Supersaturated Calcium Phosphate Solution Research Articles

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, 1.5 mM K 2HPO 4·3H 2O, 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.

Enhanced bone formation using hydroxyapatite ceramic coated with fibroblast growth factor-2

Our objective was to develop a bone substitute coated with fibroblast growth factor-2 (FGF-2) that subsequently releases FGF-2. We investigated the use of our system of bone substitutes to induce bone formation. Hydroxyapatite ceramic buttons (HAP-CBs) were coated with FGF-2 by precipitation in supersaturated calcium phosphate solution. HAP-CBs were coated with high or low doses of FGF-2, denoted as FGF-H and FGF-L. The release of FGF-2 from FGF-H and FGF-L was evaluated using its release profile and bioactivity. The efficacy of the subsequent bone formation was quantified using rats with round-shaped bone defects (5 mm in diameter) of the right parietal bone. Group 1 was treated only with HAP-CBs, group 2 with HAP-CBs and drops of FGF-2 solution, group 3 with FGF-L and group 4 with FGF-H. To detect the release of FGF-2 in vivo, the expression of bone morphogenic protein-2 (BMP-2) was measured in the defective bone tissue. FGF-2 was released in vitro from FGF-H and FGF-L, and maintained its bioactivity. Rats treated with FGF-L showed better bone formation than rats from the other groups. BMP-2 expression was detected in the defective bone tissues of group 3 at 14 days, which might indicate in vivo FGF-2 release during this period. A specific FGF-2 concentration may be needed for bone formation, and our system can release FGF-2 at adequate concentrations to induce bone formation.

Controlled formation of calcium-phosphate-based hybrid mesocrystals by organic–inorganic co-assembly

An understanding of controlled formation of biomimetic mesocrystals is of great importance in materials chemistry and engineering. Here we report that organic-inorganic hybrid plates and even mesocrystals can be conveniently synthesized using a one-pot reaction in a mixed system of protein (bovine serum albumin (BSA)), surfactant (sodium bis(2-ethylhexyl) sulfosuccinate (AOT)) and supersaturated calcium phosphate solution. The morphologies of calcium-phosphate-based products are analogous to the general inorganic crystals but they have abnormal and interesting substructures. The hybrids are constructed by the alternate stacking of organic layer (thickness of 1.31 nm) and well-crystallized inorganic mineral layer (thickness of 2.13 nm) at the nanoscale. Their morphologies (spindle, rhomboid and round) and sizes (200 nm-2 μm) can be tuned gradually by changing BSA, AOT and calcium phosphate concentrations. This modulation effect can be explained by a competition between the anisotropic and isotropic assembly of the ultrathin plate-like units. The anisotropic assembly confers mesocrystal characteristics on the hybrids while the round ones are the results of isotropic assembly. However, the basic lamellar organic-inorganic substructure remains unchanged during the hybrid formation, which is a key factor to ensure the self-assembly from molecule to micrometre scale. A morphological ternary diagram of BSA-AOT-calcium phosphate is used to describe this controlled formation process, providing a feasible strategy to prepare the required materials. This study highlights the cooperative effect of macromolecule (frame structure), small biomolecule (binding sites) and mineral phase (main component) on the generation and regulation of biomimetic hybrid mesocrystals.

Development of apatite-based composites by a biomimetic process for biomedical applications

A new biomimetic process for apatite coating on polymeric materials has been developed. In this process, the surface of a polymer is modified with amorphous calcium phosphate (ACP), and then the polymer is immersed in a supersaturated calcium phosphate solution. The new biomimetic process has the advantages of safety, simplicity, and applicability to various types and forms of polymeric materials. By adding a biomolecule (such as protein, antibacterial agent, or DNA) to the supersaturated solution, immobilization of a biomolecule into the apatite coating while retaining the intrinsic biological activity of the biomolecule is possible. As a result of this, the base polymer would possess biological activity to control cell behaviors (such as adhesion, proliferation, and differentiation), in addition to good biocompatibility owing to the apatite. Hence, the new biomimetic process and the resulting composites have a wide variety of biomedical applications including tissue engineering scaffolds, percutaneous devices, and gene delivery carriers.

Chemical analyses of hydroxyapatite formation on SAM surfaces modified with COOH, NH2, CH3, and OH functions

Hydroxyapatite formation was examined at the surface of self-assembled monolayers (SAMs) modified with four functional groups, -COOH, -NH(2), -CH(3), and -OH. For COOH-SAM and NH(2)-SAM, scanning electron spectroscopic observation showed that flake-like sheet crystals covered the whole wafer and small broccoli-like crystals were observed occasionally on the flake-like crystal base layer. For CH(3)-SAM and OH-SAM, no flake-like sheet crystals were observed; broccoli-like crystals were observed in a dispersed manner for CH(3)-SAM, but in localized spots for OH-SAM. X-ray diffraction patterns showed a strong apatite pattern oriented toward the c-axis direction for COOH-SAM. ESCA analysis revealed distinct Ca, P, O peaks for COOH-, NH(2)-, CH(3)-, and OH-SAM. Surface plasmon resonance (SPR) analysis indicated that during the supply of supersaturated calcium phosphate solution, the deposition of precipitates increased monotonically with time for COOH-SAM, increased slightly for NH(2)-SAM, but little increase in deposition was detected for CH(3)-SAM and OH-SAM.

Mesoporous bioactive glass coatings on stainless steel for enhanced cell activity, cytoskeletal organization and AsMg immobilization

Mesoporous bioactive glass (MBG) coatings with SiO2:CaO:P2O5 mol ratio of 100:0:0, 80:15:5 and 70:25:5 and a tunable pore size and pore structure were prepared on a stainless steel plate by spin-coating sol solutions containing a triblock copolymer and the inorganic precursors. The calcium content in the MBG coatings affected the mesoporous structure. With the increase in calcium content, the crystallinity of the MBG coatings increased and thus the Brunauer–Emmett–Teller (BET) surface area and pore volume decreased. The MBG coatings were evaluated on the basis of protein adsorption, cell attachment, cell proliferation, cell differentiation, cytoskeletal organization and L-ascorbic acid phosphate magnesium salt n-hydrate (AsMg) immobilization for their potential in improving implant-bone integration. The results showed that the osteoblast MC3T3-E1 cells were stimulated by the mesoporous structure and chemical composition of the MBG coatings, with enhanced cell attachment, proliferation, differentiation and better developed cytoskeleton. Moreover, AsMg was successfully immobilized on the MBG coatings by using an AsMg-containing supersaturated calcium phosphate solution. The AsMg immobilized on the MBG coatings was not denatured and showed high activity enhancing the fibroblast NIH3T3 proliferation in vitro. An appropriate range of pore size and a preferred alignment of the mesochannels of the MBG coatings on stainless steel are promising to improve the implant-bone integration.

Fabrication of biomimetic apatite coating on porous titanium and their osteointegration in femurs of dogs

In the present study, porous titanium with three-dimensionally interconnected pores and a porosity of 74.3 ± 3.8% was made by a slurry foaming method. After being pretreated with acid–alkali or alkali–heat method and then immersed into a supersaturated calcium phosphate solution to form a biomimetic apatite coating on the surface, the porous titanium samples were hemi-transcortically implanted into the femurs of dogs for two months. The experimental results showed that the surface apatite coatings deposited on acid–alkali and alkali–heat treated porous titanium had different morphology and thickness. However, histological and histomorphometric analysis confirmed that both types of apatite-coated implants had excellent osteointegration with host bone, and their osteoconduction had also no significant difference. This study proved that both acid–alkali and alkali–heat treatments might have the same efficiency in activating porous titanium, and the apatite-coated porous titanium could be potential to be used in clinic.

Zinc-containing apatite layers on external fixation rods promoting cell activity

Zinc-containing apatite layers were successfully formed on commercially available anodically oxidized Ti external fixation rods using ZnCl(2)-containing supersaturated calcium phosphate solutions. With an increase in concentration of ZnCl(2) in the supersaturated calcium phosphate solutions, the amounts of zinc that precipitated on the Ti external fixation rods increased (from 0 to 0.195 + or - 0.020 microg cm(-2)); meanwhile, the amounts of calcium and phosphorus that precipitated on the Ti external fixation rods decreased (from 11.2 + or - 1.5 and 4.8 + or - 0.5 microg cm(-2) to 2.9 + or - 1.6 and 1.3 + or - 0.9 microg cm(-2), respectively). The zinc-containing apatite layers precipitated on the Ti external fixation rods caused a significant increase in fibroblastic proliferation, osteoblastic proliferation and differentiation in vitro. The Ti external fixation rods coated with zinc-containing apatite layers are expected to be more effective in accelerating the tissue regeneration around the surgical site than those coated with an apatite layer.

Role of 20-kDa Amelogenin (P148) Phosphorylation in Calcium Phosphate Formation in Vitro

The potential role of amelogenin phosphorylation in enamel formation is elucidated through in vitro mineralization studies. Studies focused on the native 20-kDa porcine amelogenin proteolytic cleavage product P148 that is prominent in developing enamel. Experimental conditions supported spontaneous calcium phosphate precipitation with the initial formation of amorphous calcium phosphate (ACP). In the absence of protein, ACP was found to undergo relatively rapid transformation to randomly oriented plate-like apatitic crystals. In the presence of non-phosphorylated recombinant full-length amelogenin, rP172, a longer induction period was observed during which relatively small ACP nanoparticles were transiently stabilized. In the presence of rP172, these nanoparticles were found to align to form linear needle-like particles that subsequently transformed and organized into parallel arrays of apatitic needle-like crystals. In sharp contrast to these findings, P148, with a single phosphate group on serine 16, was found to inhibit calcium phosphate precipitation and stabilize ACP formation for more than 1 day. Additional studies using non-phosphorylated recombinant (rP147) and partially dephosphorylated forms of P148 (dephoso-P148) showed that the single phosphate group in P148 was responsible for the profound effect on mineral formation in vitro. The present study has provided, for the first time, evidence suggesting that the native proteolytic cleavage product P148 may have an important functional role in regulating mineralization during enamel formation by preventing unwanted mineral formation within the enamel matrix during the secretory stage of amelogenesis. Results obtained have also provided new insights into the functional role of the highly conserved hydrophilic C terminus found in full-length amelogenin.

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 °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 І gene expression. Without FGF-2, the AsMg–apatite composite layer whose ascorbate content was 3.64 ± 1.27 μg cm −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 ± 0.04 μg cm −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.

Silicate–apatite composite layers on external fixation rods and in vitro evaluation using fibroblast and osteoblast

A silicate-apatite layer was formed on commercially available anodically oxidized titanium rods using Na(2)SiO(3)-containing supersaturated calcium phosphate solutions. With the increase in the concentration of Na(2)SiO(3) in the supersaturated calcium phosphate solutions, the amounts of silicon that precipitated on the titanium rods increased from 0 to 0.07 +/- 0.02 microg/cm(2); meanwhile, the amounts of calcium and phosphorus that precipitated on the titanium rods decreased from 11.6 +/- 1.6 and 5.7 +/- 2.0 microg/cm(2) to 2.6 +/- 0.5 and 3.0 +/- 1.0 microg/cm(2), respectively. The present silicate-apatite composite layers, which demonstrated increased fibroblastic proliferation and osteoblastic proliferation and differentiation in vitro, are promising as coating layers on external fixation pins for decreasing the pin tract infection rate in vivo.

712 組織再生促進のためのシグナル担持生体材料(OS5-(2) インプラントおよび生体材料,オーガナイズドセッション)

Biomaterials loaded with signals that stimulate or induce tissue regeneration in situ are practically useful. Fibroblast growth factor-2 (FGF-2) is one of such signals. FGF-2-apatite composite layers were formed on anodically oxidized titanium external fixation pins using a supersaturated calcium phosphate solution supplemented with FGF-2.The composite layer consisted of polycrystalline poorly crystallized apatite loaded with FGF-2. The pins were percutaneously implanted in both proximal tibial metaphyses in rabbits for 4 weeks. The pins with the FGF-2-apatite composite layer showed a significantly lower pin tract infection rate (44%) than those with apatite layer (94%). Extensive angiogenesis and bone formation was observed around the pins with the composite layer. In some cases, Sharpey's-fiber-like collagen fibers were regenerated around the pins with the composite layer.

A new evaporation-based method for the preparation of biomimetic calcium phosphate coatings on metals

This study reports a new method to prepare biomimetic calcium phosphate coatings on titanium, stainless steel, CoCrMo, and tantalum. The method does not require surface etching, high supersaturation, or tight control of solution conditions. Metallic samples were dipped into a supersaturated calcium phosphate solution, withdrawn, and left to dry at room temperature. Calcium phosphate crystallites formed on and completely covered the surfaces by repeating the dip-and-dry treatment. The crystallite-covered surfaces readily grew to calcium phosphate coatings when immersed in the supersaturated solution. The mechanism of the treatment was suggested to be an evaporation-induced surface crystallization process.

Accelerating calcium phosphate growth on NaOH-treated poly-(lactic- co-glycolic acid) by evaporation-induced surface crystallization

Poly(lactic- co-glycolic acid) (PLGA) is a promising material for the regeneration of bone tissue, but its surface properties are not optimal for the application. Coating the surface of PLGA with a continuous layer of calcium phosphate is an effective approach to address the limitation. Current coating techniques for PLGA require immersion in supersaturated calcium phosphate solutions for days to weeks. In this study, we report a simple technique to accelerate the coating process to only 2 h immersion in supersaturated solutions. PLGA pellets were first treated with NaOH to increase their hydrophilicity. The NaOH-treated PLGA pellets were repeatedly dipped in a supersaturated calcium phosphate solution and dried in air. After 10 times of the dip-and-dry treatment, a layer of calcium phosphate crystallites uniformly covered the surfaces of the pellets. After the crystallite-covered pellets were immersed in the supersaturated solution for 2 h, about 5-μm thick continuous calcium phosphate coatings formed on the surfaces. The dip-and-dry technique was also applied on a variety of metals and porous structures. An evaporation-induced surface crystallization process was suggested as the mechanism for the dip-and-dry treatment.

Formation of cytochrome C–apatite composite layer on NaOH- and heat-treated titanium

A cytochrome C (cyt C) and apatite composite layer was formed on a NaOH- and heat-treated titanium substrate (Ti substrate) by immersing the Ti substrate for one day at 25 °C in supersaturated calcium phosphate solutions obtained by mixing infusion fluids. When the initial supersaturation level of the calcium phosphate solution was increased, the total amount of cyt C and apatite deposited on the Ti substrate increased. On the other hand, the cyt C content in the composite layer decreased with an increase in initial supersaturation level. The morphology of the composite layer markedly changed depending on the initial supersaturation level. Therefore, the initial supersaturation level affected the formation of the cyt C and apatite composite layer. It is expected that fibroblast growth factor-2 can be immobilized on NaOH- and heat-treated titanium substrates using the same method.

Electrochemical depositions of calcium phosphate film on commercial pure titanium and Ti–6Al–4V in two types of electrolyte at room temperature

Electrochemical depositions of calcium phosphate film on commercially pure titanium (cp-Ti) and Ti–6Al–4V in two types of electrolytes, mono-calcium phosphate monohydrate (MCPM) based aqueous solution and supersaturated calcium phosphate solution (SCPS), were carried out by the cathodic polarization. The calcium phosphate coating layer was successfully deposited on cp-Ti and Ti–6Al–4V under 3 mA/cm 2 current density for 45 min. The major phases that appeared in the film were DCPD (brushite) co-existed with octa-calcium phosphate (OCP). After soaking in revised simulated-body-fluid (r-SBF), the amorphous bone-like apatite became a single phase within a day for the specimen obtained from MCPM electrolyte. The specimens obtained from SCPS electrolyte took a week to complete the apatite precipitation in r-SBF. From the scratch test, the highest critical load of 87.2 N was obtained from the as-deposited rod-like DCPD on cp-Ti substrate. The critical load was decreasing almost 50% for all of the specimens after apatite precipitation in r-SBF. The decrease of critical load might result from loosening of the film structure during immersion of the specimens in r-SBF. The thickness of the films was 44–55 μm in average, which satisfied the qualification for using as an implant material.

Radio frequency plasma treatments on titanium for enhancement of bioactivity

Titanium and its alloys, when treated in alkali solutions, are able to form calcium phosphate coatings on their surface after immersion in supersaturated solutions. In this study, the surfaces of titanium alloy discs were modified by an alkali treatment and a radio frequency (RF) plasma procedure (150 W and 13.56 MHz) in N 2, CO 2 or N 2/O 2 (80/20%) atmospheres. After the alkali treatment, atomic force microscopy showed differences in the surface roughness of the samples. X-ray photoelectron microscopy indicated that the chemical composition of the surfaces changed after the different alkali and RF plasma treatments. The contact angles were also modified by ∼5°, making the original titanium surface more hydrophilic. Immersion in a supersaturated calcium phosphate solution was used to evaluate the bioactivity of the RF plasma-treated samples in vitro. Alkali-treated samples gave more homogeneous and thick coatings that those without alkali treatment. The use of RF plasma treatments enhanced the bioactivity of the samples, in particular for treatments performed in N 2 or N 2/O 2 atmospheres. Energy-dispersive X-ray analysis indicated that coatings had Ca/P ratios between the values of octacalcium phosphate and hydroxyapatite. X-ray diffraction confirmed the presence of these two phases in most of the coatings. This study shows that an RF plasma treatment enhanced the bioactivity of titanium surfaces.

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.

Enhancement of the bioactivity of titanium oxide nanotubes by precalcification

Titania nanotubes have been thought to be a novel material for improving bioactivity of titanium. In this work, the effect of precalcification (Pre-Ca) on the bioactivity of the TiO 2 nanotubes was investigated, which was applied by soaking the TiO 2 nanotubes in Na 2HPO 4 solution overnight and then in saturated Ca(OH) 2 solution for 5 h. It was observed that, only after 7 days immersion in a supersaturated calcium phosphate solution (SCP), hydroxyapatite (HA) was formed apparently on the surface of the precalcified TiO 2 nanotube specimens. But no obvious HA was observed on the non-precalcified specimens even up to 14 days immersion in the SCP. The bioactivity of titania nanotubes was enhanced by the precalcification procedure. Therefore, it is much easier and faster for the Ca–P coating fabrication on titanium implants with cooperation of the anodic oxidation and precalcification procedure than that with only an anodic oxidation.