of carbonate and magnesium ions in the initial crystallites at the early developmental stages of the rat calvaria.

Analysis of the contents of calcium (Ca), magnesium (Mg), phosphate, and carbonate ions in the mineral phase of rat calvaria specimens obtained at different developmental stages indicated that the mineral at the newborn stage contained a negligible amount of carbonate, but a high content of Mg. There was no significant difference in Ca and phosphate (as PO4) contents between the newborn material and that from later stages. A relatively large amount of carbonate was detected in the calvaria from 6-day-old rats, in which only immature crystals were observed, thus indicating the beginning of apatite formation. Furthermore, using laser Raman microprobe analysis we confirmed that the Raman peak at 1120 cm-1 band, indicative of a Mg-CO3 compound, appeared at the 6-day stage. We also observed that the Raman peak at 988 cm-1 found in the samples from the newborn seemed to have shifted to 963-962 cm-1 in the case of those obtained from 6-day-old rats, a shift which suggests the conversion from the non-apatitic to the apatitic form. These results indicate that carbonate ions might facilitate the initiation of crystal development by converting the inhibitory Mg ion into its inactive form (Mg-carbonate compound).

Aging↗

PubMed2003

Microstructure, mechanical, and biomimetic properties of fish scales from Pagrus major.

The fish scale of Pagrus major has an orthogonal plywood structure of stratified lamellae, 1-2 microm in thickness, consisting of closely packed 70- to 80-nm-diameter collagen fibers. X-ray diffraction, energy-dispersive X-ray analysis, and infrared spectroscopy indicate that the mineral phase in the scale is calcium-deficient hydroxyapatite containing a small amount of sodium and magnesium ions, as well as carbonate anions in phosphate sites of the apatite lattice. The tensile strength of the scale is high (approximately 90 MPa) because of the hierarchically ordered structure of mineralized collagen fibers. Mechanical failure occurs by sliding of the lamellae and associated pulling out and fracture of the collagen fibers. In contrast, demineralized scales have significantly lower tensile strength (36 MPa), indicating that interactions between the apatite crystals and collagen fibers are of fundamental importance in determining the mechanical properties. Thermal treatment of fish scales to remove the organic components produces remarkable inorganic replicas of the native orthogonal plywood structure of the fibrillary plate. The biomimetic replica produced by heating to 873 K consists of stratified porous lamellae of c-axis-aligned apatite crystals that are long, narrow plates, 0.5-0.6 microm in length and 0.1-0.2 microm in width. The textured inorganic material remains intact when heated to 1473 K, although the size of the constituent crystals increases as a result of thermal sintering.

Animals↗

PubMed1976

Dental phosphoprotein-induced formation of hydroxylapatite during in vitro synthesis of amorphous calcium phosphate.

(Ethylenedinitrilo)tetraacetic acid soluble phosphoproteins were isolated from rat incisor and bovine unerupted teeth. This material was examined for its effect on the stability of amorphous calcium phosphate in vitro. When the precipitation of amorphous calcium phosphate was attempted in the presence of small amounts of these phospho-proteins, an apatite-like mineral was observed to form, which was approximately 60% crystalline, as determined by infrared measurements. This apatite phase could not be induced by addition of phosphoprotein after the precipitation reaction. The organic phosphate bound to these phosphoproteins was shown to be directly responsible for the formation of the apatite phase, since removal of 60% of the covalently bound phosphate with alkaline phosphatase destroyed the protein's ability to induce hydroxylapatite formation. The properties of the dental phosphoproteins appear to be consistent with their possible involvement in the development of the mineral phase of dentine.

Amino Acids↗

PubMed2002

Mechanisms of uranium interactions with hydroxyapatite: implications for groundwater remediation.

The speciation of U(VI) sorbed to synthetic hydroxyapatite was investigated using a combination of U LIII-edge XAS, synchrotron XRD, batch uptake measurements, and SEM-EDS. The mechanisms of U(VI) removal by apatite were determined in order to evaluate the feasibility of apatite-based in-situ permeable reactive barriers (PRBs). In batch U(VI) uptake experiments with synthetic hydroxyapatite (HA), near complete removal of dissolved uranium (>99.5%) to <0.05 microM was observed over a range of total U(VI) concentrations up to equimolar of the total P in the suspension. XRD and XAS analyses of U(VI)-reacted HA at sorbed concentrations < or = 4,700 ppm U(VI) suggested that uranium(VI) phosphate, hydroxide, and carbonate solids were not present at these concentrations. Fits to EXAFS spectra indicate the presence of Ca neighbors at 3.81 A. U-Ca separation, suggesting that U(VI) adsorbs to the HA surfaces as an inner-sphere complex. Uranium(VI) phosphate solid phases were not detected in HA with 4700 ppm sorbed U(VI) by backscatter SEM or EDS, in agreement with the surface complexation process. In contrast, U(VI) speciation in samples that exceeded 7000 ppm sorbed U(VI) included a crystalline uranium(VI) phosphate solid phase, identified as chernikovite by XRD. At these higher concentrations, a secondary, uranium(VI) phosphate solid was detected by SEM-EDS, consistent with chernikovite precipitation. Autunite formation occurred at total U:P molar ratios > or = 0.2. Our findings provide a basis for evaluating U(VI) sorption mechanisms by commercially available natural apatites for use in development of PRBs for groundwater U(VI) remediation.

Adsorption↗

PubMed2003

Induced phytoextraction/soil washing of lead using biodegradable chelate and permeable barriers.

Chelate-induced remediation has been proposed as an effective tool for the extraction of lead (Pb) from contaminated soils by plants. However, side-effects, mainly mobilization and leaching of Pb, raise environmental concerns. Biodegradable, synthetic organic chelate ethylenediaminedisuccinic acid (EDDS), and commonly used ethylenedimanetetraacetic acid (EDTA) were used for induced phytoextraction with a test plant Brassica rapa and in situ washing of soil contaminated with 1350 mg/kg of Pb. Horizontal permeable barriers were placed 20 cm deep in soil columns and tested for their ability to prevent leaching of Pb. The reactive materials in the barriers were nutrient enriched vermiculite, peat or agricultural hydrogel, and apatite. EDTA and EDDS addition increased Pb concentrations in the test plant by 158 and 89 times compared to the control, to 817 and 464 mg/kg, respectively. In EDTA treatments, approximately 25% or more of total initial soil Pb was leached in single cycle of chelate addition. In EDDS treatments, 20% of the initial Pb was leached from columns with no barrier, while barriers with vermiculite or hydrogel and apatite decreased leaching by more than 60 times, to 0.35%. 11.6% of total initial Pb was washed from the soil above the barrier with vermiculite and apatite, where almost all leached Pb was accumulated. Results indicate that use of biodegradable chelate EDDS and permeable barriers may lead to environmentally safe induced Pb phytoextraction and in situ washing of Pb.

Aluminum Silicates↗

PubMed1998

Ultrasonic implantation of calcium metasilicate glass particles into PMMA.

Polymer materials for clinical applications should be bioactive and have a bone-bonding ability. In order to provide poly(methyl methacrylate) (PMMA) with bioactivity, granules (<45 microm) of a bioactive glass 50CaO.50SiO2 (mol %) were implanted into PMMA: they were suspended together with a piece of PMMA in a 40 tetrahydrofuran-60 ethanol (vol %) solution and ultrasonically agitated. The granules of <10 microm in size were impregnated at approximately 40-20 microm depth below the substrate surface. Two types were detected on the PMMA surface: (a) a glass-granule layer on PMMA, and (b) an inner granule layer, a PMMA layer, and an outer granule layer on the PMMA. The bioactivity of the implanted PMMA substrates was examined in vitro with a simulated body fluid (Kokubo solution). Apatite was precipitated on all glass granules and the whole substrate surfaces within 1 d. After 4 h soaking in the Kokubo solution, aggregates of apatite particles appeared on the substrate surface, independently of those on the glass granules, and they grew and proliferated on the whole subtrate surface in 7 d. Silica gel islands on PMMA due to the silicate anions from the glass were considered to induce nucleation of the apatite particles.

Journal Article↗

PubMed1999

Surface modification tailors the characteristics of biomimetic coatings nucleated on starch-based polymers.

This work describes the influence of surface pretreatments over the nucleation and growth of an apatite layer, formed by a biomimetic process, on which a bioactive glass is used as a precursor of the calcium-phosphate (Ca-P) formation on the materials surface. SEVA-C, a corn starch-based biodegradable blend, was used as substrate. The surfaces were pretreated during various periods by: (i) physical methods, namely ultraviolet radiation (u.v.), and over exposure to ethylene oxide sterilization (EtO); and (ii) chemical methods, namely potassium hydroxide (KOH) and acetic anhydride (CH3CO)2 etchings. The surface modifications, performed before the production of the biomimetic coatings, resulted in a faster formation of Ca-P nuclei during the first stages of SBF immersion, particularly in the case of the KOH etching. In this case, it was possible to observe a decrease in the average surface roughness, as measured by laser profilometry, and an increase of the hydrophilicity of the material, which was evident from a clear increment in the water-uptake ability and quantified by contact angle measurements. With this treatment it was possible not only to reduce the induction period for the formation of a well defined and dense apatite-like layer, as observed by scanning electron microscopy (SEM), but also to improve the adhesion of the Ca-P layer to the substrate, as confirmed by the adhesion strength tests. For all the studied pre-treatments, the composition of the films, analyzed by energy dispersive spectroscopy (EDS) and identified by thin-film X-ray diffraction (TF-XRD), seems to be very similar to that of human bone apatites.

Journal Article↗