In vivostudy of iron oxide-calcium phosphate composite nanoparticles for delivery to atherosclerosis.

Atherosclerosis is a macrophage-related inflammatory disease that remains a leading cause of death worldwide. Magnetic iron oxide (IO) nanocrystals are clinically used as magnetic resonance imaging contrast agents and their application as a detection agent for macrophages in arterial lesions has been studied extensively. We recently fabricated heparin-modified calcium phosphate (CaP) nanoparticles loaded with a large number of IO nanocrystals via coprecipitation from a supersaturated CaP solution supplemented with heparin and ferucarbotran (IO nanocrystals coated with carboxydextran). In this study, we further increased the content of IO nanocrystals in the heparin-modified IO-CaP composite nanoparticles by increasing the ferucarbotran concentration in the supersaturated CaP solution. The increase in nanoparticle IO content caused a decrease in particle diameter without impairing its dispersibility; the nanoparticles remained dispersed in water for up to 2 h due to electrostatic repulsion between particles due to the surface modification with heparin. The nanoparticles were more effectively taken up by murine RAW264.7 macrophages compared to free ferucarbotran without showing significant cytotoxicity. A preliminaryin vivostudy showed that the nanoparticles injected intravenously into mice delivered more IO nanocrystals to macrophage-rich carotid arterial lesions than free ferucarbotran. Our nanoparticles have potential as a delivery agent of IO nanocrystals to macrophages in arterial lesions.

In situ precipitation of amorphous calcium phosphate nanoparticles within 3D porous collagen sponges for bone tissue engineering.

Amorphous calcium phosphate (ACP) plays an important role in biomineralization within the three-dimensional (3D) collagen network in human hard tissues, and exhibits osteoconductivity. Porous collagen sponges coated with ACP nanoparticles could be considered as potential scaffolds for use in bone tissue engineering. In this study, such composite materials were fabricated via homogeneous ACP precipitation using a supersaturated calcium phosphate (CaP) solution. Homogeneous ACP precipitation was induced in situ within the sponges by a temperature-controlled coating process composed of two steps. In the first step, the CaP solution was cooled to 4 °C to suppress precipitation until the solution penetrated fully into the sponge's internal pores. In the second step, the CaP solution was warmed up to 25 °C with continuous shaking to induce ACP precipitation within the sponges. The resulting sponges were therefore coated with ACP nanoparticles on their inner and outer surfaces. A simulated body fluid (SBF) test indicated osteoconductivity of the collagen sponges coated with ACP nanoparticles. Further, ACP-coated collagen sponges immobilizing basic fibroblast growth factor (bFGF) were fabricated using the CaP solution supplemented with bFGF. The fabricated sponges allowed the sustained release of bFGF in a culture medium and enhanced proliferation of osteoblastic MC3T3-E1 cells. Such ACP-coated collagen sponges have the potential to be used as scaffolds in bone tissue engineering if pursued for further in vitro and in vivo studies.

Structural Analysis of Calcium Phosphate-Based Submicrospheres with Internally-Crystallized Iron Oxide Nanoparticles Fabricated by a Laser-Assisted Precipitation Process.

Calcium phosphate (CaP)-based submicrospheres containing magnetic iron oxide (IO) nanoparticles (IO-CaP submicrospheres) have potential for various biomedical applications. We recently achieved facile one-pot fabrication of IO-CaP submicrospheres using a laser-assisted precipitation process in which weak pulsed laser irradiation was applied to a labile CaP reaction mixture supplemented with ferrous ions under adequate pH. In this study, we performed cross-sectional transmission electron microscopy (TEM) analysis of the resulting IO-CaP submicrospheres. The cross-sectional TEM analysis revealed that the IO-CaP submicrospheres were heterogeneous in their internal nanostructures and could be categorized into two types, namely types A and B. The type A submicrospheres contained single nano-sized IO nanoparticles homogeneously dispersed throughout the CaP-based matrix. The type B submicrospheres contained larger IO nanoparticles with an irregular or spherical shape, which were mostly a few tens of nanometers in size along with one or two submicron-sized domains. These findings provide new insight into the formation mechanism of IO-CaP submicrospheres in this fabrication technique as well as future applications of the resulting IO-CaP submicrospheres.

Rapid and area-specific coating of fluoride-incorporated apatite layers by a laser-assisted biomimetic process for tooth surface functionalization.

Surface functionalization of teeth with fluoride-incorporated apatite layers displays great potential in treatments and prevention of dental disorders. In this study, we used a sintered hydroxyapatite (sHA) substrate as a model material of teeth, and established a rapid and area-specific coating technique of fluoride-incorporated apatite layers by using a laser-assisted biomimetic (LAB) process. In this technique, a sHA substrate was irradiated on the surface with a Nd:YAG pulsed UV laser for 30 min in supersaturated calcium phosphate (CaP) solutions with various fluoride concentrations. The fluoride concentration in the CaP solution was varied to control morphology, crystalline structure, and fluoride content of the resulting layers. Without fluoride in the CaP solution, an octacalcium phosphate (OCP) layer with a flake-like structure was formed on the laser-irradiated surface of the substrate. The addition of fluoride (1000 µM and 3000 µM) to the CaP solution led to the formation of fluoride-incorporated apatite layers with an enamel-like needle-like nanostructure. The fluoride-incorporated apatite layers adhered firmly to the sHA surface and reduced acid dissolution of the sHA substrate by acting as a protective covering. Additionally, the layers released fluoride ions for more than 24 h, and exhibited antibacterial activity relative to a caries-causing bacterium, namely Streptococcus mutans. Thus, our LAB process can potentially act as a new tool for functionalization of tooth surfaces. STATEMENT OF SIGNIFICANCE: We used a sintered hydroxyapatite (sHA) substrate as a model material of teeth, and established a rapid and area-specific coating technique of fluoride-incorporated apatite layers on the sHA surface by using our laser-assisted biomimetic (LAB) process. In this process, pulsed laser was utilized to accelerate seeded crystal growth in supersaturated calcium phosphate solutions supplemented with NaF. The thus-fabricated fluoride-incorporated apatite layers consisted of enamel-like needle-like nanocrystals with c-axis orientation. These fluoride-incorporated apatite layers adhered firmly to the sHA surface, reduced acid dissolution of the sHA substrate by acting as a protective covering, and exhibited antibacterial activity against Streptococcus mutans through the fluoride release. Thus, our LAB process can potentially act as a new tool for functionalization of tooth surfaces.

Physicochemical fabrication of antibacterial calcium phosphate submicrospheres with dispersed silver nanoparticles via coprecipitation and photoreduction under laser irradiation.

We achieved rapid, surfactant-free, and one-pot fabrication of antibacterial calcium phosphate (CaP) submicrospheres containing silver nanoparticles by combining physical laser and chemical coprecipitation processes. In this physicochemical process, weak pulsed laser irradiation (20min) was performed on a labile CaP reaction mixture supplemented with silver ions as a light-absorbing agent. The silver content in the submicrospheres was controlled for a wide range (Ag/P elemental ratio varied from 0.60 to 62.0) by tuning the initial concentration of silver ions (from 5 to 20mM) in the CaP reaction mixture. At the silver concentration of 5mM, we obtained unique nanocomposite particles: CaP submicrospheres (average diameter of approximately 500nm) containing metallic silver nanoparticles dispersed throughout, as a result of CaP and silver coprecipitation with simultaneous photoreduction of silver ions and spheroidization of the coprecipitates. These CaP submicrospheres containing silver nanoparticles (ca. 0.3mg silver per 1mg submicrospheres) exhibited antibacterial activity against major pathogenic oral bacteria, i.e., Streptococcus mutans, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis. Moreover, the CaP submicrospheres dissolved and neutralized the acidic environment generated by Streptococcus mutans, demonstrating their potential as acid-neutralizing and remineralizing agents. The present process and resulting antibacterial CaP-based submicrospheres are expected to be useful in dental healthcare and infection control. STATEMENT OF SIGNIFICANCE: Nano- and microsized spheres of calcium phosphate (CaP) containing silver nanoparticles have great potential in dental applications. Conventional fabrication processes were time-consuming or weak regarding the size/shape control of the spheres. In this study, we achieved a simple (one-pot), rapid (20-min irradiation), and surfactant-free fabrication of CaP submicrospheres containing silver nanoparticles by pulsed laser irradiation to a mixture of calcium, phosphate, and silver ion solutions. The resulting CaP submicrospheres contained metallic silver nanoparticles dispersed throughout in a sequence of reactions: CaP and silver coprecipitation, laser-induced melting and spheroidization of the coprecipitates, and photoreduction of silver ions. These submicrospheres showed antibacterial activity against oral bacteria and acid-neutralizing property in the bacterial suspension, and hence are worth considering for dental applications.

Laser-assisted one-pot fabrication of calcium phosphate-based submicrospheres with internally crystallized magnetite nanoparticles through chemical precipitation.

In this paper, we have further developed our simple (one-pot) and rapid (short irradiation time) laser fabrication process of submicrometer spheres composed of amorphous calcium iron phosphate. In our previous process, laser irradiation was applied to a calcium phosphate (CaP) reaction mixture supplemented with ferric ions (Fe(3+)) as a light-absorbing agent. Because the intention of the present study was to fabricate magnetite-encapsulated CaP-based submicrometer spheres, ferrous ions (Fe(2+)) were used as a light-absorbing agent rather than ferric ions. The ferrous ions served as a light-absorbing agent and facilitated the fabrication of submicrometer and micrometer spheres of amorphous calcium iron phosphate. The sphere formation and growth were better promoted by the use of ferrous ions as compared with the use of ferric ions. The chemical composition of the spheres was controllable through adjustment of the experimental conditions. By the addition of sodium hydroxide to the CaP reaction mixture supplemented with ferrous ions, fabrication of CaP-based magnetic submicrometer spheres was successfully achieved. Numerous magnetite and wüstite nanoparticles were coprecipitated or segregated into the CaP-based spherical amorphous matrix via light-material interaction during the CaP precipitation process. The magnetic properties of the magnetite and wüstite formed in the CaP-based spheres were investigated by magnetization measurements. The present process and the resulting CaP-based spheres are expected to have great potential for biomedical applications.

Improved gene transfer efficiency of a DNA-lipid-apatite composite layer by controlling the layer molecular composition.

Surface-mediated nonviral gene transfer systems using biocompatible apatite-based composite layers have potential use in tissue engineering applications. Herein, we investigated a relatively efficient system based on a DNA-lipid-apatite composite layer (DLp-Ap layer): an apatite (Ap) layer with immobilized DNA and lipid (Lp) complexes (DLp complexes). DLp-Ap layers were fabricated on substrates using supersaturated calcium phosphate coprecipitation solutions supplemented with DLp complexes, and the molecular compositions of the DLp-Ap layers were controlled by varying the net DNA concentrations and Lp/DNA ratios in the coprecipitation solutions. Increases in both the DNA concentration and Lp/DNA ratio in the coprecipitation solution increased the DLp complex content of the resulting DLp-Ap layer. However, a higher DLp complex content did not always provide increased gene transfer efficiency to the CHO-K1 cells, because there was a threshold content of approximately 10µg/cm(2). In addition, DLp-Ap layers with similar DLp complex contents exhibited different gene transfer efficiencies, most likely due to the different Lp/DNA ratios in the layers. Notably, the optimized Lp/DNA ratios in the coprecipitation solutions for maximizing the gene transfer efficiency were lower than those of the conventional particle-mediated lipofection systems. These findings will serve as a useful design guide for the preparation of DLp-Ap layers with high gene transfer efficiency.

DNA-lipid-apatite composite layers enhance gene expression of mesenchymal stem cells.

DNA-apatite composite layer (D-Ap layer) and DNA-lipid-apatite composite layer (DLp-Ap layer) were prepared on ceramic hydroxyapatite disk and scaffold using supersaturated calcium phosphate solutions supplemented with 0.5-5 µg/mL plasmid and 0-10 µL/mL lipid transfection reagent FuGENE®. Both in vitro and in vivo studies were carried out using mesenchymal stem cells (MSCs) and two kinds of gene (luciferase and bone morphogenetic protein (BMP)-2) for demonstrating potential application of the gene transfer system using the D-Ap and DLp-Ap layers in bone tissue engineering. In the in vitro study using luciferase gene, the DLp-Ap layers showed 1-2 orders of magnitudes higher gene transfer efficiency to MSCs than the D-Ap layer. In the in vivo study using BMP-2 gene, DLp-Ap layer slightly increased BMP-2 protein concentration than D-Ap layer, thereby enhancing their osteogenic differentiation than D-Ap layer. The present gene transfer system using the DLp-Ap layers, with the advantages of good biocompatibility, bone-bonding ability, and efficacy in in vitro and in vivo gene transfer to MSCs, would be useful in bone tissue engineering.

BMP-2 and ALP gene expression induced by a BMP-2 gene-fibronectin-apatite composite layer.

The bone morphogenetic protein 2 (BMP-2) gene delivery system with a gene-fibronectin (Fn)-apatite composite layer was fabricated on the surface of a hydroxyapatite ceramic scaffold. The BMP-2 gene-Fn-apatite composite layer was coated on the scaffold using a supersaturated calcium phosphate solution supplemented with BMP-2 DNA and Fn. The scaffolds were ectopically implanted into the dorsal subcutaneous tissue of rats. Four weeks after the implantation, the hydroxyapatite scaffold coated with the BMP-2 gene-Fn-apatite composite layer showed improved gene expressions of BMP-2 and alkaline phosphatase as compared with the scaffold coated with the apatite layer. Although these results suggest the possibility of ectopic bone formation induced by the present gene delivery system, further study is necessary to prove this.

Enhanced immobilization of acidic proteins in the apatite layer via electrostatic interactions in a supersaturated calcium phosphate solution.

Artificial materials coated with a protein-apatite composite layer have great potential in clinical applications as a third generation biomaterial. Such composite materials can be prepared by immersing a surface modified substrate into a supersaturated calcium phosphate solution (CP solution: 142 mM NaCl, 3.75 mM CaCl(2), 1.5mM K(2)HPO(4)·3H(2)O, buffered at pH 7.4 at 25 °C with tris(hydroxymethyl)aminomethane and HCl) supplemented with a protein. In the present study proteins of various molecular weights (MW) and isoelectric points (pI) were used to form a protein-apatite composite layer on a polymeric material to determine how the molecular properties of the protein affect the efficiency of protein immobilization (i.e. the amount of immobilized protein in the apatite layer as a percentage of the total amount of protein in solution). The results indicated that the efficiency of protein immobilization did not correlate with the MW of the protein. In contrast, the efficiency of protein immobilization was strongly related to the pI of the protein. As the pI decreased the efficiency of protein immobilization increased due to the high adsorption affinity of negatively charged acidic proteins for positively charged apatite crystals and/or apatite precursors in the CP solution. Thus, the use of acidic rather than basic proteins improves the immobilization efficiency in the present coating process.

Preparation and biological evaluation of a fibroblast growth factor-2-apatite composite layer on polymeric material.

A polymeric percutaneous device with good biocompatibility and resistance to bacterial infection is required clinically. In this study, a fibroblast growth factor-2 (FGF-2)-hydroxyapatite (HAp) composite layer (FHAp layer) was formed on the surfaces of ethylene-vinyl alcohol copolymer (EVOH) specimens using a coating process in a supersaturated calcium phosphate solution supplemented with FGF-2. FGF-2 in the FHAp layer retained its biological activity to promote proliferation of fibroblasts. The EVOH specimens coated with HAp and FHAp layers were percutaneously implanted in the scalp of rats. Not only the HAp layer but also the FHAp layer showed good biocompatibility, and FGF-2 showed no harmful effects on the skin tissue responses to the implanted specimen as long as 14 d. No significantly higher infection resistance was verified for the FHAp layer over the HAp layer, although an FHAp layer coated on a metallic percutaneous device for bone fixation demonstrated higher resistance to bacterial infection over an HAp layer in the previous study. The efficacy of FHAp layers coated on percutaneous implants in resistance to bacterial infection depends on physical factors including fixation condition, stiffness and movement of implants.

Ascorbate-apatite composite and ascorbate-FGF-2-apatite composite layers formed on external fixation rods and their effects on cell activity in vitro.

Ascorbate-apatite and ascorbate-fibroblast growth factor-2 (FGF-2)-apatite composite layers were successfully formed on anodically oxidized Ti rods clinically used for external fixation by a one-step procedure at 25 degrees C, using a metastable supersaturated calcium phosphate solution supplemented with l-ascorbic acid phosphate magnesium salt n-hydrate (AsMg) and FGF-2. The AsMg-apatite and AsMg-FGF-2-apatite composite layers were evaluated in vitro using fibroblastic NIH3T3 and osteoblastic MC3T3-E1 cells. The AsMg-FGF-2-apatite composite layer markedly enhanced the NIH3T3 cell proliferation and procollagen type capital I, Ukrainian gene expression. Without FGF-2, the AsMg-apatite composite layer whose ascorbate content was 3.64+/-1.27microgcm(-2) obviously enhanced osteoblastic proliferation and differentiation. However, the AsMg-FGF-2-apatite composite layers whose FGF-2 contents were from 0.15+/-0.03 to 0.31+/-0.04microgcm(-2) inhibited osteoblastic differentiation in vitro. Thus, the AsMg-FGF-2-apatite composite layer should be precipitated on the surface of external fixators attached to skin and soft tissue. On the other hand, the AsMg-apatite composite layer should be precipitated at the part attached to bone tissue.

Fibroblast growth factor-2-apatite composite layers on titanium screw to reduce pin tract infection rate.

Fibroblast growth factor-2 (FGF-2)-apatite composite layers were formed on anodically oxidized titanium screws to improve bone-screw interface strength and to reduce pin tract infection rate through enhanced skin tissue healing in external fixation. A calcium-containing solution supplemented with FGF-2, a phosphate-containing solution, and a sodium bicarbonate solution were mixed at a Ca/P molar ratio of 2.0 to prepare a calcium phosphate solution supersaturated with respect to calcium phosphates. Screws were individually immersed in 10 mL of the calcium phosphate solution at 37 degrees C for 2 days. Low-crystalline apatite layers incorporating FGF-2 were formed on the screw surface at FGF-2 concentrations in the supersaturated calcium phosphate solution equal to or lower than 10 mug/mL. The amounts of FGF-2 immobilized on the screws ranged from 2.3- to 2.4-mug per screw. The immobilized FGF-2 retained biological activity, as demonstrated by NIH3T3 cell proliferation. Titanium screws with the composite layer were percutaneously implanted into the bilateral proximal tibial metaphyses in rabbits for 4 weeks. The titanium screws with the composite layer formed at the optimum FGF-2 concentration showed a significantly higher bone-screw interface strength and a lower pin tract infection rate than those without the composite layer: the extraction torque and infection rates were respectively 0.230 +/- 0.073 Nm and 43.8% for the screws with the composite layer, and 0.170 +/- 0.056 Nm and 93.8% for those without the composite layer. Therefore, titanium screws with the FGF-2-apatite composite layer are useful for improving bone-screw interface strength and infection resistance in external skeletal fixation.

Fibronectin-calcium phosphate composite layer on hydroxyapatite to enhance adhesion, cell spread and osteogenic differentiation of human mesenchymal stem cells in vitro.

Fibronectin (Fn) and type I collagen (Col) were immobilized on a surface of a hydroxyapatite (HAP) ceramic by coprecipitation with calcium phosphate in a supersaturated calcium phosphate solution prepared by mixing clinically approved infusion fluids. These proteins and the calcium phosphate precipitate formed a composite surface layer. As a result, the proteins were immobilized firmly as not to be released completely for 3 d in a physiological salt solution. When human mesenchymal stem cells (hMSCs) were cultured on a HAP ceramic in a differentiation medium supplemented with dexamethasone, beta-glycerophosphate and ascorbic acid, hMSCs spread well within 1 h. The alkaline phosphatase (ALP) activity of hMSCs cultured on the Fn-calcium phosphate composite layer significantly increased compared with that of hMSCs cultured on the untreated HAP ceramic. On the other hand, Col did not increase the ALP activity of hMSCs and no synergy between Fn and Col was observed. Therefore, the Fn-calcium phosphate composite layer formed on the HAP is useful for the enhancement of the spreading and osteogenic differentiation of hMSCs in vitro.

The formation of an antibacterial agent-apatite composite coating on a polymer surface using a metastable calcium phosphate solution.

A percutaneous device with antibacterial activity and good biocompatibility is desired for clinical applications. Three types of antibacterial agent: lactoferrin (LF), tetracycline (TC), and gatifloxacin (GFLX) were immobilized on the surface of an ethylene-vinyl alcohol copolymer (EVOH) using a liquid phase coating process. In this process, an EVOH plate was alternately dipped in calcium and phosphate ion solutions, and then immersed in a metastable calcium phosphate solution supplemented with 4, 40, or 400 microg/mL of the antibacterial agent. As a result, the antibacterial agent was immobilized on the EVOH surface in the form of an antibacterial agent-apatite composite layer. The amount of immobilized antibacterial agent increased with increasing absorption affinity for apatite in the order: GFLX

Reduced platelet adhesion to titanium metal coated with apatite, albumin-apatite composite or laminin-apatite composite.

Titanium metal coated with apatite (HA-Ti), albumin-apatite composite (AA-Ti) or laminin-apatite composite (LA-Ti) was prepared by the immersion of NaOH- and heat-treated titanium metal in a calcium phosphate solution, or one supplemented with albumin or laminin. Platelet adhesion to the obtained materials under flow conditions was investigated in real time using a cone- and plate-type viscometer and fluorescence labeled platelets. Adhesion and activation of the platelets on the HA-Ti, AA-Ti and LA-Ti were definitely suppressed as compared with those on untreated titanium metal with a mirror surface. Furthermore, the numbers of platelets adhered to AA-Ti and LA-Ti are smaller than those adhered to HA-Ti, although the differences were not statistically significant. These findings suggest that HA-Ti, AA-Ti and LA-Ti, especially AA-Ti and LA-Ti, would exhibit thromboresistance that is superior to commercially pure titanium metal in terms of platelet adhesion.