In situ synthesis of magnesium-substituted biphasic calcium phosphate and in vitro biodegradation

In situ preparation of magnesium (Mg) substituted biphasic calcium phosphate (BCP) of hydroxyapatite (HAp)/β-tricalcium phosphate (β-TCP) were carried out through aqueous co-precipitation method. The concentrations of added magnesium were varied with the calcium in order to obtain constant (Ca + Mg)/P ratios of 1.602. X-ray diffraction (XRD) and Fourier transformed infrared (FTIR) spectroscopy were used to characterize the structure of synthesized magnesium substituted BCP powders. The results have shown that substitution of magnesium in the calcium deficient apatites revealed the formation of biphasic mixtures of different HAp/β-TCP ratios after heating at 1000 °C. The ratios of the formation of phase mixtures were dependent on the content of magnesium. After immersing in Hanks’ balanced salt solution (HBSS) for 1 week, 1 wt% magnesium substituted BCP powders were degraded and precipitation started to be formed with small granules consisting of number of flake-like crystal onto the surface of synthesized powders. On the other hand, in the case of pure BCP powders, the formation of new precipitates was detected after immersion in HBSS for 2 weeks. On the basis of these results, magnesium substituted BCP could be able to develop a new apatite phase on the surface in contact with physiological fluids faster than BCP does. In addition, the retention time to produce the new apatite phase in implantation operation for the BCP powder could be controlled by the amount of magnesium substitution.     

Pulsed electrodeposition for the synthesis of strontium-substituted calcium phosphate coatings with improved dissolution properties

Strontium-substituted calcium phosphate coatings are synthesized by pulsed electrodeposition on titanium alloy (Ti6Al4V) substrates. Experimental conditions of the process are optimized in order to obtain a coating with a 5% atomic substitution of calcium by strontium which corresponds to the best observations on the osteoblast cells activity and on the osteoclast cells proliferation. The physical and chemical characterizations of the obtained coating are carried out by scanning electron microscopy associated to energy dispersive X-ray spectroscopy (EDXS) for X-ray microanalysis and the structural characterization of the coating is carried out by X-ray diffraction. The in vitro dissolution/precipitation properties of the coated substrates are investigated by immersion into Dulbecco's Modified Eagle Medium (DMEM) from 1 h to 14 days. The calcium, phosphorus and strontium concentrations variations in the biological liquid are assessed by Induced Coupled Plasma - Atomic Emission Spectroscopy for each immersion time. The results show that under specific experimental conditions, the electrodeposition process is suitable to synthesize strontium-substituted calcium phosphate coatings. Moreover, the addition of hydrogen peroxide (H₂O₂) into the electrolytic solution used in the process allows us to observe a control of the strontium release during the immersion of the prosthetic materials into DMEM.     

Bioactivity of calcium phosphate coatings prepared by electrodeposition in a modified simulated body fluid

The purpose of this study was to investigate bioactivity of calcium phosphate coatings prepared by electrodeposition in a modified simulated body fluid (SBF). Calcium phosphates were electrodeposited on commercially pure titanium substrates in the modified SBF at 60 °C for 1 h maintaining the cathodic potentials of − 1.5 V, − 2 V, and − 2.5 V (vs. SCE). Subsequently, the calcium phosphate coatings were transformed into apatites during immersion in the SBF at 36.5 °C for 5 days. The apatites consisted of needle-shaped crystallites distributed irregularly with different grain sizes. As the coatings were electrodeposited at higher cathodic potential, the crystallite of the apatites got denser and the grain sizes of the apatites became bigger during subsequent immersion in the SBF. However, as the coatings were electrodeposited at higher cathodic potential, the coatings were transformed into apatites with lower crystallinity and the Ca/P atomic ratio of the apatites got higher than 1.67, that of stoichiometric hydroxyapatite, after subsequent immersion in the SBF. In addition, CO₃²⁻ ions contained in the modified SBF were incorporated in the calcium phosphate coating during electrodeposition and had an influence on transforming the calcium phosphate into bonelike apatite during subsequent immersion in the SBF showing that CO₃²⁻ incorporated in the apatites disturbed crystallization of the apatites. These results revealed that the coating electrodeposited at − 2.0 V (vs. SCE) in the modified SBF containing CO₃²⁻ ions was the most bioactive showing transformation into carbonate apatite similar to bone apatite.     

A novel wet polymeric precipitation synthesis method for monophasic β-TCP

β-Tricalcium phosphate (β-Ca₃(PO₄)₂, β-TCP) powders were synthesized using wet polymeric precipitation method for the first time to our best knowledge. The results of X-ray diffraction analysis showed the formation of almost single a Ca-deficient hydroxyapatite (CDHA) phase of a poor crystallinity already at room temperature. With continuously increasing the calcination temperature up to 800 °C the crystalline β-TCP was obtained as the main phase. It was demonstrated that infrared spectroscopy is very effective method to characterize the formation of β-Ca₃(PO₄)₂. The SEM results showed that β-Ca₃(PO₄)₂ solids were homogeneous having a small particle size distribution. The β-TCP powders consisted of spherical particles varying in size from 100 to 300 nm. Fabricated β-TCP specimens were placed to the bones of the rats and maintained for 1–2 months. The histological properties of these samples will be also investigated.     

Preparation and characterization of hydroxyapatite synthesized from oyster shell powders

The ready availability and the low cost of oyster shells, which is composed predominantly of calcium carbonate with rare impurities, along with natural wastes are attractive features for converting the biological material into hydroxyapatite (HA) powders for biomedical applications. The HA powder was synthesized using oyster shell powders and dicalcium phosphate dihydrate (CaHPO₄·2H₂O, DCPD) through ball milling and subsequently heat treatment. The HA was initiated through sintering the 1-h milled sample at 1000 °C for 1 h, while pure HA phase is formed after sintering the 10-h milled sample. The as-prepared samples, obtained after 5 or 10 h of milling and then heat-treating at 1000 °C for 1 h, contain the phase of β-tricalcium phosphate (β-TCP). Moreover, the result of FTIR analysis showed that the as-prepared HA sample is A- and B-type carbonate-containing calcium phosphates. The as-synthesize HA powder containing trace elements Mg and Sr exhibited good crystallinity (96.3%) and high phase-purity.     

A novel biodegradable nicotinic acid/calcium phosphate composite coating on Mg–3Zn alloy

A novel biodegradable composite coating is prepared to reduce the biodegradation rate of Mg–3Zn alloy. The Mg–3Zn substrate is first immersed into 0.02 mol L^? 1 nicotinic acid (NA) solution, named as vitamin B₃, to obtain a pretreatment film, and then the electrodeposition of calcium phosphate coating with ultrasonic agitation is carried out on the NA pretreatment film to obtain a NA/calcium phosphate composite coating. Surface morphology is observed by scanning electron microscopy (SEM). Chemical composition is determined by X-ray diffraction (XRD) and EDX. Protection property of the coatings is evaluated by electrochemical tests. The biodegradable behavior is investigated by immersion tests. The results indicate that a thin but compact bottom layer can be obtained by NA pretreatment. The electrodeposition calcium phosphate coating consists of many flake particles and ultrasonic agitation can greatly improve the compactness of the coating. The composite coating is biodegradable and can reduce the biodegradation rate of Mg alloys in stimulated body fluid (SBF) for twenty times. The biodegradation process of the composite coating can be attributed to the gradual dissolution of the flake particles into chippings.     

Physical–chemical and biological behavior of an amorphous calcium phosphate thin film produced by RF-magnetron sputtering

This work evaluates the thermal reactivity and the biological reactivity of an amorphous calcium phosphate thin film produced by radio frequency (RF) magnetron sputtering onto titanium substrates. The analyses showed that the sputtering conditions used in this work led to the deposition of an amorphous calcium phosphate. The thermal treatment of this amorphous coating in the presence of H₂O and CO₂ promoted the formation of a carbonated HA crystalline coating with the entrance of CO₃^2 ? ions into the hydroxyl HA lattice. When immersed in culture medium, the amorphous and carbonated coatings exhibited a remarkable instability. The presence of proteins increased the dissolution process, which was confirmed by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) analyses. Moreover, the carbonated HA coating induced precipitation independently of the presence of proteins under dynamic conditions. Despite this surface instability, this reactive calcium phosphate significantly improved the cellular behavior. The cell proliferation was higher on the Ticp than on the calcium phosphate coatings, but the two coatings increased cellular spreading and stress fiber formation. In this sense, the presence of reactive calcium phosphate coatings can stimulate cellular behavior.     

Bioactive glass-based composites for the production of dense sintered bodies and porous scaffolds

Recently several attempts have been made to combine calcium phosphates, such as β-tricalcium phosphate (β-TCP) and, most of all, hydroxyapatite (HA), with bioactive glasses of different composition, in order to develop composites with improved biological and mechanical performance. Unfortunately, the production of such systems usually implies a high-temperature treatment (up to 1300 °C), which may result in several drawbacks, including crystallization of the original glass, decomposition of the calcium phosphate phase and/or reactions between the constituent phases, with non-trivial consequences in terms of microstructure, bioactivity and mechanical properties of the final samples. In the present contribution, novel binary composites have been obtained by sintering a bioactive glass, characterized by a low tendency to crystallize, with the addition of HA or β-TCP as the second phase. In particular, the composites have been treated at a relatively low temperature (818 °C and 830 °C, depending on the sample), thus preserving the amorphous structure of the glass and minimizing the interaction between the constituent phases. The effects of the glass composition, calcium phosphate nature and processing conditions on the composite microstructure, mechanical properties and in vitro bioactivity have been systematically discussed. To conclude, a feasibility study to obtain scaffolds for bone tissue regeneration has been proposed.     

Injectable calcium phosphate–alginate–chitosan microencapsulated MC3T3-E1 cell paste for bone tissue engineering in vivo

Osteoblasts or stem cells have been delivered into injectable calcium phosphate cement (CPC) to improve its effectiveness and biological function. However, the osteogenic potential of the new construct in vivo has been rarely reported, and there are no reports on alginate–chitosan microencapsulated osteoblasts mixed with CPC. This study aimed to develop alginate–chitosan microencapsulated mouse osteoblast MC3T3-E1 cells (AC-cells), evaluate the osteogenic potential of a calcium phosphate cement complex with these AC-cells (CPC-AC-cell), and trace the implanted MC3T3-E1 cells in vivo. MC3T3-E1 cells were embedded in alginate microcapsules, cultured in osteogenic medium for 7 days, and then covered with chitosan before mixing with a paste of β-tricalcium phosphate/calcium phosphate cement (β-TCP/CPC). The construct was injected into the dorsal subcutaneous area of nude mice. Lamellar-bone-like mineralization, newly formed collagen and angiogenesis were observed at 4 weeks. At 8 weeks, areas of newly formed collagen expanded; further absorption of β-TCP/CPC and osteoid-like structures could be seen. Cell tracing in vivo showed that implanted MC3T3-E1 cells were clearly visible at 2 weeks. These in vivo results indicate that the novel injectable CPC-AC-cell construct is promising for bone tissue engineering applications.     

Chemical synthesis and characterization of magnesium substituted amorphous calcium phosphate (MG-ACP)

Amorphous calcium phosphate (ACP) was synthesized by a simple aqueous precipitation using CaCl₂ and Na₃PO₄ in the presence of MgCl₂ to ensure the formation of the ACP phase at room temperature. Magnesium substituted ACP phases corresponding to two different compositions representing the two most prominent calcium phosphate phases (hydroxyapatite: Ca + Mg/P = 1.67 and tricalcium phosphate: Ca + Mg/P = 1.5) were synthesized by this simple approach. Both compositions of ACP phases resulted in their transformation into β-tricalcium phosphate upon heat treatment in air at 600 °C. X-ray diffraction (XRD), heat treatment, scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) and Brunauer-Emmett-Teller (BET) analyses were used to characterize the phase, thermal stability, surface area, and morphology of the synthesized ACP powders corresponding to the two different nominal Ca/P compositions. Although it is known that α-TCP is the phase that appears upon heat treatment at 600 °C unsubstituted ACP, substitution of magnesium ion in ACP (both TCP and HA composition) stabilized the structure of β-TCMP phase at 600 °C. Moreover, FT-IR analysis revealed that the ACP phase regardless of the composition, exhibited characteristic bands corresponding to that of HA, with the exception of the ACP corresponding to HA composition which exhibited a prominent OH vibrational mode.     

Chemical synthesis and stabilization of magnesium substituted brushite

Hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂) is the most ubiquitous calcium phosphate phase used in implant coatings and more recently in gene/drug delivery applications due to its chemical stability under normal physiological conditions (37 °C, pH ～ 7.5, 1 atm.). However, different calcium phosphate phases, such as brushite (CaH(PO₄)?2(H₂O)) and tricalcium phosphate (Ca₃(PO₄)₂) which are thermodynamically unstable under physiological conditions are also being explored for biomedical applications. One way of stabilizing these phases under physiological conditions is to introduce magnesium to substitute for calcium in the brushite lattice. The role of magnesium as a stabilizing agent for synthesizing brushite under physiological conditions at room temperature has been studied. Chemical analysis, Fourier transform infrared spectroscopy and X-ray diffraction have also been conducted to validate the formation of magnesium substituted brushite under physiological conditions.     

Carbonate and fluoride incorporation in synthetic apatites: Comparative effect on physico-chemical properties and in vitro bioactivity in fetal bovine serum

CO₃- and/or F-substituted apatites have been considered as potential bone substitution material for dental and orthopedic applications. The objective of this study was to compare physico-chemical properties and in vitro bioactivity in fetal bovine serum (FBS) of apatites containing CO₃ and/or F. The results showed that CO₃ and F in apatites have opposite effects on crystallinity and solubility. Calcium deficient hydroxyapatite (HA) and F-substituted apatite (FA) partially transformed to beta-tricalcium phosphate (β-TCP) at temperature 950 °C. After immersion in FBS for 10 days, calcium deficient HA, FA, and CO₃-substituted apatite (CHA) and CO₃- and F-substituted apatite (CFA) pellet surfaces all showed formation of apatite.     

Effect of low temperature on formation mechanism of calcium phosphate nano powder via precipitation method

Calcium phosphate powders were synthesized with Ca/P molar ratios of initial reagents ranging from 1.660 to 1.667 using wet precipitation method. This work deals with allocating a specific temperature level to each H₃PO₄ solution and Ca(OH)₂ suspension prior to the mixing process, and studying their influences on powder composition. A high pH value of the synthesis medium and the incorporation of numerous carbonate ions into the structure were attained by dropping the temperature of the Ca(OH)₂ suspension down to 5 °C. X-ray diffractometry and FTIR spectroscopy showed that heating samples that had a medium temperature exceeding 25 °C resulted in the dominant HAp phase, regardless of the initial acid solution temperature. By maintaining the medium temperature at 5 °C, a sudden formation of the β-TCP phase occurred after thermal treatment at 1300 °C, and this trend continued with the concurrent decrease in the temperature of the initial acid solution. An interpretation of the formation mechanism under these low-temperature conditions is proposed in terms of the temperature and pH value of the medium and the state of the phosphate and carbonate ions.     

Clinical evaluation of β-TCP in the treatment of lacunar bone defects: A prospective,randomized controlled study

The aim of this study was to investigate the potential wide application of beta tricalcium phosphate (β-TCP) only for bone defects as compared to allograft. 95 patients with a solitary bone cyst were randomly assigned to the treatment. A new radiographic scoring system was employed to calculate the biodegradation of bone graft and to evaluate the influence of multiple factors. At an average of 28.43 months after surgery, a radiographic semi-quantitative analysis revealed that the degradation rates of β-TCP and the allograft were comparable (p > 0.05). Age, complication, packing methods and granule diameters have a significant influence on β-TCP degradation. The loose packing method and 3–5 mm granule size should be employed in clinical practice. A histological analysis of biopsy showed that β-TCP supported the growth of fibrous tissue, vascular tissue, as well as bone tissue into the implants. The results proved that single β-TCP is an advantageous alternative to allografts for lacunar bone defect repair and would well guide the design and clinical application of the β-TCP.     

Preparation of calcium carbonate microparticles containing organic fluorescent molecules from vaterite

The encapsulation of fluorescent organic molecules into crystalline calcium carbonate was examined using calcium carbonate microcapsule, whose crystalline phase is vaterite as a metastable phase of calcium carbonate. A calcium carbonate microcapsule with impregnated pyrene that is a water insoluble fluorescent molecule was soaked into suitable aqueous solutions to promote the phase transition of vaterite toward calcite as the stable phase of calcium carbonate. When 0.2 M calcium chloride solution was used, the largest amount of pyrene (approximately 0.06 wt%) was encapsulated into the calcite particle. Pyrene thus included was not eliminated even after thorough washing with THF. The calcite particle thus prepared produced the excimer emission of pyrene by UV irradiation. Rhodamine B was also introduced into calcium carbonate by the immersion of the microcapsule into the aqueous solutions of Rhodamine B. The fluorescence of rhodamine B was observed from the calcium carbonate particles by visible light irradiation. Acetaminophen, a common drug poorly soluble in water, was also included in the calcium carbonate particle by the same procedures as the pyrene encapsulation. As acetaminophen thus encapsulated was released by the dissolution of the calcium carbonate particle in acidic solution, the particle is expected to be applied for a dissolution-triggered drug delivery.     

Characterization of a calcium phosphate–TiO2 nanotube composite layer for biomedical applications

Well-ordered nanotube arrays of titania ~ 0.7 μm high and about 40 or 110 nm in diameter were prepared via electrochemical oxidation at constant voltage (10, 15, 20 or 25 V) in a mixture of 0.86 wt.% of NH₄F, glycerol and deionized water. The effect of annealing the nanotubes at 600 °C on their morphology and structure was examined using SEM and TEM techniques. These substrates are suitable supports for a calcium phosphate coating deposited by a simple immersion in Hank solution.The nucleation and growth of a calcium phosphate (Ca–P) coating deposited on TiO₂ nanotubes (NT) from Hanks' solution was investigated using SEM. XPS and FTIR surface analytical techniques were used to characterize the self-organized porous TiO₂ layers covered with calcium phosphate coatings before and after protein adsorption. Our results confirm that the nanotubular titania layer became stable after annealing at 600 °C, while its internal structure changed from amorphous to crystalline anatase, and eventually, a mixture of anatase and rutile. These thermally stabilized TiO₂ nanotubes significantly enhance apatite formation in Hanks' Balanced Salt Solution as compared to pure Ti covered with a native oxide layer. The Ca–P/TiO₂ NT/Ti surface adsorbs a higher amount of protein (bovine serum albumin, BSA) for a geometric surface area than does the Ti surface. The above difference in protein adsorption suggests a more promising initial cellular response for a Ca–P/TiO₂ NT/Ti composite than for a typical Ti implant surface.     

Microstructure,mechanical property and corrosion behavior of interpenetrating (HA + β-TCP)/MgCa composite fabricated by suction casting

The novel interpenetrating (HA + β-TCP)/MgCa composites were fabricated by infiltrating MgCa alloy into porous HA + β-TCP using suction casting technique. The microstructure, mechanical properties and corrosion behaviors of the composites have been evaluated by means of scanning electron microscopy (SEM), X-ray diffraction (XRD), mechanical testing, electrochemical and immersion tests. It was shown that the composites had compact structure and the interfacial bonding between MgCa alloy and HA + β-TCP scaffolds was very well. The ultimate compressive strength of the composites was about 500–1000 fold higher than that of the original porous scaffolds, and it still retained quarter-half of the strength of the bulk MgCa alloy. The electrochemical and immersion tests indicated that the corrosion resistance of the composites was better than that of the MgCa matrix alloy, and the corrosion products of the composite surface were mainly Mg(OH)₂, HA and Ca₃(PO₄)₂. Meanwhile, the mechanical and corrosive properties of the (HA + β-TCP)/MgCa composites were adjustable by the choice of HA content.     

Selective laser sintering of β-TCP/nano-58S composite scaffolds with improved mechanical properties

Nano-sized 58S bioactive glass (nano-58S) as the dispersed phase was added to β-tricalcium phosphate (β-TCP) to reinforce the mechanical properties, and then the β-TCP/nano-58S composite scaffolds were prepared via selective laser sintering (SLS). The effects of nano-58S on microstructure, mechanical properties, bioactivity, and biocompatibility of the composite scaffolds were evaluated. The results showed that nano-58S was homogeneously dispersed in the β-TCP matrix and the mechanical properties were gradually improved when the amount of nano-58S was no more than a certain value (15 wt.%). However, exceeding this value, nano-58S became the continuous phase and exhibited the brittleness of bioactive glass. Accordingly, the mechanical properties gradually decreased. The maximum fracture toughness and compressive strength were 1.347 ± 0.025 MPa · m^1/2 and 18.2 ± 0.62 MPa, respectively. In vitro tests in the simulated body fluid (SBF) demonstrated that the apatite-like layer formed faster on the composite scaffolds than on the scaffold without nano-58S, indicating that the nano-58S glass could enhance the bioactivity of the composite scaffolds. The MG-63 cells culture experiment proved that nano-58S glass could further facilitate the growth of human osteoblastic cells.     

Enhanced sintering ability of biphasic calcium phosphate by polymers used for bone scaffold fabrication

Biphasic calcium phosphate (BCP), which is composed of hydroxyapatite [HAP, Ca₁₀(PO₄)₆(OH)₂] and β-tricalcium phosphate [β-TCP, β-Ca₃(PO₄)₂], is usually difficult to densify into a solid state with selective laser sintering (SLS) due to the short sintering time. In this study, the sintering ability of BCP ceramics was significantly improved by adding a small amount of polymers, by which a liquid phase was introduced during the sintering process. The effects of the polymer content, laser power and HAP/β-TCP ratios on the microstructure, chemical composition and mechanical properties of the BCP scaffolds were investigated. The results showed that the BCP scaffolds became increasingly more compact with the increase of the poly(l-lactic acid) (PLLA) content (0–1 wt.%) and laser power (6–10 W). The fracture toughness and micro-hardness of the sintered scaffolds were also improved. Moreover, PLLA could be gradually decomposed in the late sintering stages and eliminated from the final BCP scaffolds if the PLLA content was below a certain value (approximately 1 wt.% in this case). The added PLLA could not be completely eliminated when its content was further increased to 1.5 wt.% or higher because an unexpected carbon phase was detected in the sintered scaffolds. Furthermore, many pores were observed due to the removal of PLLA. Micro-cracks and micro-pores occurred when the laser power was too high (12 W). These defects resulted in a deterioration of the mechanical properties. The hardness and fracture toughness reached maximum values of 490.3 ± 10 HV and 1.72 ± 0.10 MPa m^1/2, respectively, with a PLLA content of approximately 1 wt.% and laser power of approximately 10 W. Poly(l-lactic-co-glycolic acid) (PLGA) showed similar effects on the sintering process of BCP ceramics. Rectangular, porous BCP scaffolds were fabricated based on the optimum values of the polymer content and laser power. This work may provide an experimental basis for improving the mechanical properties of BCP bone scaffolds fabricated with SLS.     

Mechanochemical transformation of mixtures of Ca(OH)2 and (NH4)2HPO4 or P2O5

A detailed comparative study of the mechanochemical transformation of two mixtures: Ca(OH)₂–(NH₄)₂HPO₄ and Ca(OH)₂–P₂O₅, milled in a mortar dry grinder for different periods of time was carried out. The phase transformations obtained at each milling stage were studied by X-ray diffraction, infrared spectroscopy, transmission electron microscopy, differential scanning calorimetry and thermogravimetric analysis.The transformations taking place during the first periods of milling are very different for both mixtures. However, prolonged milling, over nearly the same period, causes amorphization of both mixtures. DSC analysis of the milled powders showed the temperature of crystallization of hydroxyapatite and tricalcium phosphate (β-TCP). Calcinations of all the different milled powders at 800 °C for 2 h, results in the formation of hydroxyapatite and β-TCP.