Growth Of Calcium Phosphate Research Articles

Biomimetic Coating on Porous Alumina for Tissue Engineering: Characterisation by Cell Culture and Confocal Microscopy

In this study porous alumina samples were prepared and then coated using the biomimetic coating technique using a five times Simulated Body Fluid (5.0SBF) as the growth solution. A coating was achieved after pre-treatment with concentrated acid. From elemental analysis, the coating contained calcium and phosphorous, but also sodium and chlorine. Halite was identified by XRD, a sodium chloride phase. Sintering was done to remove the halite phase. Once halite was burnt off, the calcium phosphate crystals were not covered with halite and, therefore, the apatite phases can be clearly observed. Cell culturing showed sufficient cell attachment to the less porous alumina, Sample B, that has more calcium phosphate growth, while the porous alumina, Sample A, with minimal calcium phosphate growth attained very little cell attachment. This is likely due to the contribution that calcium phosphate plays in the attachment of bone-like cells to a bioinert ceramic such as alumina. These results were repeated on both SEM and confocal microscopy analysis. Confocal microscopy was a novel characterisation approach which gave useful information and was a visual aid.

Effect of organic acids on calcium phosphate nucleation and osteogenic differentiation of human mesenchymal stem cells on peptide functionalized nanofibers.

Carboxylate-rich organic acids play an important role in controlling the growth of apatite crystals and the extent of mineralization in the natural bone. The objective of this work was to investigate the effect of organic acids on calcium phosphate (CaP) nucleation on nanofiber microsheets functionalized with a glutamic acid peptide and osteogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on the CaP-nucleated microsheets. High molecular weight poly(dl-lactide) (DL-PLA) was mixed with low molecular weight L-PLA conjugated with Glu-Glu-Gly-Gly-Cys peptide, and the mixture was electrospun to generate aligned nanofiber microsheets. The nanofiber microsheets were incubated in a modified simulated body fluid (mSBF) supplemented with different organic acids for nucleation and growth of CaP crystals on the nanofibers. Organic acids included citric acid (CA), hydroxycitric acid (HCA), tartaric acid (TART), malic acid (MA), ascorbic acid (AsA), and salicylic acid (SalA). HCA microsheets had the highest CaP content at 240 ± 10% followed by TART and CA with 225 ± 8% and 225 ± 10%, respectively. The Ca/P ratio and percent crystallinity of the nucleated CaP in TART microsheets was closest to that of stoichiometric hydroxyapatite. The extent of CaP nucleation and growth on the nanofiber microsheets depended on the acidic strength and number of hydrogen-bonding hydroxyl groups of the organic acids. Compressive modulus and degradation of the CaP nucleated microsheets were related to percent crystallinity and CaP content. Osteogenic differentiation of hMSCs seeded on the microsheets and cultured in osteogenic medium increased only for those microsheets nucleated with CaP by incubation in CA or AsA-supplemented mSBF. Further, only CA microsheets stimulated bone nodule formation by the seeded hMSCs.

Biomineralized biomimetic organic/inorganic hybrid hydrogels based on hyaluronic acid and poloxamer

A biomineralized hydrogel system containing hyaluronic acid (HA) and poloxamer composed of a poly(ethylene oxide)/poly(propylene oxide)/poly(ethylene oxide) (PEO–PPO–PEO) block copolymer was developed as a biomimetic thermo-responsive injectable hydrogel system for bone regeneration. Using HA and poloxamer macromers with polymerizable residues, organic/inorganic HA/poloxamer hydrogels with various compositions were prepared and subjected to a biomineralization process to mimic the bone extracellular matrix. An increase in HA content within the hydrogels enhanced intermolecular chelation with calcium ions, leading to an increase in nucleation and growth of calcium phosphate in the hydrogels. After the biomineralization procedure, a crystalline formation was observed within and on the surface of the hydrogel. All of the HA/poloxamer hydrogel samples exhibited relatively high water content of greater than 90% at 25°C, and the water content was influenced by the HA/poloxamer composition, biomineralization, and temperature. In particular, the HA/poloxamer hydrogel was injectable through a syringe without demonstrating appreciable macroscopic fracture at room temperature, whereas it was more opaque and adopted a more rigid structure as the temperature increased because of the increasing hydrophobicity of poloxamer. The enzymatic degradation behavior of the hydrogels depended on the concentration of hyaluronidase, HA/poloxamer composition, and biomineralization. The release kinetics of model drugs from HA/poloxamer hydrogels was primarily dependent on the drug loading content, water content, biomineralization of the hydrogels, and ionic properties of the drug. These results indicate that biomineralized HA/poloxamer hydrogel is a promising candidate material for a biomimetic hydrogel system that promotes bone tissue repair and regeneration via local delivery of drugs.

Morphological, chemical and kinetic characterisation of zein protein-induced biomimetic calcium phosphate films.

This study examines zein protein-induced growth of calcium phosphate (CaP) thin films at air-liquid interfaces. The results demonstrate that zein protein films in contact with simulated body fluids (SBF) promote the growth of hemispherical CaP particles of 150 nm radius, which combine to form a film. The CaP films formed on zein were less continuous than those obtained using a hexadecanoic acid monolayer self-assembled at the air-liquid interface. In situ ellipsometry measurements were used to follow the nucleation and growth of CaP in these systems. The kinetic parameters extracted from the ellipsometry data were dependent on the organic layer and also the SBF concentration. The mineralisation process was slower and final CaP film thicknesses smaller when zein films were used. XPS analyses revealed the presence of zein protein only on the air-facing side of the CaP films, confirming that the zein protein is involved in nucleating the unusual hemispherical CaP morphology. The zein protein-induced CaP thin films showed iridescence with a distinct range of colours as a function of CaP film thickness. Templated CaP mineralisation with zein protein is a simple and chemically facile method for synthesising CaP films with novel morphologies and controllable thicknesses.

An in situ synthesis of mesoporous SBA-16/hydroxyapatite for ciprofloxacin release: in vitro stability and cytocompatibility studies.

The present work developed a biomaterial (HA/SBA-16) based on the growth of calcium phosphate (HA) particles within an organized silica structure (SBA-16) to evaluate its application as a drug delivery system. The samples were charged with ciprofloxacin as a model drug and in vitro release assays were carried out. The samples were characterized by elemental analysis (CHN), Fourier transform infrared spectroscopy, nitrogen adsorption, scanning electron microscopy (SEM), transmission electron microscopy (TEM), small angle X-ray scattering (SAXS) and X-ray diffraction. The results obtained by TEM, SEM and SAXS reveal a well-defined cubic arrangement of a uniform spherical mesoporous structure, an intrinsic characteristic of these materials, which indicated that SBA-16 and HA/SBA-16 could potentially encapsulate bioactive molecules by means of ordered mesopores. It was found that both surface interaction and pore volume affect the rate and amount of ciprofloxacin released from the mesoporous materials. In vitro assays were performed to evaluate the adhesion, viability, and growth behavior of human adipose tissue-derived stem cells (hADSC) on SBA-16 and HA/SBA-16 nanocomposites to verify their potential as a scaffold for application in bone-tissue engineering using MTT assay and alkaline phosphatase activity tests. The results showed that the materials are promising systems for bone repair, providing a good environment for the adhesion and proliferation of rat mesenchymal stem cells and hADSC in vitro.

A novel self-assembled oligopeptide amphiphile for biomimetic mineralization of enamel.

BackgroundResearchers are looking for biomimetic mineralization of ena/mel to manage dental erosion. This study evaluated biomimetic mineralization of demineralized enamel induced by a synthetic and self-assembled oligopeptide amphiphile (OPA).ResultsThe results showed that the OPA self-assembled into nano-fibres in the presence of calcium ions and in neutral acidity. The OPA was alternately immersed in calcium chloride and sodium hypophosphate solutions to evaluate its property of mineralization. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) showed nucleation and growth of amorphous calcium phosphate along the self-assembled OPA nano-fibres when it was repetitively exposed to solutions with calcium and phosphate ions. Energy dispersive spectrometry (EDS) confirmed that these nano-particles contained calcium and phosphate. Furthermore, electron diffraction pattern suggested that the nano-particles precipitated on OPA nano-fibres were comparable to amorphous calcium phosphate. Acid-etched human enamel slices were incubated at 37°C in metastable calcium phosphate solution with the OPA for biomimetic mineralization. SEM and X-ray diffraction indicated that the OPA induced the formation of hydroxyapatite crystals in organized bundles on etched enamel. TEM micrographs revealed there were 20–30 nm nano-amorphous calcium phosphate precipitates in the biomimetic mineralizing solution. The particles were found separately bound to the oligopeptide fibres. Biomimetic mineralization with or without the oligopeptide increased demineralized enamel microhardness.ConclusionsA novel OPA was successfully fabricated, which fostered the biomimetic mineralization of demineralized enamel. It is one of the primary steps towards the design and construction of novel biomaterial for future clinical therapy of dental erosion.

Chitosan-coated electrospun PLA fibers for rapid mineralization of calcium phosphate

In this work, hydroxyapatite (HA) mineralized on chitosan (CS)-coated poly(lactic acid) (PLA) nanofiber mat was prepared and compared in terms of mineralization characteristics. Significant calcium phosphate crystals formed on various concentrations of CS-coated PLA fiber mat with better uniformity after 2h of incubation in 10 times simulated body fluid (10× SBF). X-ray diffraction results further indicated that the composition of the deposited mineral was a mixture of dicalcium phosphate dehydrates and apatite. Chitosan, a cationic polysaccharide, can promote more nucleation and growth of calcium phosphate under conditions of 0.4% chitosan concentrations. These results indicated that HA-mineralized on CS-coated PLA fiber mat can be prepared directly via simply using CS coating followed by SBF immersion, and the results also suggest that this composite can mimic structural, compositional, and biological functions of native bone and can serve as a good candidate for bone tissue engineering (BTE).

Rapid Biomimetic Mineralization of Hydroxyapatite-g-PDLLA Hybrid Microspheres

Hydroxyapatite-graft-poly(D,L-lactide) (HA-g-PDLLA) nanoparticles were synthesized here to fabricate hybrid microspheres with diameter in the range of 150-200 μm by emulsion solvent evaporation techniques. The as-obtained microspheres were treated with alkaline solution in order to selectively degrade the PDLLA layer which covered on the surface of hybrid microspheres and instead to generate a dense coating of HA nanoparticles. The hybrid microspheres with enriched HA nanoparticles on the surface were further immersed in simulated body fluid (SBF) solution to evaluate the bone-forming ability of the bioactive hybrid microspheres via the in vitro biomimetic mineralization process. The resultant microspheres were analyzed by using X-ray photoelectron spectroscopy (XPS), X-ray powder diffraction (XRD), scanning electron microscopy (SEM), and thermogravimetric analysis (TGA) to understand the nucleation and growth of bioactive calcium phosphate (Ca-P) crystals as a function of surface treatment. Results in this work clearly demonstrated that the existing HA nanoparticles on the surface of hybrid microspheres after alkaline treatment greatly affect the growth of the bone-like Ca-P crystals in SBF solutions. The biomimetic hybrid microspheres were found to be excellent candidates for use as injectable scaffolds for bone tissue engineering.

Growth of Calcium Phosphate Coating on Ti-7.5Mo Alloy after Anodic Oxidation

Titanium and its alloys are widely used as biomaterials due to their mechanical, chemical and biological properties. To enhance the biocompatibility of titanium alloys, various surface treatments have been proposed. In particular, the formation of titanium oxide nanotubes layers has been extensively examined. Among the various materials for implants, calcium phosphates and hydroxyapatite are widely used clinically. In this work, titanium nanotubes were fabricated on the surface of Ti-7.5Mo alloy by anodization. The samples were anodized for 20 V in an electrolyte containing glycerol in combination with ammonium fluoride (NH4F, 0.25%), and the anodization time was 24 h. After being anodized, specimens were heat treated at 450 °C and 600°C for 1 h to crystallize the amorphous TiO2 nanotubes and then treated with NaOH solution to make them bioactive, to induce growth of calcium phosphate in a simulated body fluid. Surface morphology and coating chemistry were obtained respectively using, field-emission scanning electron microscopy (FEG-SEM), AFM and X-ray diffraction (XRD). It was shown that the presence of titanium nanotubes induces the growth of a sodium titanate nanolayer. During the subsequent in-vitro immersion in a simulated body fluid, the sodium titanate nanolayer induced the nucleation and growth of nanodimensioned calcium phosphate. It was possible to observe the formation of TiO2 nanotubes on the surface of Ti-7.5Mo. Calcium phosphate coating was greater in the samples with larger nanotube diameter. These findings represent a simple surface treatment for Ti-7.5Mo alloy that has high potential for biomedical applications.

Surface characteristics and biological response of titanium oxide layer formed via micro-arc oxidation in K3PO4 and Na3PO4 electrolytes

Abstract The present work demonstrated the influence of phosphate electrolytes on surface characteristics and biological response of the titanium oxide layer formed via micro-arc oxidation coating. For this purpose, the present coatings were carried out on titanium samples using two different electrolytes containing Na3PO4 and K3PO4. The surface roughness of the oxide layer formed in K3PO4 electrolyte was higher than that in the Na3PO4 electrolyte. This was attributed to the higher fraction of the micropores in the oxide layer prepared by the K3PO4 electrolyte than that by the Na3PO4 electrolyte. From the results of simulated body fluid test, the precipitation and growth of amorphous calcium phosphate on the oxide layer formed in the K3PO4 electrolyte was superior to that in the Na3PO4 electrolyte due to the enhanced surface roughness. In addition, in vitro cell adhesion was triggered in case of the oxide layer formed in the K3PO4 electrolyte.

Incorporation of tyrosine phosphate into tetraphenylethylene affords an amphiphilic molecule for alkaline phosphatase detection, hydrogelation and calcium mineralization.

Simple conjugation of tyrosine-phosphate with tetraphenylethylene generates a new amphiphile, which not only undergoes enzymatic dephosphorylation to generate a relative hydrophobic residue for alkaline phosphatase (ALP) detection with significant enhancement of the fluorescence signals, but also self-assembles in water to result in a novel supramolecular hydrogel with gelation-enhanced fluorescence emission features upon the changes of pH. In addition, the highly ordered micelle nanostructures self-assembled from this amphiphile exhibit the ability to serve as efficient templates to promote the nucleation and growth of calcium phosphate. Since amino acids and peptides are an important class of bioactive entities whose functions range from biomolecular recognition to supramolecular self-assembly, this study demonstrates the potential to generate an amphiphile with a novel molecular architecture from the TPE and amino acid conjugate with multifunctional properties.

Biodegradable polymer-lipid monolayers as templates for calcium phosphate mineralization.

A combination of poly([R]-3-hydroxy-10-undecenoate) (PHUE), a biodegradable polymer from the group of polyhydroxyalkanoates (PHAs), and lipids of different head groups was used to support the growth of calcium phosphate, the main component of mammalian bones. Crystallization took place under two-dimensional films (Langmuir monolayers). The addition of a negatively charged lipid, 1,2-dioleoyl-sn-glycero-3-phospho-l-serine, to a PHUE film led to the formation of lipid domains (rich in negative charge), and resulted in excellent mineralization control: crystals with uniform size and morphology were formed. The results show that carefully optimized combinations of materials can lead to better control of calcium phosphate crystallization compared to one-component organic scaffolds.

Laser-Assisted Biomimetic Process for Calcium Phosphate Coating on a Hydroxyapatite Ceramic

The present authors recently developed a new calcium phosphate (CaP) coating technique on an ethylene-vinyl alcohol copolymer substrate utilizing a laser-assisted biomimetic (LAB) process. In the present study, the LAB process was applied to a sintered hydroxyapatite (sHA) substrate for CaP coating. The LAB process was carried out by irradiating the sHA substrate immersed in a supersaturated CaP solution with a low-energy Nd-YAG pulsed laser. Within 30 min of irradiation, contiuous CaP layers with different morphologies were successfully formed on the laser-irradiated sHA surface. A submicron cavernous structure of the CaP layer was developed into a micron flake-like structure as the laser power increased from 1 to 3 W. This result suggests that the secondary nucleation and growth of CaP crystals were accelerated by laser irradiation in a power-dependent manner. Laser absorption by the sHA substrate and the resulting increase in ambient temperature locally near the surface should be responsible for the accelerated CaP nucleation and growth. The present CaP coating technique using the LAB process is simple and quick, hence it would be useful in orthopedic and dental applications as an on-demand surface-functionalization method for biomaterials consisting of sHA.

Growth of calcium phosphates on magnesium substrates for corrosion control in biomedical applications via immersion techniques

Magnesium (Mg) has been suggested as a revolutionary biodegradable replacement for current permanent metals used in orthopedic applications. Current investigations concentrate on the control of the corrosion rate to match bone healing. Calcium phosphate coatings have been a recent focus of these investigations through various coating protocols. Within this investigation, an in situ crystallization technique was utilized as an inexpensive and relatively simple method to produce a brushite and monetite coating on pure Mg. Coatings were characterized using energy dispersive spectroscopy, glancing angle X-ray diffraction and field emission scanning electron microscopy. Corrosion protection properties of the coatings were assessed in physiological buffers, Earles balanced salt solution, minimum essential media, and minimum essential media containing serum albumin, over a 4-week period. Using this novel coating protocol, our findings indicate brushite and monetite coated Mg to have significant corrosive protective effects when compared with its uncoated counterpart whilst maintaining high coating substrate adhesion, homogeneity, and reproducibility.

Preparation of pH-sensitive CaP nanoparticles coated with a phosphate-based block copolymer for efficient gene delivery

We have synthesized a phosphate-based block copolymer, PEG-b-PMOEP (poly(ethylene glycol)-b-poly(2-methacryloyloxyethyl phosphate)), with a narrow molecular weight distribution (PD = 1.06) by atomic transfer radical polymerization (ATRP), and have constructed calcium phosphate nanoparticles (CaPNs) coated with the block copolymer as an efficient and safe intracellular gene delivery carrier. The phosphate-mimic PMOEP block could be incorporated into the calcium phosphate (CaP) core to entrap pDNA, with the PEG block forming a shell to prevent uncontrolled growth of CaP precipitates and aggregates in physiological fluids. The CaPNs showed high colloidal stability at pH 7.4, but released entrapped pDNA at an endosomal pH of 5.0 through a pH-dependent protonation of phosphate moieties for efficient endosomal escape. The PEG-b-PMOEP/CaP/pDNA nanoparticles, which were formed simply by mixing, exhibited great potential as gene delivery carriers for future gene therapy applications due to their high transfection efficiency, low toxicity, and good stability under physiological conditions.

Aortic Valve-derived Calcifyng Nanoparticles: No Evidence of Life

Introduction and objectivesCalcifying nanoparticles, also known as “nanobacteria,” are very small bacteria-like structures (0.1-0.5μm) with the ability to facilitate the precipitation and growth of calcium phosphate in pathological conditions and have been associated with aortic valve calcification. The status of nanobacteria is controversial; some have proposed that they are a new class of living organism while others describe calcifying nanoparticles as mineralo-fetuin complexes. The objective of the present study is to elucidate if calcifying nanoparticles are living entities, based on whether or not they have metabolic activity, a characteristic of life, irrespective of their composition. MethodsCalcifying nanoparticles were grown from 6 different valves randomly chosen among 84 consecutively explanted aortic valves, as described in the literature. The 1H-NMR spectra were acquired from calcifying nanoparticles culture media to assess metabolic changes and the presence of 16sRNA in the culture media was investigated by real-time polymerase chain reaction. ResultsAfter 6 weeks in culture, calcifying nanoparticles could be seen clearly attached to the surface of culture flasks. All samples were negative for 16sRNA, discarding the presence of known bacteria. 1H-NMR spectra showed no difference between calcifying nanoparticles and 6-week-old sterile culture media maintained under the same conditions. ConclusionsOur results show that calcifying nanoparticles cannot be considered as living organisms.

Nanopartículas derivadas de válvula aórtica: sin evidencias de vida

Introduction and objectivesCalcifying nanoparticles, also known as “nanobacteria,” are very small bacteria-like structures (0.1-0.5μm) with the ability to facilitate the precipitation and growth of calcium phosphate in pathological conditions and have been associated with aortic valve calcification. The status of nanobacteria is controversial; some have proposed that they are a new class of living organism while others describe calcifying nanoparticles as mineralo-fetuin complexes. The objective of the present study is to elucidate if calcifying nanoparticles are living entities, based on whether or not they have metabolic activity, a characteristic of life, irrespective of their composition. MethodsCalcifying nanoparticles were grown from 6 different valves randomly chosen among 84 consecutively explanted aortic valves, as described in the literature. The 1H-NMR spectra were acquired from calcifying nanoparticles culture media to assess metabolic changes and the presence of 16sRNA in the culture media was investigated by real-time polymerase chain reaction. ResultsAfter 6 weeks in culture, calcifying nanoparticles could be seen clearly attached to the surface of culture flasks. All samples were negative for 16sRNA, discarding the presence of known bacteria. 1H-NMR spectra showed no difference between calcifying nanoparticles and 6-week-old sterile culture media maintained under the same conditions. ConclusionsOur results show that calcifying nanoparticles cannot be considered as living organisms.Full English text available from:www.revespcardiol.org

Controlled growth of calcium phosphate using phosphatidylcholine-modified porous titania as reaction compartments

Phosphatidylcholine-modified porous titania was employed as reaction compartments for the controlled growth of calcium phosphate. The process of calcium phosphate precipitation was investigated in detail using in situ quartz crystal microbalance (QCM), scanning electron microscopy (SEM) and X-ray diffraction (XRD) techniques. The obtained results showed that the nucleation and growth of calcium phosphate could take place within porous titania modified by neutral phosphatidylcholine in simulated body fluid (SBF), and that two different stages were present in the process of calcium phosphate precipitation, indicating two different kinetic processes. At the nucleation stage, the Ca2+ ions in SBF solutions bound firstly into the phosphatidylcholine-modified porous titania, and then the free PO43− ions in SBF solutions were attracted to the reaction compartments, causing the formation of calcium phosphate nuclei. With the increase of immersion time, a large amount of Ca2+ and PO43− ions in supersaturated SBF solutions deposited spontaneously into the phosphatidylcholine-modified porous titania to form calcium phosphate precipitation. In terms of the in situ frequency shifts, the reaction rate constants of calcium phosphate (K1 and K2) were estimated, respectively, at two different stages, and the results were (6.20±0.31)×10−4s−1 and (1.25±0.13)×10−5s−1, respectively, in 2.0 SBF solutions, indicating that the reaction rate at the nucleation stage was higher than that at the growth stage, and that the growth stage was the rate-limiting step.

Calcium phosphate growth at electropolished titanium surfaces.

This work investigated the ability of electropolished Ti surface to induce Hydroxyapatite (HA) nucleation and growth in vitro via a biomimetic method in Simulated Body Fluid (SBF). The HA induction ability of Ti surface upon electropolishing was compared to that of Ti substrates modified with common chemical methods including alkali, acidic and hydrogen peroxide treatments. Our results revealed the excellent ability of electropolished Ti surfaces in inducing the formation of bone-like HA at the Ti/SBF interface. The chemical composition, crystallinity and thickness of the HA coating obtained on the electropolished Ti surface was found to be comparable to that achieved on the surface of alkali treated Ti substrate, one of the most effective and popular chemical treatments. The surface characteristics of electropolished Ti contributing to HA growth were discussed thoroughly.

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.