Apatite Nanocrystals Research Articles

Electrospun nanostructured fibers of collagen-biomimetic apatite on titanium alloy.

Titanium and its alloys are currently the mainly used materials to manufacture orthopaedic implants due to their excellent mechanical properties and corrosion resistance. Although these materials are bioinert, the improvement of biological properties (e.g., bone implant contact) can be obtained by the application of a material that mimics the bone extracellular matrix. To this aim, this work describes a new method to produce nanostructured collagen-apatite composites on titanium alloy substrate, by combining electrospinning and biomimetic mineralization. The characterization results showed that the obtained mineralized scaffolds have morphological, structural, and chemical compositional features similar to natural bone extracellular matrix. Finally, the topographic distribution of the chemical composition in the mineralized matrix evaluated by Fourier Transform Infrared microspectroscopy demonstrated that the apatite nanocrystals cover the collagen fibers assembled by the electrospinning.

Nanocrystalline carbonate-apatites: role of Ca/P ratio on the upload and release of anticancer platinum bisphosphonates

In the present study two nanocrystalline apatites have been investigated as bone-specific drug delivery devices to be used for treatment of bone tumors either by local implantation or by injection. In order to assess how the Ca/P ratio can influence the adsorption and release of anticancer platinum-bisphosphonate complexes, two kinds of apatite nanocrystals having different Ca/P ratios but similar morphologies, degree of crystallinity, and surface areas have been synthesized and characterized. The two platinum-bisphosphonate complexes considered were the bis-{ethylenediamineplatinum(ii)}-2-amino-1-hydroxyethane-1,1-diyl-bisphosphonate and the bis-{ethylenediamineplatinum(ii)}medronate. The Ca/P ratio plays an important role in the adsorption as well as in the release of the two drugs. In fact, the apatite with a higher Ca/P ratio showed greater affinity for both platinum complexes. Also the chemical structure of the two Pt complexes appreciably affects their affinity towards as well as their release from the two kinds of apatites. In particular, the platinum complex whose bisphosphonate contains a free aminic group showed greater upload and smaller release. The cytotoxicity of the Pt complexes released from the apatite was tested against human cervical, colon, and lung cancer cells as well as against osteosarcoma cells. In agreement with previous work, the Pt complexes released were found to be more cytotoxic than the unmodified complexes.

Mineral ecophysiological data provide growing evidence for microbial activity in banded-iron formations

The phosphorus composition of banded-iron formations (BIFs) has been used as a proxy for Precambrian seawater composition and the paleoeredox state of Earth's surface environment. However, it is unclear whether the phosphorus in BIFs originally entered the sediment as a sorbed component of the iron oxyhydroxide particles, or whether it was incorporated into the biomass of marine phytoplankton. We conducted high-resolution mineral analyses and report here the first detection of an Fe(III) acetate salt, as well as nanocrystals of apatite in association with magnetite, in the 2.48 Ga Dales Gorge Member of the Brockman Iron Formation (a BIF), Hamersley, Western Australia. The clusters of apatite are similar in size and morphology to biogenic apatite crystals resulting from biomass decay in Phanerozoic marine sediments, while the formation of an Fe(III) acetate salt and magnetite not only implies the original presence of biomass in the BIF sediments, but also that organic carbon likely served as an electron donor during bacterial Fe(III) reduction. This study is important because it suggests that phytoplankton may have played a key role in the transfer of phosphorus (and other trace elements) from the photic zone to the seafloor.

Fabrication of Mesoporous Hydroxycarbonate Apatite/Polyaniline Composite Coatings on Ti6Al4V Substrates

Abstract Hydroxycarbonate apatite/polyaniline composite coatings (HCp/PANI coatings) with mesoporous structure were fabricated by the electrophoretic deposition of calcium carbonate particles and polyaniline (PANI) nanofibers on Ti6Al4V substrates followed by treatment with phosphate buffer solutions (PBS) at 37 °C. After soaking in PBS, the perfect crack-free CaCO 3 /PANI coatings are converted to HCp/PANI coatings with platelet-like structure via a dissolution-precipitation reaction. The mesopores and the macropores (or apertures) are formed within and among the platelets, respectively. Simulated body fluid (SBF) immersion tests reveal that the porous structure can improve the in vitro apatite forming ability of coatings. In SBF, the bone-like apatite nanocrystals are firstly deposited around the mesopores, and then fill the macropores with the increase of immersion time. Moreover, the superior in vitro apatite forming ability of HCp/PANI coatings is related to the PANI nanofibers. The functional groups (H 2 PO 4 ) in PANI not only increase the local supersaturation, but also induce the heterogeneous nucleation and the growth of bone-like apatites.

Biomimetic Precipitation of Uniaxially Grown Calcium Phosphate Crystals from Full-Length Human Amelogenin Sols

Human dental enamel forms over a period of 2 – 4 years by substituting the enamel matrix, a protein gel mostly composed of a single protein, amelogenin with fibrous apatite nanocrystals. Self-assembly of a dense amelogenin matrix is presumed to direct the growth of apatite fibers and their organization into bundles that eventually comprise the mature enamel, the hardest tissue in the mammalian body. This work aims to establish the physicochemical and biochemical conditions for the synthesis of fibrous apatite crystals under the control of a recombinant full-length human amelogenin matrix in combination with a programmable titration system. The growth of apatite substrates was initiated from supersaturated calcium phosphate solutions in the presence of dispersed amelogenin assemblies. It was shown earlier and confirmed in this study that binding of amelogenin onto apatite surfaces presents the first step that leads to substrate-specific crystal growth. In this work, we report enhanced nucleation and growth under conditions at which amelogenin and apatite carry opposite charges and adsorption of the protein onto the apatite seeds is even more favored. Experiments at pH below the isoelectric point of amelogenin showed increased protein binding to apatite and at low Ca/P molar ratios resulted in a change in crystal morphology from plate-like to fibrous and rod-shaped. Concentrations of calcium and phosphate ions in the supernatant did not show drastic decreases throughout the titration period, indicating controlled precipitation from the protein suspension metastable with respect to calcium phosphate. It is argued that ameloblasts in the developing enamel may vary the density of the protein matrix at the nano scale by varying local pH, and thus control the interaction between the mineral and protein phases. The biomimetic experimental setting applied in this study has thus proven as convenient for gaining insight into the fundamental nature of the process of amelogenesis.

Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA

Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particle-forming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA.

Multi-phase biocomposite material in-situ fabricated by using hydroxyapatite and amorphous nanosilica

Abstract In the present study hydroxyapatite (HA) and HA/nanosilica (NS) slurries were separately prepared by adding precipitated HA to distilled water and to colloidal silica suspension, respectively and their rheological properties were compared to each other. Both slurries where then dried, powdered, compacted as disks and sintered at 1 100–1 300°C to evaluate and compare their physical, mechanical and some biological properties. The results showed that the HA/NS slurry was more stable and thixotropic than the pure HA slurry. X-ray diffraction analysis showed that the sintered HA/NS was a multi-phase material composed of apatite, tricalcium phosphate, cristobalite and amorphous glass, depending on sintering temperature. Both mechanical and biological properties of the composite were considerably better than those of sintered HA. Bioactivity of the composite was confirmed by precipitation of apatite nanocrystals onto the surfaces of the sample after soaking in simulated body fluid. The results of cell culture tests showed the same proliferation rate of rat calvaria osteoblasts on both sintered HA and composite with higher alkaline phosphatase activity for the latter. The results indicated that the composite with improved mechanical and biological properties may act more successfully than HA as a bone scaffold material.

Biomimetic Self-Assembling Copolymer−Hydroxyapatite Nanocomposites with the Nanocrystal Size Controlled by Citrate

Citrate binds strongly to the surface of calcium phosphate (apatite) nanocrystals in bone and is thought to prevent crystal thickening. In this work, citrate added as a regulatory element enabled molecular control of the size and stability of hydroxyapatite (HAp) nanocrystals in synthetic nanocomposites, fabricated with self-assembling block copolymer templates. The decrease of the HAp crystal size within the polymer matrix with increasing citrate concentration was documented by solid-state nuclear magnetic resonance (NMR) techniques and wide-angle X-ray diffraction (XRD), while the shapes of HAp nanocrystals were determined by transmission electron microscopy (TEM). Advanced NMR techniques were used to characterize the interfacial species and reveal enhanced interactions between mineral and organic matrix, concomitant with the size effects. The surface-to-volume ratios determined by NMR spectroscopy and long-range 31P{1H} dipolar dephasing show that 2, 10, and 40 mM citrate changes the thicknesses of the HAp crystals from 4 nm without citrate to 2.9, 2.8, and 2.3 nm, respectively. With citrate concentrations comparable to those in body fluids, HAp nanocrystals of sizes and morphologies similar to those in avian and bovine bones have been produced.

In situ remineralizaiton of partially demineralized human dentine mediated by a biomimetic non-collagen peptide

Biomineralization is a well-regulated process mediated by many extracellular matrix proteins. Some collagenous proteins act as frameworks for depositing minerals in a preferred orientation while non-collagenous proteins (BSP, BPP, OC, etc.) function as regulators of the mineralization process, either as nuclei or inhibitors of hydroxyapatite. Biomimetic remineralization strategies should reproduce the dimension and structural hierarchy of apatite within a collagen matrix. Here we synthesized a peptide with sequence (EEEEEEEEDSpESpSpEEDR) mimicking the function of BSP and DMP1 as calcium phosphate- and collagen-binding matrix protein analogues. More metastable calcium phosphate nanoprecursors were combined with the collagen matrix in acid-demineralized dentine pre-incubated with peptides during the process of remineralization in the phosphate-containing fluid. With the presence of our synthetic peptides, these nanoprecursors were transformed into polyelectrolyte-stabilized apatite nanocrystals that assembled at the “hole zones” (gap remineralization) and along the surface of the dentine collagen fibrils (surface remineralization). The transition from nanocrystals to larger apatite platelets was promoted by synthetic peptidesvia the formation of mesocrystal intermediates. It was concluded that deliberately designed peptide (EEEEEEEEDSpESpSpEEDR) improved in situ remineralization of acid-etched dentine.

How to control the size and morphology of apatite nanocrystals in bone

Biomineralized materials are highly organized composites with hierarchical structures, in which the basic building blocks (apatite crystals) are generally in the nanometer size range to ensure optimum physical and biological functions (1). The biological mechanisms of tissue development have attracted a great deal of recent attention in fields ranging from biology and chemistry to materials science and bioengineering (2, 3). It is well known that the organic component of bone acts as an important regulator of lattice orientation, particle size, and size distribution in biomineralization processes, but the molecular recognition details at inorganic crystal interfaces are poorly understood (4). Recently, most investigations have been focused on the effects of macromolecules such as carboxylate-rich proteins on hydroxyapatite nucleation and growth because complex interactions and changes of conformation are involved (5, 6). The role of small organic molecules such as citrate was deemphasized and was assumed to be simply that of adsorption on the crystal growth steps, resulting in inhibition of crystal growth (7, 8). However, there is still much to be learned about the mechanisms of crystal size control in biological systems. New insight about the role of citrate is emerging, as illustrated by a study in PNAS (9), in which strongly bound citrate molecules were identified as playing critical roles in interfering with crystal thickening and stabilizing apatite nanocrystal sizes in bone.

Amelogenin as a promoter of nucleation and crystal growth of apatite

Human dental enamel forms over a period of 2–4 years by substituting the enamel matrix, a protein gel mostly composed of a single protein, amelogenin with fibrous apatite nanocrystals. Self-assembly of amelogenin and the products of its selective proteolytic digestion are presumed to direct the growth of apatite fibers and their organization into bundles that eventually comprise the mature enamel, the hardest tissue in the mammalian body. This work aimed to establish the physicochemical and biochemical conditions for the growth of apatite crystals under the control of a recombinant amelogenin matrix (rH174) in combination with a programmable titration system. The growth of apatite substrates was initiated in the presence of self-assembling amelogenin particles. A series of constant titration rate experiments was performed that allowed for a gradual increase of the calcium and/or phosphate concentrations in the protein suspensions. We observed a significant amount of apatite crystals formed on the substrates following the titration of rH174 sols that comprised the initial supersaturation ratio equal to zero. The protein layers adsorbed onto the substrate apatite crystals were shown to act as promoters of nucleation and growth of calcium phosphates subsequently formed on the substrate surface. Nucleation lag time experiments have showed that rH174 tends to accelerate precipitation from metastable calcium phosphate solutions in proportion to its concentration. Despite their mainly hydrophobic nature, amelogenin nanospheres, the size and surface charge properties of which were analyzed using dynamic light scattering, acted as a nucleating agent for the crystallization of apatite. The biomimetic experimental setting applied in this study proves as convenient for gaining insight into the fundamental nature of the process of amelogenesis.

Strongly bound citrate stabilizes the apatite nanocrystals in bone.

Nanocrystals of apatitic calcium phosphate impart the organic-inorganic nanocomposite in bone with favorable mechanical properties. So far, the factors preventing crystal growth beyond the favorable thickness of ca. 3 nm have not been identified. Here we show that the apatite surfaces are studded with strongly bound citrate molecules, whose signals have been identified unambiguously by multinuclear magnetic resonance (NMR) analysis. NMR reveals that bound citrate accounts for 5.5 wt% of the organic matter in bone and covers apatite at a density of about 1 molecule per (2 nm)(2), with its three carboxylate groups at distances of 0.3 to 0.45 nm from the apatite surface. Bound citrate is highly conserved, being found in fish, avian, and mammalian bone, which indicates its critical role in interfering with crystal thickening and stabilizing the apatite nanocrystals in bone.

A chemical phosphorylation-inspired design for Type I collagen biomimetic remineralization

Objectives Type I collagen alone cannot initiate tissue mineralization. Sodium trimetaphosphate (STMP) is frequently employed as a chemical phosphorylating reagent in the food industry. This study examined the feasibility of using STMP as a functional analog of matrix phosphoproteins for biomimetic remineralization of resin-bonded dentin. Methods Equilibrium adsorption and desorption studies of STMP were performed using demineralized dentin powder (DDP). Interaction between STMP and DDP was examined using Fourier transform-infrared spectroscopy. Based on those results, a bio-inspired mineralization scheme was developed for chemical phosphorylation of acid-etched dentin with STMP, followed by infiltration of the STMP-treated collagen matrix with two etch-and-rinse adhesives. Resin–dentin interfaces were remineralized in a Portland cement-simulated body fluid system, with or without the use of polyacrylic acid (PAA) as a dual biomimetic analog. Remineralized resin–dentin interfaces were examined unstained using transmission electron microscopy. Results Analysis of saturation binding curves revealed the presence of irreversible phosphate group binding sites on the surface of the DDP. FT-IR provided additional evidence of chemical interaction between STMP and DDP, with increased in the peak intensities of the P O and P–O–C stretching modes. Those peaks returned to their original intensities after alkaline phosphatase treatment. Evidence of intrafibrillar apatite formation could be seen in incompletely resin-infiltrated, STMP-phosphorylated collagen matrices only when PAA was present in the SBF. Significance These results reinforce the importance of PAA for sequestration of amorphous calcium phosphate nanoprecursors in the biomimetic remineralization scheme. They also highlight the role of STMP as a templating analog of dentin matrix phosphoproteins for inducing intrafibrillar remineralization of apatite nanocrystals within the collagen matrix of incompletely resin-infiltrated dentin.

Medical Potentialities of Biomimetic Apatites through Adsorption, Ionic Substitution, and Mineral/Organic Associations: Three Illustrative Examples

AbstractBiomimetic calcium phosphate apatites are particularly adapted to bio‐medical applications due to their biocompatibility and high surface reactivity. In this contribution we report three selected examples dealing with mineral/organic interactions devoted to convey new functionalities to apatite materials, either in the form of dry bioceramics or of aqueous colloids. We first studied the adsorption of risedronate (bisphosphonate) molecules, which present potential therapeutic properties for the treatment of osteoporosis. We then addressed the preparation of luminescent Eu‐doped apatites for exploring apatite/collagen interfaces through the FRET technique, in view of preparing “advanced” biocomposites exhibiting close spatial interaction between apatite crystals and collagen fibers. Finally, we showed the possibility to obtain nanometer‐scaled apatite‐based colloids, with particle size tailorable in the range 30–100 nm by controlling the agglomeration state of apatite nanocrystals by way of surface functionalization with a phospholipid moiety. This paper is aimed at illustrating some of the numerous potentialities of calcium phosphate apatites in the bio‐medical field, allowing one to foresee perspectives lying well beyond bone‐related applications.

Bone-like nanocomposites based on self-assembled protein-based matrices with Ca2+ capturing capability

In the present work, bone-like nanocomposites have been successfully synthesized based on the mineralization of self-assembled protein-based microgels. Such mircogels were achieved by the in vitro reconstitution of collagen monomeric solutions in the presence of alginate in a microemulsion system. Microstructural observations revealed that the collagen-alginate composite beads possessed a nanofibrous three dimensional (3D) interconnected porous microstructure. The obtained microgels were pre-incubated in calcium-containing solution to capture Ca(2+) ions, and subsequently immersed in phosphate-containing solution to initiate the formation of hydroxyapatite (HA) by an alternative incubating procedure. It was observed that a substantial amount of bone-like apatite nanocrystals were orderly and homogeneously deposited throughout the porous fibrillar networks. Herein, the collagen-alginate composite microgels served as a mineralization template for the synthesis of HA-polymer nanocomposites, which could be ideal vehicles potentially for cell carriers, bone repair and proteins and drugs delivery in tissue regeneration.

Mineralisation of reconstituted collagen using polyvinylphosphonic acid/polyacrylic acid templating matrix protein analogues in the presence of calcium, phosphate and hydroxyl ions

The complex morphologies of mineralised collagen fibrils are regulated through interactions between the collagen matrix and non-collagenous extracellular proteins. In the present study, polyvinylphosphonic acid, a biomimetic analogue of matrix phosphoproteins, was synthesised and confirmed with FTIR and NMR. Biomimetic mineralisation of reconstituted collagen fibrils devoid of natural non-collagenous proteins was demonstrated with TEM using a Portland cement-containing resin composite and a phosphate-containing fluid in the presence of polyacrylic acid as sequestration, and polyvinylphosphonic acid as templating matrix protein analogues. In the presence of these dual biomimetic analogues in the mineralisation medium, intrafibrillar and extrafibrillar mineralisation via bottom-up nanoparticle assembly based on the non-classical crystallisation pathway could be identified. Conversely, only large mineral spheres with no preferred association with collagen fibrils were observed in the absence of biomimetic analogues in the medium. Mineral phases were evident within the collagen fibrils as early as 4 h after the initially-formed amorphous calcium phosphate nanoprecursors were transformed into apatite nanocrystals. Selected area electron diffraction patterns of highly mineralised collagen fibrils were nearly identical to those of natural bone, with apatite crystallites preferentially aligned along the collagen fibril axes.

Fabrication and Drug Delivery of Ultrathin Mesoporous Bioactive Glass Hollow Fibers

AbstractUltrathin mesoporous bioactive glass hollow fibers (MBGHFs) fabricated using an electrospinning technique and combined with a phase‐separation‐induced agent, poly(ethylene oxide) (PEO), are described. The rapid solvent evaporation during electrospinning and the PEO‐induced phase separation process demonstrated play vital roles in the formation of ultrathin bioactive glass fibers with hollow cores and mesoporous walls. Immersing the MBGHFs in simulated body fluid rapidly results in the development of a layer of enamel‐like apatite mesocrystals at the fiber surfaces and apatite nanocrystals inside the hollow cores. Drug loading and release experiments indicate that the drug loading capacity and drug release behavior of the MBGHFs strongly depends on the fiber length. MBGHFs with fiber length >50 µm can become excellent carriers for drug delivery. The shortening of the fiber length reduces drug loading amounts and accelerates drug release. The MBGHFs reported here with sophisticated structure, high bioactivity, and good drug delivery capability can be a promising scaffold for hard tissue repair and wound healing when organized into 3D macroporous membranes.

Preparation and Physicochemical Characteristics of Luminescent Apatite-Based Colloids

Luminescent colloidal nanosystems based on europium-doped biomimetic apatite were prepared and investigated. The colloids were synthesized by soft chemistry in the presence of a phospholipid moiety, 2-aminoethylphosphoric acid (AEP), with varying europium doping rates. Physicochemical features, including compositional, structural, morphological, and luminescence properties, were examined. Experimental evidence showed that suspensions prepared from an initial Eu/(Eu + Ca) molar ratio up to 2% consisted of single-phased biomimetic apatite nanocrystals covered with AEP molecules. The mean particle size was found to depend closely on the AEP content, enabling the production of apatite colloids with a controlled size down to ca. 30 nm. The colloids showed luminescence properties typical of europium-doped systems with narrow emission bands and long luminescence lifetimes of the order to the millisecond, and the data suggested the location of Eu3+ ions in a common crystallographic environment for all the colloids. These systems, stable over time and capable of being excited in close-to-visible or visible light domains, may raise interest in the future in the field of medical imaging.

Mesoporous structure and evolution mechanism of hydroxycarbonate apatite microspheres

Monodisperse mesoporous hydroxycarbonate apatite microspheres (MHAMs) were fabricated by soaking calcium carbonate microspheres (CCMs) in a cetyltrimethylammonium bromide (CTAB)/Na 2HPO 4/cyclohexane/ n-butanol emulsion system. After soaking CCMs in the emulsion system at 20 °C, hydroxycarbonate apatite nanocrystals nucleate heterogeneously on the surfaces of CCMs via a dissolution–precipitation reaction. The as-formed nanocrystals aggregate to form mesopores with the pore size of ∼ 3.9 nm and ∼ 9.0 nm. The smaller mesopores are derived from the direct aggregation of the nanocrystals, which do not change obviously with the reaction time. In contrast, the larger mesopores are formed by using CTAB micelles as templates, and their pore size decreases from ∼ 9.0 nm to ∼ 7.4 nm with increasing the reaction time from 6 h to 1 day. After reaction for 3 or 5 days, the larger mesopores disappear because of unstability of the CTAB micelles. If reaction temperature is kept at 50 °C, the conversion rate of CCMs to MHAMs is greater than that at 20 °C, and the corresponding mesoporous structure is unimodal with the pore size of ∼ 3.9 nm. Simulated body fluid immersion tests reveal that MHAMs have a great in vitro bioactivity, which is attributed to the mesoporous structure and B-type CO 3 2− substitution of MHAMs.

Modification of bone-like apatite nanoparticle size and growth kinetics by alizarin red S

The formation of nanocrystals in biomineralization such as in bone occurs under the influence of organic molecules. Prompted by this fact, the effect of alizarin red S, a dye used in in vivo bone labeling methods, on bone-like carbonated apatite nanocrystal formation was investigated as a function of alizarin red S additive concentration. The obtained nanoparticles were investigated by powder X-ray diffraction (XRD), FTIR as well thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) while the kinetics of nanoparticle formation was investigated by in situ pH and synchrotron XRD measurements. Increasing alizarin red S concentration lead to amorphous particles over a threshold concentration and to smaller crystallites in a dose-dependent fashion. Alizarin red S induced a macroscopic lattice strain that scaled linearly with the alizarin red S concentration; this effect is reminiscent of that seen in biogenic calcium carbonates. TGA showed that the amorphous particles contained significantly more water than the crystalline samples and the DSC data showed that crystallization occurs after loss of most of the included organic material. The in situ studies showed that the formation of apatite goes via the very rapid formation of an amorphous precursor that after a certain nucleation time crystallizes into apatite. This nucleation time increased exponentially with alizarin red S concentration showing that this additive strongly stabilizes the amorphous precursor phase.