Facile synthesis,in vitro biocompatibility,and antibacterial activity of porous hollow hydroxyapatite microspheres

In this study, porous hollow hydroxyapatite (HAp) microspheres are prepared using chitosan microspheres as novel sacrificial templates and their microstructure, biocompatibility, and drug delivery properties are evaluated. Scanning electron microscope (SEM) observations show that HAp microspheres are spherical in morphology with a diameter of 100–300 μm and have a porous and core–shell structure. X-ray diffractometer patterns show that HAp microspheres consist of apatite phase. MTT assay indicates that HAp microspheres are biocompatible and have no significant cytotoxicity. SEM observations show that HAp microspheres support attachment and proliferation of osteoblast MC3T3-E1 cells. After being soaked in the solution of tetracycline hydrochloride (TH, model drug), HAp microspheres adsorb TH with an adsorption capacity of 47% to derive TH-loaded HAp microspheres. When exposed to two types of representative bacteria: Escherichia coli and Staphylococcus aureus, TH-loaded HAp microspheres maintain the biological activity of TH to inhibit the growth of bacteria.     

Preferential occupancy of metal ions in the hydroxyapatite solid solutions synthesized by hydrothermal method

The solid solutions in the systems of Ca-Cd HAp [Ca_10−xCd_xHAp (x = 0–10)], Ca-Sr HAp [Ca_10−xSr_xHAp (x = 0–10)] and Ca-Pb HAp [Ca_10−xPb_xHAp (x = 0–10)], were successfully synthesized at 200 °C for 12 h under hydrothermal conditions. The site of the metal ions in the solid solutions was analyzed by the Rietveld method. The results of the Rietveld analysis indicated that the metal ions of Pb²⁺, Sr²⁺, and Cd²⁺ all preferentially occupied M (2) sites in the apatite structure. The preferential occupancy of the metal ions in M (2) sites were explained mainly by their ionic radius and electronegativity.     

Fabrication of silver-doped apatite powders from silver-substituted octacalcium phosphate powders via solid–solid phase-conversion process

The excellent biocompatibility of apatite (hydroxyapatite, HAp; carbonate apatite, CO₃Ap) materials makes them suitable candidates as bone substitutes. However, they have no antibacterial ability. Meanwhile, silver (Ag) exhibits excellent antibacterial properties across a wide antibacterial spectrum. However, soluble Ag salts exhibit cytotoxicity and poor aesthetic properties. We dope Ag into an apatite unit lattice in order for the composite material to exhibit antibacterial contact abilities while simultaneously limiting the release of Ag⁺, which is the primary cause of the unwanted color changes and cytotoxicity. When a crystal structure in which silver ions are substituted for Ca in octacalcium phosphate (OCP) (Ag-OCP) is immersed in water and/or (NH₄)₂CO₃-containing solutions, Ag-OCP is converted into an apatite containing Ag via a solid–solid phase-transformation process. The Ag contents of the apatite and precursor Ag-OCP are the same. The CO₃ content of apatite samples depends on the (NH₄)₂CO₃ concentration of the treated solutions. A single-pot, single-step treatment enables the synthesis of both Ag-containing HAp and CO₃Ap. Further, these Ag-containing HAp and CO₃Ap samples show little color change from that of the precursor Ag-OCP.     

The effect of phosphate source on the sintering of carbonate substituted hydroxyapatite

Carbonate hydroxyapatite (CHAp) was synthesized by a wet precipitation method. In the synthesis, calcium oxide, H₃PO₄ and (NH₄)₂HPO₄ were applied as reactants. NH₄HCO₃ in the amount of 0.05–0.20 mol per 0.3 mol of H₃PO₄ or (NH₄)₂HPO₄ was used as the source of CO₃^2? groups. Sintering was carried out using powders and uniaxially pressed samples of carbonate and non-carbonate hydroxyapatites. The influence of synthesis conditions as well as sintering atmosphere on sinterability and phase composition of the final ceramic materials was evaluated. It was found that using properly synthesized CHAp powders and CO₂ atmosphere during heat-treatment it was possible to obtain dense CHAp ceramics with 1% of CaCO₃ as the secondary phase at the temperature 300–350 °C lower than that required for non-carbonate hydroxyapatite. Chemical stability and bioactive potential of CHAp–CaCO₃ ceramics were confirmed by in vitro tests.     

Fabrication of Hydroxyapatite Hollow Microspheres by the Composite‐Hydroxide Approach

Hydroxyapatite (HAp) hollow microspheres with hierarchically porous structure and nanorice‐like architecture units were synthesized by the composite‐hydroxide approach. NH₄H₂PO₄ was first reacted with hydroxides to form a hydroxide‐insoluble M₃PO₄ (the term M represents either Na or K). The diffusing M₃PO₄ at the liquid–solid interface then reacted with the Ca(OH)₂, forming insoluble HAp hollow spheres by Kirkendall process. Results show that when reaction time is increased from 3 to 12 h, the average diameter of the microspheres increased from 2.64 to 4.16 μm. In addition, the size homogeneity of the hollow microspheres was improved gradually due to Ostwald ripening. The prepared hollow microspheres were treated by ultrasound, and nanorice‐like component units with a large mass of mesopores (<10 nm) were displayed. These prepared HAp hollow microspheres might be expected to apply to ion adsorption and drug delivery.     

Modeling and optimization of combustion synthesis for hydroxyapatite production

In this study, the combustion synthesis of hydroxyapatite was studied and optimized under controlled experimental conditions. The hydroxyapatite content, crystallinity and crystallite size were monitored under changes in type of fuel, pH or red/ox ratio, muffle temperature, and reaction time. The products were characterized by X-ray diffraction, refinement of crystalline phases by the Rietveld method, scanning electron microscopy, and infrared spectroscopy with the Fourier transform. The decomposition of hydroxyapatite, which was the major phase, produced beta tricalcium phosphate in all samples, as indicated by their diffractograms. Infrared spectroscopy analysis revealed the presence of b-type carbonate groups in the structure; thus, the synthesized compound has the following chemical formula: Ca_10-x (PO₄)_6-x (CO₃)_x (OH)_2-x, where 0 < x < 2, which corresponds to carbonated hydroxyapatite (CHAp). The combustion process was modeled using a first-degree polynomial, and we found that the interaction between time and temperature to be the most influential parameter. Optimization indicated that processing conditions at 650 °C for 30 min, using urea at pH = 2 and ϕ_e = 1.134, produced the best result in terms of HAp composition, yielding 89.25% wt.     

Processing of hydroxyapatite and its composites using ceramic fused filament fabrication (CF3)

In this article, we report the fabrication of hydroxyapatite (HAp) and its composites with 7.75 vol% Si₃N₄ (HAp10SN) using ceramic fused filament fabrication (CF³). Homogeneous feedstock with 40 vol% ceramic powder was prepared and used to extrude filaments for further printing using a desktop printer. Our results showed that the addition of Si₃N₄ to HAp increases the feedstock viscosity. However, the filaments and CF³ parts made using HAp and HAp10SN feedstocks exhibited comparable densities without gross defects. We have obtained relatively smoother CF³ parts with HAp10SN than pure HAp, which is attributed to their high feedstock viscosity and formation of liquid phase during sintering. Sintering at 1250 °C for 4 h in air, after thermal debinding, resulted in a relative density of ~85% with HAp and tricalcium phosphate (TCP) as major constituents. Sintered HAp10SN samples also revealed almost 70% reduction in the grain size and 4-fold increase in the hardness compared to pure HAp. Our results indicate that the CF³ processed HAp10SN samples containing ~15% porosity, Si₃N₄ particles and Si-substituted HAp/TCP have strong potential as bone replacements.     

Protein release behavior from carbonate apatite hydrogel

Incorporation of protein in poorly crystalline carbonate apatite hydrogel, the protein release behavior from the cake and the dissolution of the cake in aqueous solution were studied. Poorly crystalline apatite was prepared by precipitation of saturated solution of calcium and phosphate in air or under N₂ atmosphere. The carbonate content and particle size of obtained hydrogel depends on the maturation period and atmospheric condition. The mixture of apatite hydrogel and protein was dried for 4 days in air at 40% of relative humidity to yield cakes. The quantity of loaded cytochrome c in apatite hydrogel formed in air was 0.490 wt.%, while that in apatite hydrogel formed under N₂ atmosphere was 0.305 wt.%. But the quantity of loaded albumin in apatite hydrogel formed in air was less than that in apatite hydrogel formed under N₂ atmosphere. For the cakes containing protein immersed in aqueous solution, the protein release was less than the decrease in weight of apatite cake, which may be associated with apatite recrystallization and protein re-incorporation. The quantity of released protein may depend on the quantity of loaded protein in apatite hydrogel.     

Improved properties of hydroxyapatite–carbon nanotube biocomposite: Mechanical,in vitro bioactivity and biological studies

The present work describes a simple shear mixing technique for developing a hydroxyapatite (HAp)–carbon nanotube (CNT) nanocomposite and the effect of reinforcement on the physical, mechanical, in vitro bioactivity and biological properties of HAp. XRD and FTIR confirmed that the main phase of the composites is HAp. HRTEM images demonstrated the formation of a two-dimensional nanocomposite structure, whereas FESEM images indicated the formation of nanosized HAp grains featuring sporadically distributed CNT molecules. No major phase changes in HAp were observed with up to 5% added CNT. However, adding more than 1% CNTs caused an increase in internal crystal strain and increased substitution of CO₃²⁻ for OH⁻ and PO₄³⁻ groups in pure HAp. The average crystallite size increased from ~46 nm to ~100 nm with only 0.5% added CNT, remained nearly unaffected up to 2% CNTs thereafter and suddenly decreased at 5% CNTs (~61 nm). The FESEM and HRTEM images clearly showed the attachment of MWCNT chains on HAp grains, which directly affected the samples' fracture toughness and flexural strength. Of the samples, 1% showed maximum values of K_1C, whereas 5% showed maximum values of HV and three-point bending flexural strength. The in vitro bioactivity indicated increased apatite formation on the sample surface up to 1% CNTs after 24 weeks. However, adding 2% and 5% CNTs resulted in a manifold increase in apatite formation up to 12 weeks, after which dissolution increased up to 24 weeks, possibly due to increased substitution of CO₃²⁻ for OH⁻ and PO₄³⁻ groups. This result is confirmed by the FTIR studies. For all added CNT contents, all samples exhibited high haemocompatibility. However, there was a compromise between the observed mechanical properties and in vitro bioactivity studied up to 24 weeks, and care must be taken before selecting any final application of the nanocomposites.     

Comparative study of surface properties of Mg-substituted hydroxyapatite bioceramic microspheres

Mg-substituted hydroxyapatite (HAp) bioceramic microspheres were prepared by spray drying and subsequent processing at 1173, 1273 and 1373 K. Influence of various Mg substitution levels (up to 0.84 ± 0.10 wt%) on physicochemical properties of the HAp bioceramic microspheres was evaluated. Obtained results were used for the elucidation of the compositional and structural characteristics of the microspheres in conjunction with adsorption of protein, namely, bovine serum albumin (BSA). The primary difference among the microspheres processed at various temperature was the presence or absence of the micropores (<2 nm in diameter) and mesopores (between 2 and 50 nm). Presence of the micro- and mesopores resulted in higher specific surface area (SSA), enhanced solubility, i.e., ion release, and, accordingly, increase in the amount of BSA adsorbed on the microspheres. Furthermore, the BSA adsorption capacity of the microspheres decreased with increasing Mg content despite of higher SSA.     

Synthesis of Eu-doped hydroxyapatite whiskers and fabrication of phosphor layer via electrophoretic deposition process

Highly biocompatible and efficiently luminescent whiskers of the hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂, HAp) doped with various concentrations (0-5 at.%) of europium were prepared by hydrothermal synthesis and the Eu-doped Hap-coating layers onto the surface of titanium substrate were fabricated by the electrophoretic deposition (EPD) process for fluorescent probe application. The maximum doping concentration of Eu accommodating into the host lattice of HAp was detected as ~1.5 at.% and all the hydrothermally synthesized Eu-doped HAp whiskers were found to have high crystallinity and orientation growth along the c-axis by X-ray diffraction (XRD) identification. The valence of the doped Eu was identified as trivalent and divalent coexistence at a concentration percentage of Eu³⁺: Eu²⁺ = 78%: 22% by X-ray photoelectron spectroscopy (XPS) spectra. The replacement site of the doped Eu ions in the crystal structure of HAp host was clarified by Rietveld refinement. The whisker morphology of the hydrothermally synthesized particle was demonstrated by field-emission scanning electron microscopy (FE-SEM) observation and their component elements were analyzed by energy dispersive X-ray (EDX) mapping. The photoluminescence (PL) emissions of the Eu-doped HAp whiskers and fabricated their coating layers were both revealed mainly at ~615 nm (⁵D₀ → ⁷F₂) and ~697 nm (⁵D₀ → ⁷F₄), which is a wavelength that easily transmitting through living system for biological imaging. The PL emission are falling in the region of reddish orange and belonging to color temperature below 1500 K. Decay time and internal and external quantum efficiencies (QEs) were also measured to reveal them depending on the doping concentration of Eu. The hydrothermally prepared Eu-doped HAp whiskers would be aimed at biomedical application, due to their promising fluorescent function of probe for in vivo imaging in medical diagnose by utilizing the superior biocompatibility of the HAp host and highly efficient luminescent property of the Eu activator.     

The Role of MgAl2O4 Powder Packing on Phase Stability of Hydroxyapatite During Sintering

MgAl₂O₄ (spinel) was utilized as a packing powder in the sintering of hydroxyapatite (HAp) and the composite of HAp/3 mol% Y₂O₃‐stabilized tetragonal zirconia (3Y‐TZP). The influence of spinel on phase stability of HAp was investigated using X‐ray diffraction (XRD), scanning electron microscopy (SEM), Raman spectroscopy, and electron probe microanalysis (EPMA) to reveal the reaction in the vicinity of the interface between HAp and spinel. When covered with spinel powder, decomposition temperatures for both HAp monolith and HAp in the composite were raised from 1360°C to 1470°C and from 930°C to 1280°C, respectively. SEM images supported the role of spinel on retardation of the decomposition, showing a dense cross section of the monolith after sintering for 2 h at 1400°C with the spinel as opposed to a porous feature without the covering. XRD results indicated that the increase in the decomposition temperatures was accompanied by a decrease in the a‐axis dimension of the hexagonal structure of HAp, probably as a result of the substitution of F^? for OH^?. EPMA revealed that negligible reaction occurred between HAp and spinel even at 1500°C, but the Ca²⁺ in HAp diffused about 20 μm into 3Y‐TZP to form a cubic zirconia solid solution at 1275°C, resulting in the decomposition. The involvement of F^? ion in the contraction of a‐axis parameter and the consequent phase stability were manifested by an increase in the Raman band of the symmetric stretching of the P–O bonds at 962.3 cm^?1 and the appearance of a band for fluoroapatite at 3538 cm^?1.     

Preparation and characterization of magnetic poly(styrene–glycidyl methacrylate) microspheres for highly efficient protein adsorption by two‐stage dispersion polymerization

Micrometer‐sized superparamagnetic poly(styrene–glycidyl methacrylate)/Fe₃O₄ spheres were synthesized by two‐stage dispersion polymerization with modified hydrophobic Fe₃O₄ nanoparticles, styrene (St), and glycidyl methacrylate (GMA). The morphology and properties of the magnetic Fe₃O₄–P (St‐GMA) microspheres were examined by scanning electron microscopy, transmission electron microscopy, vibrating sample magnetometry, thermogravimetric analysis, and attenuated total reflectance. The average size of the obtained magnetic microspheres was 1.50 μm in diameter with a narrow size distribution, and the saturation magnetization of the magnetic microspheres was 8.23 emu/g. The magnetic Fe₃O₄–P (St‐GMA) microspheres with immobilized iminodiacetic acid–Cu²⁺ groups were used to investigate the adsorption capacity and selectivity of the model proteins, bovine hemoglobin (BHb) and bovine serum albumin (BSA). We found that the adsorption capacity of BHb was as high as 190.66 mg/g of microspheres, which was 3.20 times greater than that of BSA, which was only 59.64 mg/g of microspheres as determined by high‐performance liquid chromatography. With a rather low nonspecific adsorption, these microspheres have great potential for protein separation and purification applications. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43005.     

Synthesis and characterization of star-shaped poly (lactide-co-glycolide) and its drug-loaded microspheres

The star-shaped poly (lactide-co-glycolide) (PLGA) was synthesized via the ring-opening polymerization of d,l-lactide and glycolide, with pentaerythritol as a multifunctional initiator and stannous 2-ethyl hexanoate as a catalyst. The structures of these polymers were characterized by ¹³C-NMR spectroscopy, while the molecular weight and polydispersity index (PDI) were determined by gel permeation chromatography (GPC). The glass transition temperature (T _g) of copolymer was determined by differential scanning calorimetry (DSC). Bovine serum albumin (BSA) loaded microspheres were fabricated using star-shaped PLGA by a W/O/W double emulsion solvent evaporation method. The results of characterization demonstrated that the particle size of the PLGA microspheres were about 80–150 μm, the maximum loading capacity and encapsulation efficiency of BSA-loaded microspheres were 67.51 μg/(mg microspheres) and 78.39%, respectively, which were better than linear PLGA. The in vitro release profiles of BSA in phosphate buffer saline (PBS) lasted for 37 h. Drug release profiles can be affected by polymer molecular weight and the ratio of polymer to drug. The maximum release percentage was 80%.     

Controlled synthesis and morphological evolution of dendritic porous microspheres of calcium phosphates

Dendritic porous microspheres (DPMs) consisted of octacalcium phosphate (OCP) nucleus and carbonated hydroxyapatite (CHAp) nanoflakes have been synthesized under good control by a template-induced method in a system of constant temperature and pressure. Batch experiments were carried out to investigate the effect of the template Na₂EDTA and the organic conditioner glycerol on the dendritic porous morphology. Corresponding morphological evolution process was also researched. The results revealed that the template Na₂EDTA altered the natural growth habit of CHAp and induced the first dendritic growth of CHAp nanoflakes on the surface of OCP, and organic conditioner glycerol controlled the second dendritic growth of CHAp nanoflakes on the bigger ones. Higher concentrations of glycerol resulted in higher surface fractal dimension, smaller pore diameter, and greater specific surface area.     

Facile and eco-friendly fabrication of hierarchical superhydrophobic coating from eggshell biowaste

This study reports a facile and sustainable approach to fabricate superhydrophobic coating from eggshell biowaste. The coating was prepared by ball milling chicken eggshells, composed of hydrophilic calcium carbonate (CaCO₃), to microsized particles followed by surface hydrophobilizing with stearic acid (C₁₇H₃₅COOH) to form low surface energy nanosized calcium stearate ((C₁₇H₃₅COO)₂Ca) through the esterification of hydroxyl groups (-OH) absorbed on a surface of CaCO₃ with carboxyl groups (–COOH) of stearic acid. Then, a layer of modified eggshell particles dispersed in polystyrene (PS) binder was dip-coated on a substrate. A coated surface with water contact angles of 151° ± 1° on glass and 153° ± 1° on cotton fabric substrates was achieved when a 4:1 weight ratio of the modified eggshell:PS was used. The uniform distribution of the modified eggshell particles throughout the coating led to a surface with high degree of hierarchical micro-nanoscale roughness which resulted in superhydrophobicity. The superhydrophobic eggshell coating showed good environmental stability, self-cleaning, and oil/water separation properties. These results suggest that eggshell biowaste can be utilized for superhydrophobic applications.     

Preparation and properties of bimodal porous apatite ceramics through slip casting using different hydroxyapatite powders

A bimodal porous hydroxyapatite (HAp) body with high flexural strength was prepared through slip casting. The effect of different particle sizes on the flexural strength and microstructure of three different types of hydroxyapatite (HAp) powders was studied. The powder characteristic of laboratory-synthesized HAp powder (L-HAp) was obtained through a wet-milling method, drying and heating of a mixture of calcium hydrogen phosphate di-hydrate and calcium carbonate. The median particle size of L-HAp was 0.34 μm, and the specific surface area was 38.01 m²/g. The commercial HAp had median particle sizes for the K-HAp (Kishida chemical Co. Ltd., K-HAp) and T-HAp (Taihei chemical Co. Ltd., T-HAp) of 1.13 and 3.65 μm, and specific surface areas of 11.62 and 6.23 m²/g, respectively. The different powder characteristics affected the slip characteristics, and the flexural strength and microstructure of the sintered porous HAp bodies were also different. The flexural strengths of the porous HAp ceramics prepared by heating at 1200 °C for 3 h in air were 17.59 MPa for L-HAp with a porosity of 60.48%, 3.92 MPa for commercial K-HAp with a porosity of 79.37%, and 4.55 MPa for commercial T-HAp with a porosity of 76.46%.     

Enhanced in vitro inhibition of MCF-7 and magnetic properties of cobalt incorporated calcium phosphate (HAp and β-TCP) nanoparticles

The Co²⁺ (0.1 M) incorporated hydroxyapatite (HAp) and beta tricalcium phosphate (β-TCP) nanoparticles were synthesized by the microwave assisted technique and sintering of HAp respectively. The samples were thermally treated at temperatures ranging from 200 to 1000°C. The partial substitutions of Co²⁺ at the Ca²⁺ site of HAp were confirmed from the slight shift (～0.2°) in the (002) and (211) XRD peaks. The morphology of the nanoparticles was transformed from nanospheres to twinned particles on thermal treatment. In addition, the particle size of Co-600 was increased (from ～50 nm to ～100 nm) due to the recrystallization process. Further, the thermal treatment enhanced the crystallinity (41.15 to 90.16%), retentivity (M_r) and coercivity (H_c) of the nanoparticles. The cobalt incorporated HAp and β-TCP possessed paramagnetic property. The excellent bioactivity of β-TCP has been confirmed by the mineralization in simulated body fluid (SBF). Compared to HAp, β-TCP possessed better compatibility towards C2C12 cells on cobalt incorporation as evidenced by the in vitro cell viability. Moreover, both HAp and β-TCP have significantly inhibited the growth of MCF-7 on increasing the interaction time (72 h). Hence, the inhibition characteristics of Co²⁺ incorporated calcium phosphate (CaP) towards MCF-7 (without affecting the normal cells) demonstrate its competency as a potential material for cancer therapy over the already existing nanoparticles.     

A highly sensitive method for detection of protein based on inhibition of Ru(bpy)32+/TPrA electrochemiluminescent system

The detection of proteins is very important in the study of biochemistry procedure, and electrochemiluminescence method is a very practical tool for the study of protein folding, structure and quantification. In this work, bovine serum albumin (BSA) and casein were found to be able to significantly quench the electrochemiluminescence (ECL) of Ru(bpy)₃²⁺/TPrA system, based on which a highly sensitive approach for the detection of protein was proposed. Under the optimized conditions, the logarithmic plot of the inhibited ECL versus the concentration of BSA and casein were linear over the ranges of 1–30 μg/L and 0.1–1.6 μg/L, respectively. The corresponding limit of detection (LOD) was 0.45 μg/L for BSA and 0.026 μg/L for casein (S/N = 3). UV, ECL and fluorescence methods were used to investigate the mechanism of the inhibited ECL of Ru(bpy)₃²⁺/TPrA/BSA system. A mechanism based on the formation of protein-Ru(bpy)₃²⁺ super molecule was proposed, which would prevent Ru(bpy)₃²⁺ from reaching the working electrode surface so that induced ECL quenching.