Amorphous Calcium Phosphate Particles Research Articles

Inhibiting Pathological Calcium Phosphate Mineralization: Implications for Disease Progression.

Pathological calcifications, especially calcium phosphate microcalcifications (MCs), appear in most early breast cancer lesions, and their formation correlates with more aggressive tumors and a poorer prognosis. Hydroxyapatite (HA) is a key MC component that crystallizes in the tumor microenvironment. It is often associated with malignant breast cancer lesions and can trigger tumorigenesis in vitro. Here, we investigate the impact of additives on HA crystallization and inhibition, and how precancerous breast cells respond to minerals that are deposited in the presence of these additives. We show that nonstoichiometric HA spontaneously crystallizes in a solution simulating the tumor microenvironmental fluids and exhibits lump-like morphology similar to breast cancer MCs. In this system, the effectiveness of poly(aspartic acid) and poly(acrylic acid) (PAA) to inhibit HA is examined as a potential route to improve cancer prognosis. In the presence of additives, the formation of HA lumps is associated with the promotion or only minimal inhibition of mineralization, whereas the formation of amorphous calcium phosphate (ACP) lumps is followed by inhibition of mineralization. PAA emerges as a robust HA inhibitor by forming spherical ACP particles. When precancerous breast cells are exposed to various HA and ACP minerals, the most influential factors on cell proliferation are the mineral phase and whether the mineral is in the form of discrete particles or particle aggregates. The tumorigenic effects on cells, ranging from cytotoxicity and suppression of proliferation to triggering of proliferation, can be summarized as HA particles < HA aggregates < ACP particles < ACP aggregates. The cellular response to minerals can be attributed to a combination of factors, including mineral phase, crystallinity, morphology, surface texture, aggregation state, and surface potential. These findings have implications for understanding mineral-cell interactions within the tumor microenvironment and suggest that, in some cases, the byproducts of HA inhibition can contribute to disease progression more than HA itself.

3D printing of drug-eluting bioactive multifunctional coatings for orthopedic applications.

Three-dimensional (3D) printing is implemented for surface modification of titanium alloy substrates with multilayered biofunctional polymeric coatings. Poly(lactic-co-glycolic) acid (PLGA) and polycaprolactone (PCL) polymers were embedded with amorphous calcium phosphate (ACP) and vancomycin (VA) therapeutic agents to promote osseointegration and antibacterial activity, respectively. PCL coatings revealed a uniform deposition pattern of the ACP-laden formulation and enhanced cell adhesion on the titanium alloy substrates as compared to the PLGA coatings. Scanning electron microscopy and Fourier-transform infrared spectroscopy confirmed a nanocomposite structure of ACP particles showing strong binding with the polymers. Cell viability data showed comparable MC3T3 osteoblast proliferation on polymeric coatings as equivalent to positive controls. In vitro live/dead assessment indicated higher cell attachments for 10 layers (burst release of ACP) as compared to 20 layers (steady release) for PCL coatings. The PCL coatings loaded with the antibacterial drug VA displayed a tunable release kinetics profile based on the multilayered design and drug content of the coatings. Moreover, the concentration of active VA released from the coatings was above the minimum inhibitory concentration and minimum bactericidal concentration, demonstrating its effectiveness against Staphylococcus aureus bacterial strain. This research provides a basis for developing antibacterial biocompatible coatings to promote osseointegration of orthopedic implants.

Eggshell-derived amorphous calcium phosphate: Synthesis, characterization and bio-functions as bone graft materials in novel 3D osteoblastic spheroids model

A multitude of autogenous/allogeneic and semi-synthetic bone graft materials have been developed to reconstruct the defective bone tissue but with high bio-cost and potential environmental pollution. With high calcium content and several trace elements, chicken eggshells are no longer considered as wastes but attractive sources of high-value-added biomaterials. This study used chicken eggshells and synthetic hydroxyapatite (HAp) to synthesize amorphous calcium phosphate (ACP) bone graft materials, namely Control and Eggshell. The physiochemical characteristics, biosafety, and immunocompatibility of synthetic ACP particles were inspected. Their osteogenic activity was further investigated in a novel osteoblastic spheroids model. Eggshell ACP particles exhibited ideal cytocompatibility compared to the control ACP and were more resistant to re-crystallization. In osteoblastic spheroids, Eggshell ACP mediated typical osteogenic mRNA profiles of MC-3T3-E1 cells, accompanied by the increased formation of mineralized nodules and boosted synthesis of ECM proteins represented by OPN and collagen I. This study establishes a promising technique to synthesize stable, safe, and osteoinductive ACP graft particles from eggshell waste. Furthermore, the osteoblastic spheroids constructed in the present study provide a more practical model for biomaterial research, which reflect the three-dimensional interaction between host bone tissue and graft materials more realistically.

Biominerals Added Bioresorbable Calcium Phosphate Loaded Biopolymer Composites

Nanocrystalline calcium phosphate (CP) bioceramic coatings and their combination with biopolymers are innovative types of resorbable coatings for load-bearing implants that can promote the integration of metallic implants into human bodies. The nanocrystalline, amorphous CP particles are an advantageous form of the various calcium phosphate phases since they have a faster dissolution rate than that of crystalline hydroxyapatite. Owing to the biomineral additions (Mg, Zn, Sr) in optimized concentrations, the base CP particles became more similar to the mineral phase in human bones (dCP). The effect of biomineral addition into the CaP phases was thoroughly studied. The results showed that the shape, morphology, and amorphous characteristic slightly changed in the case of biomineral addition in low concentrations. The optimized dCP particles were then incorporated into a chosen polycaprolactone (PCL) biopolymer matrix. Very thin, non-continuous, rough layers were formed on the surface of implant substrates via the spin coating method. The SEM elemental mapping proved the perfect incorporation and distribution of dCP particles into the polymer matrix. The bioresorption rate of thin films was followed by corrosion measurements over a long period of time. The corrosion results indicated a faster dissolution rate for the dCP-PCL composite compared to the dCP and CP powder layers.

How to build a bone? - Hydroxyapatite or Posner’s clusters as bone minerals

For decades, hydroxyapatite has been considered the main mineral phase of mammalian tooth enamel and bone that crystallizes from a precursor phase, amorphous calcium phosphate. Amorphous calcium phosphate, in turn, is proposed to be made from small clusters of ions known as Posner’s clusters, with a size of 0.95 ​nm and chemical formula of Ca9(PO4)6. In isolation, Posner’s clusters are extremely unstable and spontaneously aggregate and nucleate to the thermodynamically stable phase of hydroxyapatite within a few seconds. In this critical review, we will discuss the progress made in identifying the role of Posner’s clusters in the bone mineralization process, visualization of the clusters and associated challenges, and potential pathways to utilize them in bone tissue engineering strategies. We hypothesize, based on collective data in the literature and our recent experimental data, that Posner’s cluster agglomerates and amorphous calcium phosphate particles are in fact the main mineral component of bone, and hydroxyapatite crystals are, in part, artefacts of sample processing.

Effectiveness of antiscalants in preventing calcium phosphate scaling in reverse osmosis applications

Antiscalants are well known to prevent the precipitation of carbonate and sulphate scales of calcium in reverse osmosis (RO) applications, but according to literature their inhibitory ability against calcium phosphate is not clear. The objective of this study was to investigate if antiscalants, without acid addition, can prevent calcium phosphate scaling in RO systems. Eight calcium phosphate antiscalants from different manufacturers spanning a range of concentrations were tested in batch (in glass reactors) experiments to inhibit the formation of calcium phosphate in synthetic concentrate corresponding to 85% recovery (Ca2+ = 765 mg/L, PO43− = 13–15 mg/L and pH = 7.6) of a groundwater RO in the Netherlands. Additionally, once-through lab-scale RO tests were conducted where an RO element was fed with synthetic concentrate and the performance of antiscalants was evaluated from the rate of flux-decline in the RO element. Without antiscalant addition, a substantial flux-decline was observed due to the deposition of amorphous calcium phosphate (ACP) on the RO membrane. The tested antiscalants were unable to inhibit the formation of ACP and were incapable of preventing the deposition of the formed ACP particles, since with each antiscalant, the flux of the RO element decreased at least 15% in a 3-h period. Briefly, the available antiscalants, tested in this study, did not provide acceptable inhibition of calcium phosphate scaling in RO applications.

Phase transition of amorphous calcium phosphate to calcium hydrogen phosphate dihydrate in simulated body fluid

We study the initial process of the rapid Ca-P crystallization in SBF by ex situ and in situ observation. To reduce the crystallization rate, calcite are present in the system. Ex situ measurements revealed that amorphous calcium phosphate (ACP) immediately precipitated on SiO2 substrate. The inside of transformed to the calcium hydrogen phosphate. In situ observation indicated that the formation of the DCPD started at day 7 and the DCPD region gradually increased inside ACP particles. Our results showed a solid-solid phase transition from ACP to DCPD. We propose the new pathway of Ca-P crystallization.

Development of Apatite Nuclei Precipitated Carbon Nanotube-Polyether Ether Ketone Composite with Biological and Electrical Properties

We aimed to impart apatite-forming ability to carbon nanotube (CNT)-polyether ether ketone (PEEK) composite (CNT-PEEK). Since CNT possesses electrical conductivity, CNT-PEEK can be expected to useful not only for implant materials but also biosensing devices. First of all, in this study, CNT-PEEK was treated with sulfuric acid to form fine pores on its surface. Then, the hydrophilicity of the substrate was improved by oxygen plasma treatment. After that, the substrate was promptly immersed in simulated body fluid (SBF) which was adjusted at pH 8.40, 25.0 °C (alkaline SBF) and held in an incubator set at 70.0 °C for 1 day to deposit fine particles of amorphous calcium phosphate, which we refer to as ‘apatite nuclei’. When thus-treated CNT-PEEK was immersed in SBF, its surface was spontaneously covered with hydroxyapatite within 1 day by apatite nuclei deposited in the fine pores and high apatite-forming ability was successfully demonstrated. The CNT-PEEK also showed conductivity even after the above treatment and showed smaller impedance than that of the untreated CNT-PEEK substrate.

Synthesis, microstructure and mechanical properties of tricalcium phosphate–hydroxyapatite (TCP/HA) composite ceramic

Tricalcium phosphate-hydroxyapatite (TCP/HA) is an important composite ceramic ubiquitously applied in biomedicine research. In this work, a novel design and synthesizing method for preparation of TCP/HA composites is proposed consisting in suspension mixing amorphous calcium phosphate (ACP) and hydroxyapatite (HA) ceramic powders with different weight ratios, and their compaction under a uniaxial pressure of 200 MPa and sintering at 1150 °C for 2 h. The obtained sintered bodies exhibited optimum hardness and fracture toughness of 5 GPa and 1.27 MPa m1/2, and values significantly improved as compared with those obtained with the traditional mechanical mixing method. The mechanical enhancement mechanism was mainly attributed to the good ACP and HA phase dispersion and ACP crystallization. SEM and HRTEM observations showed the suspension mixing method to allow amorphous spherical ACP and crystalline rod-like HA particles with size around 50 nm to uniformly disperse at the nano scale in the composite powder, which contributes to the excellent sinter ability. Furthermore, XRD results indicate that after sintering, nearly 100% of amorphous spherical ACP transformed to the crystalline β-TCP, which results in the nanoparticle shrinkage, porosity reduction and composite densification.

Multifunctional Mineral Hydrogels: Potential in Artificially Intelligent Skins and Drug Delivery.

Hydrogels have received considerable attention due to their potential applications in the fields of drug delivery, tissue engineering, and stimuli-responsive devices. Nonetheless, it is still a great difficulty in designing hydrogels with multifunctional characteristics including excellent antibacterial activity and appropriate mechanical and remarkable sensing properties. In the present study, a novel type of organic–inorganic adhesive is demonstrated, which comprises inorganic matter of amorphous calcium phosphate particles and organic substances of poly(acrylic acid) and chitosan. The hydrogel possesses excellent biocompatible and antibacterial activity, unique viscoelastic properties, high quantity of drug load, and remarkably sensitive pressure sensing, which have potential use as antibacterial biomaterials, artificially intelligent skins, and drug delivery carriers.

In vivo and in vitro bioactivity of a “precursor of apatite” treatment on polyetheretherketone

We recently developed a surface treatment, “precursor of apatite” (PrA), for polyetheretherketone (PrA-PEEK) via a simple, low-temperature process aiming to achieve stronger and faster adhesion to bone. The treatment involves three steps: H2SO4 immersion, exposure to O2 plasma discharge, and alkaline simulated body fluid (alkaline SBF) treatment. This method produces homogeneous fine particles of amorphous calcium phosphate on the PEEK, and we confirmed that PrA-PEEK had excellent apatite formation ability in an SBF immersion test. In the present study using PEEK implants in rabbit tibia, mechanical tests, and histological and radiological analyses revealed that PrA provided the PEEK substrate with excellent bone-bonding properties and osteo-conductivity at early stages (4 and 8 weeks), extending to 16 weeks. In vitro study using MC3T3-E1 cells revealed via XTT assay that PrA on the PEEK substrate resulted in no cytotoxicity, though PrA treatment seemed to suppress gene expression of integrin β-1 and Alp after 7-day incubation as shown by real-time PCR. On the whole, PrA treatment succeeded in giving in vivo bone-bonding properties to the PEEK substrate, and the treatment is a safe and promising method that can be applied in clinical settings. There was an inconsistency between in vivo and in vitro bioactivity, and this discrepancy indicated that apatite formation does not always need activation of osteoblasts at very early stage and that optimal conditions at cell and organism level may be different. Statement of SignificancePolyetheretherketone (PEEK) is an attractive engineering polymer used for spine and dental surgery. To further improve clinical outcome of PEEK-based materials, we developed “Precursor of apatite” (PrA) treatment on the PEEK surface to confer bone-bonding properties. The advantages of this treatment are that it does not require high-temperature processing or special chemicals, and it is inexpensive. The present study clarified excellent in vivo bone-bonding property of PrA treatment. In addition, the results revealed important insights indicating that optimal conditions, especially wettability and crystallinity in calcium phosphate, differ at cell and organism levels. Moreover, our results indicated that prediction of in vivo bioactivity should be done in combination with multiple in vitro tests.

Amorphous Calcium Phosphate Formation and Aggregation Process Revealed by Light Scattering Techniques

Amorphous calcium phosphate (ACP) attracts attention as a precursor of crystalline calcium phosphates (CaPs) formation in vitro and in vivo as well as due to its excellent biological properties. Its formation can be considered to be an aggregation process. Although aggregation of ACP is of interest for both gaining a fundamental understanding of biominerals formation and in the synthesis of novel materials, it has still not been investigated in detail. In this work, the ACP aggregation was followed by two widely applied techniques suitable for following nanoparticles aggregation in general: dynamic light scattering (DLS) and laser diffraction (LD). In addition, the ACP formation was followed by potentiometric measurements and formed precipitates were characterized by Fourier transform infrared spectroscopy (FTIR), powder X-ray diffraction (PXRD), transmission electron microscopy (TEM), and atomic force microscopy (AFM). The results showed that aggregation of ACP particles is a process which from the earliest stages simultaneously takes place at wide length scales, from nanometers to micrometers, leading to a highly polydisperse precipitation system, with polydispersity and vol. % of larger aggregates increasing with concentration. Obtained results provide insight into developing a way of regulating ACP and consequently CaP formation by controlling aggregation on the scale of interest.

A Kinetic Model for Hydroxyapatite Precipitation in Mineralizing Solutions

Numerous studies have shown that there is an amorphous calcium phosphate (ACP) phase preceding the precipitation of crystalline hydroxyapatite (HA) in calcium phosphate solutions. It has also been shown that the addition of magnesium to the solutions has a stabilizing effect by inhibiting the transformation of ACP to HA. The stabilizing effect of Mg2+ is attributed to the stronger bonds between water molecules and the magnesium ions adsorbed on the surface of the ACP particles, making it harder for them to dehydrate. However, the kinetics of the reactions between calcium and phosphate ions to form ACP and then HA crystals, and the effects of varying concentrations of Mg on the kinetics have not been studied theoretically in detail. In this study, we develop and validate a kinetic model for analyzing such reactions. The pertinent rate constants are derived by calibrating the model against temporal changes in Ca2+ concentration reported by others. The predicted onset and growth of HA crystallization for sol...

Formation Process of Hydroxyapatite Granules in Agarose Hydrogel by Electrophoresis

Biomimetic mineralization is useful in obtaining organic–inorganic composite and inorganic materials with controlled sizes and morphologies. The formation of hydroxyapatite (HA) granules with porous structures can be achieved by using electrophoresis processes inside an agarose gel in which the compulsory migration of calcium and phosphate ions are driven by an electric field, causing precipitation of the granules. In the present study, we examined the formation process of HA granules through electrophoresis and the subsequent aging process. Spherical particles of amorphous calcium phosphate (ACP) precipitated, increasing their size with increased running time during electrophoresis; then the ACP granules gradually transformed to the HA granules during the aging process in ultrapure water. When the ACP granules were large, one HA granule was formed from one ACP granule. One HA granule was formed from multiple ACP granules for small-sized ACP granules. The size of ACP granules depends on the gel concentrat...

Fabrication of Bioactive Fiber-reinforced PEEK and MXD6 by Incorporation of Precursor of Apatite.

We aimed to develop an effective process to provide bioactivity to carbon fiber-reinforced polyetheretherketone (PEEK), glass fiber-reinforced PEEK and glass fiber-reinforced poly(m-xylyleneadipamide)-6 (MXD6), possessing similar elastic modulus to cortical bone in this study. First, we formed fine pores on the surface of each substrate by a short-time sulfuric acid treatment. Second, in order to provide hydrophilic property, we treated the surfaces of each substrate with oxygen plasma. Finally, we deposited fine particles of amorphous calcium phosphate (PrAp) in the pores by soaking each substrate in SBF adjusted at pH 8.40, 25.0°C, and subsequently kept at 70.0°C for 24 h. By this treatment, we obtained the bioactive fiber-reinforced polymers. By soaking thus-obtained each material in SBF, apatite formation was induced on the whole surface of each substrate within 1 day by PrAp deposited in the pores and high apatite-forming ability was performed on each material. The adhesive strength between the apatite layer showed high value by mechanical anchoring effect generated by the apatite formed in the pores. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2254-2265, 2018.

Self-Etch Adhesive as a Carrier for ACP Nanoprecursors to Deliver Biomimetic Remineralization.

Lab biomineralization should be carried out in an actual clinical practice. This study evaluated self-etch adhesive as a carrier for amorphous calcium phosphate (ACP) nanoprecursors to continuously deliver biomimetic remineralization of self-assembly type I collagen and demineralized dentin. Si-containing ACP particles (Si-ACP) stabilized with polyaspartic acid (PAsp) were synthesized and characterized by transmission electron microscopy (TEM), scanning electron microscopy-energy-dispersive X-ray spectroscopy, Fourier transform infrared analysis, X-ray powder diffractometry, and X-ray phototelectron spectroscopy. The biomimetic remineralization of single-layer reconstituted type I collagen fibrils and demineralized dentin was analyzed by using two one-bottle self-etch dentin adhesives (Clearfil S3 Bond (S3), Kurraray-Noritake; Adper Easy One (AEO), 3 M ESPE) as a carrier loaded (or not, in the case of the control) with 25 wt % of Si-ACP particles. In vitro cytotoxicity assessed by the Cell Counting Kit-8 indicated that the Si-ACP particles had no adverse effect on cell viability. The capacity for Ca and P ions release from cured Si-ACP-containing adhesives (S3, AEO) was evaluated by inductively coupled plasma-atomic emission spectrometry, revealing the successively increasing release of Ca and P ions for 28 days. The intra- and extrafibrillar remineralization of type I collagen and demineralized dentin was confirmed by TEM and selected-area electron diffraction when the adhesives were used as a carrier loaded with Si-ACP particles. Therefore, we propose self-etch adhesive as a novel carrier for ACP nanoprecursors to continuously deliver biomimetic remineralization.

Rechargeable calcium phosphate orthodontic cement with sustained ion release and re-release.

White spot lesions (WSL) due to enamel demineralization are major complications for orthodontic treatments. Calcium phosphate (CaP) dental resins with Ca and P ion releases are promising for remineralization. However, previous Ca and P releases lasted for only weeks. Experimental orthodontic cements were developed using pyromellitic glycerol dimethacrylate (PMGDM) and ethoxylated bisphenol A dimethacrylate (EBPADMA) at mass ratio of 1:1 (PE); and PE plus 10% of 2-hydroxyethyl methacrylate (HEMA) and 5% of bisphenol A glycidyl dimethacrylate (BisGMA) (PEHB). Particles of amorphous calcium phosphate (ACP) were incorporated into PE and PEHB at 40% filler level. Specimens were tested for bracket-enamel shear bond strength, water sorption, CaP release, and ion recharge and re-release. PEHB+40ACP had higher bracket-enamel bond strength and ion release and rechargeability than PE+40ACP. ACP incorporation into the novel orthodontic cement did not adversely affect the bracket-enamel bond strength. Ion release and re-release from the novel ACP orthodontic cement indicated favorable release and re-release patterns. The recharged orthodontic cement could release CaP ions continuously for four weeks without further recharge. Novel rechargeable orthodontic cement containing ACP was developed with a high bracket-enamel bond strength and the ability to be repeatedly recharged to maintain long-term high levels of CaP ion releases.

An Evaluation of Bioactive Glass/Calcium Phosphate Cement Composite, Synthesized via Sol-Gel Method

In this study four types of calcium phosphate cement/bioactive glass composites has been synthesized via mixing and sol-gel method and the effect of hydroxyapatite/tricalcium phosphate ratio to its mechanical properties and setting time was investigated. The prepared samples were characterized using X-ray diffraction (XRD), foureir transform infra-red spectroscopy (FTIR), scanning electron microscopy (SEM), mechanical testing and setting time measurement. XRD analysis showed amorphous structure of the prepared bioactive glass. But the patterns of the prepared composite had sharp peaks because of their crystalline structure. FTIR analysis indicated that the composites had carbonated calcium phosphate structure. SEM micrographs illustrated amorphous calcium phosphate particles with irregular shapes. With increasing the HA/TCP ratio, Young's modulus and compressive strength of the composites increased from 179 to 453 MPa and from 20 to 38 MPa respectively. The setting time of the samples decreased with increasing the HA/TCP ratio from 22 to 18 minutes.

MMP20 Proteolysis of Native Amelogenin Regulates Mineralization In Vitro

Recent studies have shown that native phosphorylated full-length porcine amelogenin (P173) and its predominant cleavage product (P148) can inhibit spontaneous calcium phosphate formation in vitro by stabilizing an amorphous calcium phosphate (ACP) precursor phase. Since full-length amelogenin undergoes proteolysis by matrix metalloproteinase 20 (MMP20, enamelysin) soon after secretion, the present study was conducted to assess the effect of amelogenin proteolysis on calcium phosphate formation. Calcium and phosphate were sequentially added to protein solutions without and with added MMP20 (ratio = 200:1) under physiological-like conditions of ionic strength (163 mM) in 50 mM Tris-HCl (pH 7.4) at 37 °C. Protein degradation with time was assessed by gel-electrophoresis, and mineral products formed were characterized by transmission electron microscopy (TEM). MMP20 was found to cleave P173 to primarily generate P148, along with P162, P46-148, and P63/64-148. In sharp contrast, MMP20 did not cleave P148. In addition, the formation of well-aligned bundles of enamel-like hydroxyapatite (HA) crystals was promoted in the presence of P173 with added MMP20, while only ACP particles were seen in the absence of MMP20. Although P148 was found to have a somewhat lower capacity to stabilize ACP and prevent HA formation compared with P173 in the absence of MMP20, essentially no HA formation was observed in the presence of somewhat higher concentrations of P148 regardless of MMP20 addition, due to the lack of observed protein proteolysis. Present findings suggest that ACP transformation to ordered arrays of enamel crystals may be regulated in part by the proteolysis of full-length native amelogenin, while the predominant amelogenin degradation product in developing enamel (e.g., P148) primarily serves to prevent uncontrolled mineral formation during the secretory stage of amelogenesis.

Bone mineralization pathways during the rapid growth of embryonic chicken long bones

The uptake and transport of ions from the environment to the site of bone formation is only partially understood and, for the most part, based on disparate observations in different animals. Here we study different aspects of the biomineralization pathways in one system, the rapidly forming long bones of the chicken embryo. We mainly used cryo-fixation and cryo-electron imaging to preserve the often unstable mineral phases in the tissues. We show the presence of surprisingly large amounts of mineral particles located inside membrane-delineated vesicles in the bone forming tissue between the blood vessels and the forming bone surface. Some of these particles are also located inside mitochondrial networks. The surfaces of the forming bones in the extracellular space contain abundant aggregates of amorphous calcium phosphate particles, but these are not enveloped by vesicle membranes. In the bone resorbing region, osteoclasts also contain many particles in both mitochondrial networks and within vesicles. Some of these particles are present also between cells. These observations, together with the previously reported observation that CaP mineral particles inside membranes are present in blood vessels, leads us to the conclusion that important components of the bone mineralization pathways in rapidly forming chicken bone are dense phase mineral particles bound within membranes. It remains to be determined whether these mineral particles are transported to the site of bone formation in the solid state, fluid state or dissolve and re-precipitate.