Formation of macropores in calcium phosphate cement implants

A calcium phosphate cement (CPC) was shown to harden at ambient temperatures and form hydroxyapatite as the only end-product. Animal study results showed that CPC resorbed slowly and was replaced by new bone. For some clinical applications, it would be desirable to have macropores built into the CPC implant to obtain a more rapid resorption and concomitant osseointegration of the implant. The present study investigated the feasibility of a new method for producing macropores in CPC. Sucrose granules, NaHCO₃, and Na₂HPO₄ were sieved to obtain particle sizes in the range of 125 m to 250 m. The following mixtures of CPC powder (an equimolar mixture of tetracalcium phosphate, Ca₄(PO_{4 2}O, and dicalcium phosphate anhydrous, CaHPO₄) and one of the above additive granules were prepared: control–no additive; mixture A–0.25 mass fraction of sucrose; mixture B–0.25 mass fraction of NaHCO₃; mixture C–0.25 mass fraction of Na₂HPO₄, and mixture D–0.33 mass fraction of Na₂HPO₄. Cement samples were prepared by mixing 0.3 g of the above mixtures with 0.075 ml of the cement liquid (1 mol/l Na₂HPO₄). After hardening, the specimens were placed in water for 20 h at about 60 °C to completely dissolve the additive crystals. Well-formed macropores in the shapes of the entrapped crystals were observed by scanning electron microscope (SEM). The macroporosities (mean±standard deviation; n = 6) expressed as volume fraction in % were 0, 18.9 ± 1.7, 26.9 ± 1.6, 38.3 ± 4.4 and 50.3 ± 2.7 for the control, A, B, C and D, respectively. The diametral tensile strengths (mean±standard deviation; n = 3) expressed in MPa were 10.1 ± 0.7, 3.7 ± 0.3, 2.4 ± 0.2, 1.5 ± 0.5 and 0.4 ± 0.1, respectively, for the five groups. The results showed that macropores can readily be formed in CPC implants with the use of water-soluble crystals. The mechanical strength of CPC decreased with increasing macroporosity. © 2001 Kluwer Academic Publishers     

Formation of monetite nanoparticles and nanofibers in reverse micelles

Reverse micelles solution of water and cyclohexane containing either cetyltrimethylammonium bromide (CTAB) or polyoxyethylene-8-dodecyl ether (C₁₂E₈) surfactants and n-pentanol as co-surfactant have been used as organized reaction microenvironments for monetite (dicalcium phosphate anhydrous, DCPA) precipitation. Well-crystallized monetite nanoparticles with various morphologies such as spheres, nanofibers and bundles of nanowires were obtained in CTAB reverse micelles solution. The molar ratio of water and surfactant (W _o) and the molar ratio of co-surfactant and surfactant (P _o) have great influence on the structure and morphology of the final products. A generalized mechanism for the growth of monetite in reverse micelles is proposed, in which the interaction between the surfactant molecules and PO₄³⁻ ions leads to the formation of a surfactant/CaHPO₄ complex. It is because of this central complex that the further fusion with reactant ions containing reverse micelles will occur only in one direction. Changing the content of water and co-surfactant has great influence on the morphology of reverse micelles and on the interaction between the surfactant/CaHPO₄ complex leading to a fine tuning of the morphology of products. By contrast, lacking of this interaction in the C₁₂E₈ system only tablet amorphous calcium phosphate can be formed.     

Positively charged calcium phosphate/polymer nanoparticles for photodynamic therapy

The charge of nanoparticles influences their ability to pass through the cellular membrane, and a positive charge should be beneficial. The negative charge of calcium phosphate nanoparticles with an inner shell of carboxymethyl cellulose (CMC) was reversed by adding an outer shell of poly(ethyleneimine) (PEI) into which the photoactive dye 5,10,15,20-tetrakis(3-hydroxyphenyl)-porphyrin (mTHPP) was loaded. The aqueous dispersion of the nanoparticles was used for photodynamic therapy with HT29 cells (human colon adenocarcinoma cells), HIG-82 cells (rabbit synoviocytes), and J774A.1 cells (murine macrophages). A high photodynamic activity (killing) together with a very low dark toxicity was observed for HIG-82 and for J774.1 cells at 2 μM dye concentration. The killing efficiency was equivalent to the pure photoactive dye that, however, needs to be administered in alcoholic solution.     

Processing of nanocrystalline hydroxyapatite particles via reverse microemulsions

Nanocrystalline hydroxyapatite (HAP) particles were synthesized at room temperature using reverse microemulsions, in which cyclohexane was used as the organic phase, mixed surfactant with TX-100 and 1- pentanol, and CaCl₂ solution as aqueous phase. The reactor systems with aqueous/organic volumetric ratios 1:10, 1:5, 2:5, and 1:2 were carefully selected for the microemulsion processing by the pseudo-ternary phase diagram and the electric conductivity measurement of the emulsion. The as-obtained HAP nanoparticles with carbonate substitution and broadening X-ray diffraction (XRD) traces were similar to the fine powder of human bone, despite of the aqueous/organic volumetric ratio in the emulsion. No obvious other’s phase occurred after as-obtained particles calcined under different temperature till 700 °C. In the emulsion-derived precursors, the HAP particles based on spherical morphology were prepared into the size between 15 ∼ 30 nm as a low volumetric ratio of 1:10 or 1:5 was applied. As the volumetric ratio increased to 2:5, the HAP particles with rod-like shape of (140∼280) × (10∼80) nm were formed. Practical implication of the results is that the nanocrystalline bone-like hydroxyapatite can be obtained via the emulsion processing at room temperature without further calcinations.     

Formation mechanism of calcium phosphate coating on micro-arc oxidized magnesium

A calcium phosphate coating was prepared on the surface of micro-arc oxidized magnesium by a chemical method. The microstructure evolution of the coating was characterized by X-ray diffraction, Fourier-transformed infrared spectroscopy and scanning electron microscopy. The results showed that Ca₁₀(PO₄)₆(OH)₂ (HA) was firstly formed on the surface of micro-arc oxidized magnesium, followed by flake-like CaHPO₄·2H₂O (DCPD). The solution pH and Ca²⁺ concentration had intense influence on the formation of calcium phosphate coating. Acidic Ca²⁺ enrichment solution was favourable for HA formation on the surface of micro-arc oxidized magnesium. High concentration of HPO₄²⁻ and low concentration of Ca²⁺ in acidic solution improved the formation of DCPD coating.     

Increased osteoblast density in the presence of novel calcium phosphate coated magnetic nanoparticles

Bone diseases (including osteoporosis, osteoarthritis and bone cancer) are of great concern to the medical world. Drugs are available to treat such diseases, but often these drugs are not specifically targeted to the site of the disease and, thus, lack an immediate directed therapeutic effect. The optimal drug delivery system should enhance healthy bone growth with high specificity to the site of bone disease. It has been previously shown that magnetic nanoparticles can be directed in the presence of a magnetic field to any part of the body, allowing for site-specific drug delivery and possibly an immediate increase in bone density. The objective of the present study was to build off of this evidence and determine the density of osteoblasts (bone forming cells) in the presence of various uncoated and coated magnetic nanoparticles that could eventually be used in drug delivery applications. Results showed that some magnetic nanoparticles (specifically, γ-Fe(2)O(3)) significantly promoted osteoblast density (that is, cells per well) after 5 and 8 days of culture compared to controls (no particles). These magnetic nanoparticles were further coated with calcium phosphate (CaP; the main inorganic component of bone) to tailor them for treating various bone diseases. The coatings were conducted in the presence of either bovine serum albumin (BSA) or citric acid (CA) to reduce magnetic nanoparticle agglomeration, a common problem resulting from the use of nanoparticles which decreases their effectiveness. Results with these coatings showed that magnetic nanoparticles, specifically (γ-Fe(2)O(3)), coated in the presence of BSA significantly increased osteoblast density compared to controls after 1 day. In this manner, this study provided unexpected evidence that CaP-coated γ-Fe(2)O(3) magnetic nanoparticles increased osteoblast density (compared to no particles) and, thus, should be further studied to treat numerous bone diseases.     

Formation of carbonate apatite on calcium phosphate coatings containing silver ions

The prerequisite for bioactive materials to bond to living bone is the formation of biologically equivalent carbonate apatite on their surfaces in the body. Calcium phosphate ceramic surfaces can be transformed to a biological apatite through a series of surface reactions including dissolution–precipitation and ion exchange. In the present work, apatite coatings with different crystallinity, compositions and crystal sizes, including a well-crystallized hydroxyapatite coating, were synthesized electrochemically and doped with silver ions in silver nitrate solution at room temperature. The formation of a new carbonate apatite on the surface of these coatings was investigated in an acellular simulated body fluid with ion concentrations comparable with those of human blood plasma, using scanning electron microscopy and Fourier transform-infrared spectroscopy. The results show that small quantities of silver ions incorporated into apatite coatings may have a strong stimulatory effect on the formation of carbonate apatite without adversely affecting the chemical stability of these coatings.     

Modification of porous calcium phosphate surfaces with different geometries of bioactive glass nanoparticles

In this study, the effects of bioactive glass nanoparticles' (nBGs) size and shape incorporated into hydroxyapatite/β-tricalcium phosphate (BCP) scaffolds were investigated. We prepared a highly porous (> 85%) BCP scaffold and coated its surface with a nanocomposite layer consisted of polycaprolactone (PCL) and rod (~ 153 nm in height and ~ 29 nm in width) or spherical (~ 33 nm and 64 nm in diameter) nBGs. Osteogenic gene expression by primary human osteoblast-like cells (HOB) was investigated using quantitative real time polymerase chain reaction (q-RT-PCR). We demonstrated for the first time that in vitro osteogenesis is dramatically affected by the shape of the nBGs, whereby rod shaped nBGs showed the most significant osteogenic induction, compared to spherical particles (regardless of their size). Importantly, the good biological effect observed for the rod shaped nBGs was coupled by a marked increase in the modulus (~ 48 MPa), compressive strength (~ 1 MPa) and failure strain (~ 6%), compared to those for the BCP scaffolds (~ 4 MPa, ~ 1 MPa and ~ 0.5% respectively). The findings of this study demonstrated that the shape of the nBGs is of significant importance when considering bone regeneration.     

微乳液法制备纳米羟基磷灰石的机理

采用二已基琥珀酰磺酸钠(aerosol OT,AOT)作为表面活性剂,正辛醇作为助表面活性剂,异辛烷为油相,将Ca(H2PO4)2·H2O水溶液和Ca(OH)2饱和溶液作为水相制成反相微乳液,并使两种微乳液反应,制备出呈单分散的球形纳米羟基磷灰石颗粒.通过系统性试验绘制了AOT/异辛烷/Ca(H2PO4)2·H2O水溶液和AOT/异辛烷/Ca(OH)2饱和溶液微乳液的局部三元拟相图,确定了该体系的反相微乳液单相区.采用动态激光散射原理分析了上述微乳液的胶束直径,确定了胶束直径与体系含水量的线性关系,并计算了微乳液胶束的有关参数.分析了羟基磷灰石的反应机理和颗粒的形成过程,并采用紫外可见光吸收光谱,研究了体系含水量对羟基磷灰石超细颗粒形成过程的影响.     

Effect of strontium ions substitution on gene delivery related properties of calcium phosphate nanoparticles

Gene therapy has been considered a strategy for delivery of therapeutic nucleic acids to a specific site. Calcium phosphates are one gene delivery vector group of interest. However, low transfection efficiency has limited the use of calcium phosphate in gene delivery applications. Present work aims at studying the fabrication of strontium substituted calcium phosphate nanoparticles with improved gene delivery related properties. Strontium substituted calcium phosphate was prepared using a simple sol gel method. X-ray diffraction analysis, Fourier transform infrared spectroscopy, transmission electron microscopy, specific surface area analysis, zeta potential measurement and ion release evaluation were used to characterize the samples. This characterization showed strontium and carbonate co-substituted calcium phosphate which resulted in nano size particles with low crystallinity, high specific surface area, positive surface charge, and a high dissolution rate. These improved properties could increase the DNA concentration on the vector as well as the endosomal escape of the complex that leads to higher transfection efficiency of this novel gene delivery vector.     

Phase transformation of calcium phenyl phosphate in calcium hydroxyapatite

Calcium phenyl phosphate (CaPP) was synthesized from a mixture of Ca(OH)₂ and phenyl phosphate (C₆H₅PO₄H₂) in an aqueous media. XRD pattern of CaPP exhibited five diffraction peaks at 2θ = 6.6, 13.3, 20.0, 26.8 and 33.7°. The d-spacing ratio of these peaks was ca. 1:1/2:1/3:1/4:1/5. The molar ratios of Ca/P and phenyl/P of CaPP were 1.0 and 0.92, respectively, and the chemical formula of the material was expressed as (C₆H₅PO₄)_0.92(HPO₄)_0.08Ca·1.3H₂O, similar to that of dicalcium phosphate dihydrate (CaHPO₄·2H₂O: DCPD). These results allowed us to infer that CaPP is composed of a multilayer alternating bilayer of phenyl groups of the phosphates and DCPD-like phase. The structure of the material was essentially not altered after aging at pH 9.0-11.0 and 85 °C in an aqueous media. While, after aging at pH ≤8.0, the diffraction peaks of CaPP were suddenly weakened and disappeared at pH 7.0. Besides, new peaks due to calcium hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂: Hap) appeared and their intensity was strengthened with decreasing the solution pH. TEM observation revealed that the Hap particles formed at pH 6.0 are fibrous with ca. 1.5 μm in length and ca. 0.2 μm in width. From these results, it is presumed that the layered CaPP was dissolved, hydrolyzed and reprecipitated to fibrous Hap particles at pH ≤8.0 and 85 °C in aqueous media. This phase transformation of CaPP in Hap resembled to the formation mechanism of Hap in animal organism.     

Design and development of bioinspired calcium phosphate nanoparticles of MTX: pharmacodynamic and pharmacokinetic evaluation

The aim of this investigation is the management of rheumatoid arthritis (RA) by developing methotrexate-loaded calcium phosphate nanoparticles (MTX-CAP-NP) and to evaluate pharmacokinetic and pharmacodynamic behavior in adjuvant induced arthritis model. The nanoparticles were synthesized by wet precipitation method and optimized by Box-Behnken experimental design. MTX-CAP-NPs were characterized by TEM, FTIR, DSC and XRD studies. The particle size, zeta potential and entrapment efficiency of the optimized nanoparticles were found to be 204.90?±?64?nm, ?11.58?±?4.80?mV, and 88.33?±?3.74%, respectively. TEM, FTIR, DSC and XRD studies revealed that the developed nanoparticles were nearly spherical in shape and the crystalline structure of CAP-NP was not changed after MTX loading. The pharmacokinetic studies revealed that MTX-CAP-NP enhanced bioavailability of MTX by 2.6-fold when compared to marketed formulation (FOLITRAX-10). Under pharmacodynamic evaluation, arthritic assessment, radiography and histopathology studies revealed that CAP has ability to regenerate cartilage and bone therefore, together with MTX, MTX-CAP-NPs have shown significant reduction in disease progression. The overall work demonstrated that the developed nanodelivery system was well tolerated and more effective than the marketed formulation.     

Synthesis of magnetic nanoparticles in bicontinuous microemulsions. Effect of surfactant concentration

Precipitation was accomplished at 80 °C for magnetic nanoparticles in bicontinuous microemulsions that were stabilized with different concentrations of a surfactants mixture of dodecyltrimethylammonium bromide/didodecyldimethylammonium bromide (3/2, w/w). These nanoparticles were characterized by X-ray Diffraction, Scanning Transmission Electronic Microscopy (STEM), and Vibrating Sample Magnetometry (VSM), which demonstrated that they were composed of magnetite or a mixture of magnetite-maghemite. The particles were found to have average diameters between 6.9 and 7.9 nm with relatively narrow particle size distribution and showed possible superparamagnetic behavior. In addition, we observed an inverse dependence of particle size on surfactant concentration. Yields obtained in these precipitation reactions were found to be up to three times higher than those typically reported in specialized literature about precipitation of magnetic nanoparticles in reverse microemulsions.     

Controlled synthesis of calcium carbonate nanocrystals with multi-morphologies in different bicontinuous microemulsions

The nucleation and growth of calcium carbonate nanocrystals were studied in two types of bicontinuous microemulsions, consisted of P-octyl polyethylene glycol phenylether (OP)/n-amyl alcohol/cyclohexane/water, and the above microemulsion containing dl-aspartic acid (dl-Asp). The produced CaCO₃ nanocrystals were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectrometer (FT-IR) and X-ray diffraction (XRD). The results indicated that OP and dl-Asp used as soft template could control synthesis of CaCO₃ nanocrystals well. The various shapes of CaCO₃ nanocrystals, such as solid sphere, network, whisker, rod, and hollow sphere were successfully prepared by altering the concentration of the reactants, adding dl-Asp, and adjusting the pH values of dl-Asp/CaCl₂ aqueous solution. Sole calcite phase was obtained in OP bicontinuous microemulsion. The presence of dl-Asp was helpful for the formation of thermodynamically unstable vaterite phase. In OP/dl-Asp bicontinuous microemulsion, the higher pH value of dl-Asp/CaCl₂ aqueous solution is, the more vaterite will be formed. A nucleation-limited growth and limited aggregation (NGA) model was used to explain growth mechanism of the network-like calcium carbonate in this study.     

Dialysate calcium and calcium/phosphate balance in hemodialysis

The changing pattern of pharmaceutical use in dialysis patients has resulted in several alterations to dialysate calcium concentration over the past 40 years. Non‐calcium–containing phosphate binders and calcimimetics are the most recent examples of drugs that influence the overall calcium balance in dialysis patients. Renal osteodystrophy, vascular disease, and mortality are believed to be linked in patients with chronic kidney disease (CKD), although to date most of the evidence is based only on statistical associations. The precise pathophysiology of vascular calcification in end‐stage renal disease is unknown, but risk factors include age, hypertension, time on dialysis, and, most significantly, abnormalities in calcium and phosphate balance. Prospective studies are required before “cause and effect” can be established with certainty, but it is an active metabolic process with inhibitors and promoters. Serum calcium levels are clearly influenced by dialysate calcium and may therefore play an important role in influencing vascular calcification. Clinical management of hyperphosphatemia is being made easier by the introduction of potent non‐calcium–based oral phosphate binders such as lanthanum carbonate. Short‐term and long‐term studies have demonstrated its efficacy and safety. Vitamin D analogs have been a disappointment in the control of serum parathyroid hormone (PTH) levels, but evidence is emerging that vitamin D has other important metabolic effects apart from this, and may confer survival advantages to patients with CKD. Calcimimetics such as cinacalcet enable much more effective and precise control of PTH levels, but at the cost of a major financial burden. While it is unreasonable to expect that any one of these recent pharmacological developments will be a panacea, they provide researchers with the tools to begin to examine the complex interplay between calcium, phosphate, vitamin D, and PTH, such that further progress is fortunately inevitable.     

A novel method to prepare magnetic nanoparticles: precipitation in bicontinuous microemulsions

A novel method to prepare iron oxide nanoparticles by precipitation in bicontinuous microemulsions is reported. Precipitation reactions were carried out in microemulsions stabilized with a mixture of dodecyltrimethylammonium bromide/didodecyldimethylammonium bromide (3/2, w/w). Nanoparticles were characterized by X-ray diffraction, transmission electronic microscopy (TEM) and vibrating sample magnetometry (VSM), and consisted of magnetite or a mixture of magnetite and maghemite. The particles have an average diameter of 8 nm with a relatively narrow particle size distribution and show possible superparamagnetic behavior. Noticeably higher yields of precipitate are observed for this new approach in comparison with those typicall obtained using reverse microemulsions.     

Si complexes in calcium phosphate biomaterials

Silicon complexes in silicon doped calcium phosphate bioceramics have been studied using ²⁹Si magic angle spinning nuclear magnetic resonance spectroscopy with the objective of identifying the charge compensation mechanisms of silicon dopants. Three different materials have been studied: a multiphase material composed predominantly of a silicon stabilized α-tricalcium phosphate (α-TCP) phase plus a hydroxyapatite (HA) phase, a single phase Si-HA material and a single phase silicon stabilized α-TCP material. NMR results showed that in all three materials the silicon dopants formed Q¹ structures in which two silicate tetrahedra share an oxygen, creating an oxygen vacancy which compensated the substitution of two silicon for phosphorus. This finding may explain the phase evolution previously found where silicon stabilized α-TCP is found at low temperature after sintering.     

Formation of porous calcium phosphate films on partially stabilized zirconia substrates by the spray-pyrolysis technique

Porous calcium phosphate films could be formed on partially stabilized zirconia (3YZ) substrates by a spray-pyrolysis technique. The use of calcium metaphosphate as a binder was effective to enhance the binding strengths of these films to the substrates. The crystalline phase in the resulting films was mainly -calcium orthophosphate. This phase was thermally stabilized by solid solution with Y³⁺. The thickness of the film (30–150 m) was dependent upon the spraying time; the pore size was about 15 m. The films were still present on the substrate after Scotch tape (810) was adhered to the film side and then taken off from the substrate. The films prepared in this study were found to bind strongly to the substrate.     

Porous calcium phosphate ceramics prepared by coating polyurethane foams with calcium phosphate cements

Porous calcium phosphates have important biomedical applications such as bone defect fillers, tissue engineering scaffolds and drug delivery systems. While a number of methods to produce the porous calcium phosphate ceramics have been reported, this study aimed to develop a new fabrication method. The new method involved the use of polyurethane foams to produce highly porous calcium phosphate cements (CPCs). By firing the porous CPCs at 1200 °C, the polyurethane foams were burnt off and the CPCs prepared at room temperature were converted into sintered porous hydroxyapatite (HA)-based calcium phosphate ceramics. The sintered porous calcium phosphate ceramics could then be coated with a layer of the CPC at room temperature, resulting in high porosity, high pore interconnectivity and controlled pore size.