Calcium Phosphate Crystal Growth Research Articles

Crystal growth of calcium phosphates from aqueous solutions in the presence of strontium

In the present work, the effect of the presence of strontium ions in supersaturated calcium phosphate solutions on the rates of crystal growth of octacalcium phosphate (Ca8H2(PO4)6·5H2O, OCP) and of hydroxyapatite (Ca5(PO4)3OH, HAP) was investigated at 37°C and at constant solution supersaturation. The presence of strontium reduced crystal growth rates of both calcium phosphate phases. The reduction of the OCP crystal growth was ca. 40% for strontium concentration of 1mM while for HAP was significantly higher, reaching 70% for the same concentration of strontium in the solution. Higher retardation of the rates of HAP crystal growth was achieved at higher supersaturations in the presence of 1mM strontium concentration. Moreover, strontium ions were shown to stabilize or delay the transformation of the unstable OCP that tends to hydrolyze to the thermodynamically more stable HAP. Strontium ions were incorporated into both OCP and HAP growing on the respective seed crystals.

Experimental Investigation on Mechanism of Hydrophilic Acrylic Intraocular Lens Calcification

To construct a model simulating intraocular lens (IOL) opacification attributable to the formation of calcium phosphate deposits and to investigate the kinetics of deposit formation. Prospective laboratory investigation. Department of Ophthalmology, Medical School and Department of Chemical Engineering, Laboratory of Inorganic and Analytical Chemistry, University of Patras, Greece. Three hydrophilic acrylic IOLs (26% water content) were placed inside a 10-mL double-walled thermostated reactor simulating the anterior chamber. Simulated aqueous humor was injected continuously into the reactor using a pump with variable speed. The observation of IOLs was carried out in situ daily by optical microscopy. Scanning electron microscopy and energy-dispersive radiographic spectroscopy were used for the identification of the morphologic features and the composition of the deposits. The lenses were removed and inspected 5, 9, and 12 months after the initiation of the experiment. Investigation showed deposits of calcium phosphate crystallites in the interior of opacified IOLs. However, these deposits were not observed on the surface of the IOLs. In agreement with earlier reports by our group and in the literature, IOL opacification is the result of calcification. It is suggested that the surface hydroxyl groups of the polyacrylic polymeric components of the IOLs are capable of inducing surface nucleation and crystal growth of calcium phosphates. However, most important is the finding that the calcification of IOLs is initiated from their interior through the development of sufficiently high local supersaturation, realized through the diffusion of calcium and phosphate ions.

Nano/micro‐sized calcium phosphate coating on carbon/carbon composites by ultrasonic assisted electrochemical deposition

AbstractNano/micro‐sized calcium phosphate (CaP) coating was prepared on carbon/carbon (C/C) composites by ultrasonic assisted electrochemical deposition. The coating obtained at different deposition voltages contained a mixture of hydroxyapatite (HA) and brushite (DCPD). The homogenous coating prepared at 2.4 V consisted of nano‐sized and needle‐like HA embedded in micro‐sized and plate‐like DCPD. An interlocking structure was formed along the depth direction of the coating. The internal stress may be released effectively through the interlocking structure of the coating. And the plate‐like crystals of the coating were inset in the grooves on the surface of C/C composites. This led to a better adhesive strength of the coating. Meanwhile, the formed interlocking structure could help enhance cohesive strength of the coating. It was found that the growth of CaP crystals in the coating under the voltage of 2.4 V consisted of the plate‐like crystals deposited initially. Then the plate‐needle‐like crystals of submicron size formed among the plate‐like crystals and developed needle‐like ones. The CaP‐coated C/C composites might improve the biological properties of coating for its unique morphology, structures and strong adhesion to the C/C substrate. Copyright © 2011 John Wiley & Sons, Ltd.

Biomimetic Calcium Phosphate Crystal Mineralization on Electrospun Cellulose-Based Scaffolds

Novel cellulose based-scaffolds were studied for their ability to nucleate bioactive calcium phosphate crystals for future bone healing applications. Cellulose-based scaffolds were produced by electrospinning cellulose acetate (CA) dissolved in a mixture of acetone/dimethylacetamide (DMAc). The resulting nonwoven CA mats containing fibrils with diameters in the range of 200 nm to 1.5 μm were saponified by NaOH/ethanol for varying times to produce regenerated cellulose scaffolds. Biomimetic crystal growth nucleated from the fiber surface was studied as a function of surface chemistry. Regenerated cellulose scaffolds of varying treatments were soaked in simulated body fluid (SBF) solution. Scaffolds that were treated with CaCl(2), a mixture of carboxymethyl cellulose (CMC) and CaCl(2), and NaOH and CaCl(2), were analyzed using X-ray photoelectron spectroscopy, X-ray powder diffraction, and scanning electron microscopy to understand the growth of bioactive calcium phosphate (Ca-P) crystals as a function of surface treatment. The crystal structure of the nucleated Ca-P crystals had a diffraction pattern similar to that of hydroxyapatite, the mineralized component of bone. The study shows that the scaffold surface chemistry can be manipulated, providing numerous routes to engineer cellulosic substrates for the requirements of scaffolding.

PEGylated Calcium Phosphate Nanocomposites as Smart Environment‐Sensitive Carriers for siRNA Delivery

A novel inorganic–organic hybrid nanocomposite is formed in situ using a simple and straightforward method. Conjugate of short interfering RNA (siRNA) duplex with poly(ethylene glycol) via a disulfide linkage (PEG–SS–siRNA) is demonstrated to regulate the crystal growth of calcium phosphate (CaP), yielding a monodispersed nanocomposite. The resultant nanocomposite can be utilized as nanocarriers for siRNA delivery. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Study on the Effects of Type I Collagen Combined with Noncollagenous Proteins on Hydroxyapatite Formation in vitro using SPM and GIXD

AbstractThe effects of the components of extracellular matrix on the bone formation and the kinetics of crystal growth of calcium phosphate have remained unknown. In this paper, we reported a method to investigate the role of Type I collagen and the interactions with other ECM proteins such as fibronectin and elastin during biomimic mineralization process in vitro. The early stage of mineralization was characterized by scanning probe microscopy (SPM) and shear modulation force microscopy (SMFM). The late stage of mineralization was investigated by synchrotron grazing incident x-ray diffraction (GIXD). The results demonstrate the cooperative interaction between type I collagen and noncollagenous proteins such as fibronectin or elastin could be essential for the biomineralization.

In VitroEffects of Cementum Protein 1 (CEMP1) on Calcium Phosphate Crystal Formation and its Role During the Mineralization Process

ABSTRACTCementum contains specific molecules that could serve to identify, isolate and characterize the cementoblast lineage and to determine the cellular and molecular mechanisms that regulate the cementogenesis process, since it plays a key role during the periodontal regeneration process. One of these molecules is the human cementum protein 1 (CEMP1); which has a molecular weight of 25,9 kDa.In vitroexperiments have shown that CEMP1 promotes cellular adhesion and differentiation. In addition, this protein has been implied in regulating the degree of deposition, composition and morphology of hydroxyapatite crystals formed by putative cementoblastin vitro. Therefore, it is possible that CEMP1 promotes the formation, growth and regulates the morphology of hydroxyapatite crystalsin vitro.We have produced a human recombinant CEMP1 (hrCEMP1) in a prokaryotic system. ThehrCEMP1 purification was realized using the column NiTA HisPrep FF/16. Assays of calcium phosphate crystal growth were realized by means of capillary counterdiffusion system. Our results demonstrated thathrCEMP1 promotes octacalcium phosphate crystal nucleation and possesses high affinity for hydroxyapatite. We infer thathrCEMP1 plays a key role during the regeneration of mineralized tissues.

Bioactive Porous Titanium: An Alternative to Surgical Implants

Porous titanium implants have been used to improve implant-bone attachment by the ingrowth of bone tissue within the porous structure. Despite the efficient bone adhesion of porous titanium implants, chemical bonds are required at bone-implant interface. These implants can become bioactive by a biomimetic precipitation process. The aim of this work was to enhance the bioactivity of pure porous titanium implants by biomimetic process. The samples immersed in a simulated body fluid promoted the nucleation and growth of calcium phosphate (Ca-P) crystals, such as hydroxyapatite (Hap), on the material surface. Scanning electron microscopy, energy dispersive spectroscopy, and Fourier transform infrared spectroscopy analyses revealed that a Ca-P deposition occurred without the need of pretreatments to improve the surface bioactivity. This present study indicates the potential for growing a bone-like Hap layer on porous titanium implants by biomimetic processes.

Effects of pH and initial Ca2+−H2PO4 − concentration on fibroin mineralization

In the present study, the effects of pH and initial Ca2+−H2PO4 − (Ca-P) concentration on fibroin mineralization were studied. The crystal growth of calcium phosphates was regulated by regenerated silk fibroin for 8 h (at pH 4.0, 7.0 and 10.0, respectively). Meanwhile, different concentrations of Ca2+ were employed at a certain pH value, keeping the initial Ca-P molar ratio constant at 1.67, i.e., the stoichiometry of hydroxyapatite [Ca10(PO4)6(OH)2, HAP]. The products were characterized by X-ray diffractometry (XRD), scanning electron microscopy (SEM) and thermogravimetric analysis (TGA). The results demonstrated that, compared to pH 4.0 and 10.0, pH 7.0 promoted the transformation of brushite (CaHPO4·2H2O, DCPD) to HAP. In the composites of mineralized fibroin, DCPD is the main inorganic phase at both relatively low and high pH, while HAP is the main inorganic phase at pH 7.0. Additionally, the initial Ca-P concentration does not affect the kind of inorganic phase in the synthesized mineralized fibroin, but induce to different contents of inorganic mineral and different morphology of DCPD at pH 4.0 and pH 10.0.

The effect of Leucine on the crystal growth of calcium phosphate

Crystallization kinetics of hydroxyapatite, HAP, in the presence of Leucine, a natural amino acid with hydrophobic side groups, were investigated at conditions of sustained supersaturation, 37 degrees C, pH 7.40, ionic strength 0.15 M. In the presence of Leucine, the crystal growth rates of HAP decreased markedly. Tauhis action is due to adsorption and subsequent blocking of the active growth sites onto the surface of the HAP crystals. The kinetic results revealed that a Langmuir-type adsorption isotherm is followed and an affinity constants of k(aff )=20.26 x 10(2) L/mol for HAP crystal surface were calculated. The apparent order of the crystal growth reaction was found to be equal to two, suggesting a surface diffusion controlled mechanism.

Fetuin-A, Valve Calcification, and Diabetes

In the current issue of Circulation , Ix et al demonstrate an inverse correlation between mitral and aortic valve calcification and serum fetuin-A levels in a cross-sectional study of 970 patients with coronary artery disease and without renal disease.1 Increased serum fetuin was significantly associated with diabetes mellitus, hypertriglyceridemia, serum albumin, and body mass index, with a weaker association with serum C-reactive protein, low-density lipoprotein cholesterol, and serum calcium. The link between aortic stenosis and fetuin held only in those without diabetes.1 The data highlight the complex interactions of fetuin with inflammation, insulin resistance, and tissue calcification. Furthermore, the results, though intriguing, underscore our lack of understanding of the cellular mechanisms of calcification in diverse pathological substrates, such as the degenerating aortic leaflet and mitral annulus. Article p 2533 Fetuin is a member of the cystatin superfamily of cysteine protease inhibitors, originally discovered as the major component of fetal bovine serum. It is a carrier for growth factors, binds to and inactivates transforming growth factor (TGF)–β and bone morphogenic protein, and is a major component of mineralized bone.2 Fetuin-A is also an acute-phase glycoprotein, produced in adults primarily in the liver, and is a powerful circulating inhibitor of hydroxyapatite formation. Mice that lack fetuin-A exhibit extensive soft-tissue calcification, which is accelerated on a mineral-rich diet, which suggests that fetuin-A acts to inhibit calcification systemically.3 In addition to its effects on mineralization, fetuin-A inhibits insulin receptor autophosphorylation and tyrosine kinase activity in vitro and in vivo. Fetuin-null knockout mice demonstrate improved insulin sensitivity and resistance to diet-induced obesity, and it has been postulated that the absence of fetuin in mice may contribute to the improvement of insulin sensitivity associated with aging.4 Clinically, serum fetuin-A is decreased in patients with moderate to severe chronic kidney disease, especially …

Calcium phosphate crystal growth under controlled environment through urea hydrolysis

In this study, octacalcium phosphate (OCP, Ca 8 (HPO 4) 2(PO 4) 4 5H 2O) micro-fibers were successfully synthesized and isolated purely from the aqueous solution in the environment controlled by urea hydrolysis. During the process, the OCP micro-fibers were suspended in the middle of the reaction solution and weaved a thin film after isolation from the solution. The as-synthesized OCP fibers had the length larger than ∼200 μm, the width equal to ∼2 μm and the aspect ratio as high as 100. Various characterizations proved that the OCP fibers were well crystallized and contained no other impurities that were critical for the materials used in biomedical applications. This study revealed that using urea hydrolysis to control the reaction ambient was an effective way to produce pure OCP without any impurities. This study also demonstrated that dicalcium phosphate anhydrous (DCPA, CaHPO 4) pre-precipitation was the necessary step for OCP fiber growth, yet itself was another useful bioceramics. OCP fibers could be potentially used as the woven porous bioceramics or form fiber-reinforced composite biomaterials.

Site-Specific In Vivo Calcification and Osteogenesis Stimulated by Bone Sialoprotein

Bone sialoprotein (BSP) is one of the major non-collagenous glycosylated phosphoproteins of the extracellular matrix in bone. In vitro studies suggest that BSP may play important roles in the initiation and/or growth of calcium-phosphate crystals. To investigate the potential role of BSP in more complex in vivo environments, we implanted purified bovine BSP with type-I collagen as a carrier into surgically created rat calvarial defects and thoracic subcutaneous pouches. The responses to the implants were assessed by histochemistry, immunohistochemistry, in situ hybridization, quantitative real-time PCR, and biochemical analyses. BSP-collagen, but not collagen alone, elicited mineral deposition in the matrix of proliferating cells near the dura at days 4-5 followed by osteoblast differentiation and synthesis of new bone in the mid-portion of the calvarial defects. In contrast, implantation of BSP-collagen into subcutaneous pouches did not induce calcification or osteogenesis over the same experimental period. We explored the underlying mechanisms for the site-specific responses to BSP-collagen implants and found that higher levels of calcium content and alkaline phosphatase activity at the cranial site at days 2-5 were associated with the BSP-mediated calcification. We also found that BSP stimulated osteoblast differentiation through up-regulation of cbfa1 and osterix, key transcription factors of osteoblast differentiation, which occurred in the calvarial defects but not in the subcutaneous tissue. These results demonstrate that BSP stimulates calcification and osteogenesis in a site-specific manner, and that local environment and the specificities of responding cells may play critical roles in the function of BSP in vivo.

Calcium phosphate crystal growth under controlled atmosphere in electrochemical deposition

Cathodic deposition of calcium phosphate (Ca–P) on titanium was studied under controlled atmospheres. Electrochemical deposition was conducted in a sealed electrochemical cell with a controlled flow of nitrogen, carbon dioxide or compressed air. The Ca–P phases of the depositions on titanium were examined by X-ray diffraction, Fourier transform–infrared spectroscopy, scanning electron microscopy and transmission electron microscopy. Experimental results showed that hydroxyapatite (HA) deposition occurs under a pure nitrogen atmosphere, while octacalcium phosphate (OCP) deposition occurs under a carbon dioxide atmosphere. The atmosphere in the electrochemical cell effectively maintained constant levels of pH value in the electrolyte during the deposition process. A HA/OCP layered structure on titanium was obtained by controlling the atmosphere. The electrochemical deposition process of Ca–P can be explained by the thermodynamic and kinetics theory of the Ca–P precipitation from an aqueous supersaturated solution.

Optimization of the mineral content in polymeric gels: The effect of calcium to phosphate molar ratio

The influence of calcium to phosphate (Ca/P) molar ratio on the extent of mineralization in a model (poly)acrylamide gel was investigated under simulated physiological conditions. We hypothesized that the optimal growth of hydroxyapatite crystals will take place at the stoichiometric Ca/P molar ratio of 1.67. Phosphate ions were incorporated during the polymerization of the gel and mineralization was initiated by submersion of the gel in calcium acetate solution. Ca/P molar ratios were varied in the range of 0.5–5.0. The mineralized gel was characterized by Raman spectroscopy, scanning electron microscopy (SEM) and mineral weight fraction analysis via ashing. Raman spectra captured across the bulk of the gels indicated the presence of mineral at the core section. The phosphate symmetric stretching peak was observed in the range of 955–960 cm −1 which is characteristic of hydroxyapatite. SEM images showed that crystals formed at Ca/P=2.0 were denser and larger in size than at other molar ratios. In agreement with SEM images, the dry weight fraction of mineral reached the maximum at the molar ratio of 2.0 and the extent of mineralization rapidly declined as the molar ratio diverged from 2.0. Also, the crystallinity of the mineral was optimum at the molar ratio of 2.0. Thus it appears that for effective mineralization, the molar ratio of the two ions needs to be in excess of the stoichiometric requirement, suggesting that ions are expended in processes other than the formation and growth of hydroxyapatite crystals. Therefore, the optimal level of mineralization in biomimetic-based growth of calcium phosphate crystals in sol–gel environment requires consideration of a range of molar ratios as opposed to using the molar ratios corresponding to that of the crystal species intended to grow.

Regulation of Vascular Calcification

Vascular calcification is prevalent in aging as well as a number of pathological conditions, and it is now recognized as a strong predictor of cardiovascular events in the general population as well as diabetic and end-stage renal disease patients. Vascular calcification is a highly regulated process involving inductive and inhibitory mechanisms. This article focuses on two molecules, phosphate and osteopontin, that have been implicated in the induction or inhibition of vascular calcification, respectively. Elevated phosphate is of interest because hyperphosphatemia is recognized as a major nonconventional risk factor for cardiovascular disease mortality in end-stage renal disease patients. Studies to date suggest that elevated phosphate stimulates smooth muscle cell phenotypic transition and mineralization via the activity of a sodium-dependent phosphate cotransporter. Osteopontin, however, appears to block vascular calcification most likely by preventing calcium phosphate crystal growth and inducing cellular mineral resorption.

Free‐Standing Nanogold Membranes as Supports for the Growth of Calcium Phosphate Crystals

Current strategies for bone tissue regeneration focus on the development of implantable matrices that mimic biological tissues. Inorganic composites are of special interest for bone substitute applications. It is necessary to create an artificial three-dimensional scaffold-like porous material with certain geometrical structure to induce bone growth. We report here the growth of calcium phosphate crystals on free-standing carboxylic acid functionalized gold nanoparticle membranes. The gold nanoparticle membrane is synthesized by the spontaneous reduction of aqueous chloroaurate ions by a diamine molecule at a liquid-liquid interface. This membrane is robust and malleable, and most importantly, the gold nanoparticles in the membrane may be functionalized with suitable ligands. In this study, the amino acids aspartic acid and cysteine together with an aromatic bifunctional molecule, anthranilic acid, were used to modify the surface of the gold nanoparticles in the membrane. The free carboxylic acid groups on the gold nanoparticles further to functionalization with these molecules were then used to bind Ca(2+) ions and reacted with phosphate ions to yield calcium phosphate. The nature of the nanogold surface modifier directed the formation of either crystalline hydroxyapatite or amorphous calcium phosphate. The nanogold membrane thus suggests potential biomedical application as biocompatible implants and grafts.

The effect of recombinant mouse amelogenins on the formation and organization of hydroxyapatite crystals in vitro

Amelogenin is the most abundant protein in developing dental enamel. It is believed to play an important role in the regulation of the growth and organization of enamel crystals. Amelogenin, unlike many other proteins found in biominerals, is mostly hydrophobic except for a 13 amino acid hydrophilic C-terminal domain. To clarify the role of amelogenin in enamel mineralization, we designed calcium phosphate crystal growth experiments in the presence of recombinant amelogenins with or without the charged C-terminal domain. The shape and organization of the crystals were examined by TEM in bright field and diffraction modes. It was found that both full-length and truncated amelogenin inhibit crystal growth in directions normal to the c-axis. At the same time, crystallites organized into parallel arrays only in the presence of the full-length amelogenin in monomeric form. Pre-assembled amelogenins had no effect on crystals organization. These results imply that the hydrophobic portion of amelogenin plays a role in an inhibition of crystal growth, whereas the C-terminal domain is essential for the alignment of crystals into parallel arrays. Our data also suggest that nascent enamel structure emerges as a result of cooperative interactions between forming crystals and assembling proteins.

The mineralization process of calcium phosphate induced by the LB films of porphyrin

The Langmuir–Blodgett (LB) films of zinc(II)5,10,15,20-tetra[4-oxy(2-stearic acid) porphyrin] (ZnTSPP) was used as ordered molecular template to induce the growth of calcium phosphate crystals. This process at the surface of LB films was monitored in situ by Inverted Phase Contrast Microscopy (IPCM). It was found that the crystal morphologies changed from amorphous to regular crystal with the prolonging of the time. The transformation from unstable octacalcium phosphate (OCP) to the most stable hydroxyapatite (HAp) was confirmed. This process is step by step, which is different from the traditional epitaxial process.

Ultrastructural study of calculus–enamel and calculus–root interfaces

The attachment of dental calculus to the tooth (enamel or cementum) surface affects the ease or difficulty of its removal. Understanding the ultrastructural features of the calculus-tooth interface will help in the development of efficient strategies for efficient removal of dental calculus. The aim of this study was to determine the ultrastructural characteristics of the calculus-tooth interface in relation to the occurrence of calculus fracture. Investigation of the ultrastructural characteristics of the calculus-tooth interface was made on eight human molars with mature supragingival and subgingival calculus using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and fourier transform infra-red (FT-IR) spectroscopy. Fractures were shown by SEM to consistently occur within the calculus itself, but not at the calculus-tooth interface. Higher magnification revealed that the enamel apatite crystals (in the case of supragingival calculus) or the cementum apatite crystals (in the case of subgingival calculus) appeared intimately connected with the calculus crystals at the calculus-enamel or calculus-cementum interface. TEM micrographs confirmed this intimate direct connection or fusion (epitaxial growth) of calculus crystals with enamel and cementum apatite crystals. FT-IR showed lower concentrations of organic phase attributed to microorganisms and higher concentrations of collagen at the calculus-cementum interface compared to that in the calculus away from the interface. Difficulty in complete calculus removal from tooth surfaces (especially from cementum or dentin) may be due in part to the intimate contact between the calculus and the tooth, due to the chemical bonding between the calculus crystals and the tooth apatite crystals and occasional fusion (i.e., epitaxial growth) of the calculus calcium phosphate crystals with the enamel, dentin or apatite crystals. This cohesive bonding results in fracture planes occurring within the calculus instead of at the calculus-tooth interface.