Osteoclastic cell behaviors affected by the α-tricalcium phosphate based bone cements

Calcium phosphate cements (CPCs) have recently gained great interest as injectable bone substitutes for use in dentistry and orthopedics. α-tricalcium phosphate (α-TCP) is a popularly used precursor powder for CPCs. When mixed with appropriate content of liquid and kept under aqueous conditions, α-TCP dissolves to form a calcium-deficient hydroxyapatite and then hardens to cement. In this study, α-TCP based cement (CP) and its composite cement with chitosan (Ch-CP) were prepared and the osteoclastic responses to the cements and their elution products were evaluated. Preliminary evaluation of the cements revealed that the CP and Ch-CP hardened within ~10 min at an appropriate powder-to-liquid ratio (PL) of 3.0. In addition, CP and Ch-CP were transformed into an apatite phase following immersion in a saline solution. Moreover, the osteoblastic cells were viable on the cements for up to 10 days. Mouse-derived bone marrow cells were isolated and activated with osteoclastic differentiation medium, and the effects of the CP and Ch-CP substrates and their ionic eluants on the osteoclastic activity were investigated. Osteoclastic cells were viable for up to 14 days on both types of cements, maintaining a higher cell growth level than the control culture dish. Multi-nucleated osteoclastic cells that were tartrate-resistant acid phosphatase (TRAP)-positive were clearly observed when cultured on the cement substrates as well as treated with the cement eluants. The TRAP activity was found to be significantly higher in cells influenced by the cement substrates and their eluants with respect to the control culture dish (Ch-CP > CP ≫ control). Overall, the osteoclastic differentiation was highly stimulated by the α-TCP based experimental cements in terms of both the substrate interaction and their elution products.     

Reactivity of α-tricalcium phosphate

The reactivity of -tricalcium phosphate (-TCP) in forming hydroxyapatite (HAp) at 37°C was investigated. The effects of synthesis route, HAp seeding and the presence of calcium salts on the mechanism and extent of HAp formation were examined by pH measurements and/or isothermal calorimetric analyses. A synthesis temperature at the lower end in the temperature range of 1100–1300°C and the reaction of -TCP with a high specific surface area greatly improved rate and extent of HAp formation. The time for complete reaction decreased from 18 h to 14 h, when the reaction was carried out in the presence of 1 wt% of HAp seeds; the hydrolysis mechanism did not change. At HAp seeds proportion of 5 wt% and 10 wt%, transformation occurred without a nucleation period. The calcium salt additives studied were anhydrous and dihydrate form of dicalcium phosphate (CaHPO₄ and CaHPO₄ · 2H₂O), calcium carbonate (CaCO₃), and calcium sulfate hemihydrate (CaSO₄ · 1/2H₂O). All the additives delayed HAp formation as determined by the isothermal calorimetric analyses. Their retarding effects in decreasing order are CaCO₃, CaSO₄ · 1/2H₂O, DCPD, DCP. CaCO₃ almost completely retarded HAp formation. After 24 h, hydrolysis was complete only for pure -TCP and for the -TCP-DCP blend. Reaction was complete in other formulations before 48 h except for the CaCO₃-containing blend. In all mixtures conversion to HAp occurred without forming any intermediates. However gypsum formed in the mixture containing CaSO₄ · 1/2H₂O. All the -TCP-additive mixtures, excluding -TCP-CaCO₃, reached nominally the same strength value after 24 h of reaction as governed by the transformation of -TCP to HAp. For phase-pure -TCP, the average tensile strength changed from 0.36 ± 0.03 MPa to 7.26 ± 0.6 MPa. Upon hydrolysis only the CaSO₄ · 1/2H₂O-containing mixture exhibited slightly higher strength averaging 8.36 ± 0.9 MPa.     

Preparation of octacalcium phosphate by the hydrolysis of α-tricalcium phosphate

A new simple method of preparation for the thermodynamically unstable octacalcium phosphate [Ca₈H₂(PO₄)₆·5H₂O; OCP] has been developed using the hydrolysis of -Ca₃(PO₄)₂ instead of the conventional hydrolysis of CaHPO₄·2H₂O. The hydrolysis experiments were carried out by treating an -Ca₃(PO₄)₂(1 g)-H₂O(50 m) suspension for 3 h at temperatures in the range 40 to 80° C and at pHs in the range 3 to 7.5. The formation of OCR was limited to within a narrow region between formation regions of other phosphates. Favourable conditions for OCP preparation were, for example, 70° C, pH4.5 to 5.0 and 60° C, pH5.0. Particles of OCP were composed of tight aggregates of strip-like microcrystals growing probably along the [0 0 1] and (1 0 0) plane of the OCP structure. Nearly stoichiometric OCP was obtained under the most suitable conditions with good reproducibility. Pyrolytic processes of OCP were approximately consistent with the data published so far. However, the temperatures of the appearance and disappearance of pyrolytic crystalline phases and ionic species deviated slightly from the published data. Thermal dehydration up to 150° C without destruction of OCP and decomposition reactions above 300° C resulted in changes in surface area and average pore radius of OCP.     

Enhanced hydrated properties of α-tricalcium phosphate bone cement mediated by loading magnesium substituted octacalcium phosphate

A novel Mg-containing α-tricalcium phosphate (α-TCP) bone cement with excellent wetting compressive strength and suitable setting time has been developed in this work. Mg-substituted octacalcium phosphate (Mg-OCP) crystals with the special morphology was prepared by homogeneous precipitation method, and used to modify the α-TCP bone cement. Mg²⁺ was successfully introduced into OCP structure, and the filaments/sheet-like morphologies of OCP were obtained by changing the Mg²⁺ concentration. As added in the α-TCP cement matrix, the effects of Mg-OCP on the wetting compressive strength, setting time and microstructure of the cement system were studied. The addition of Mg-OCP shortened the setting time of cement pastes with the minimum setting time of 15 min, and enhanced the wetting compressive strength of the hydrated cement products with the highest wetting compressive strength of 36.86 MPa. This work highlights the special morphologies of Mg-OCP induced by Mg²⁺ substitution, which made an effect on the hydration reaction of α-TCP cement; Furthermore, Mg-OCP was supposed to improve the condensation capacities and mechanical properties of α-TCP cement system as a novel cement admixture.     

Effect of iron on the setting properties of α-TCP bone cements

New ceramic materials with the ability to set like cement, after mixing a powder phase made of one and/or several of these new reactants and a liquid phase, have been obtained within the ternary system “CaO-P₂O₅-FeO”. These new reactants have magnetic properties, i.e. cement made from them maintains its magnetic property during the whole setting and hardening. These new materials can be of use, for example, in dental applications, in the treatment of certain types of bone cancer and, in general, in the fields of Biomaterials and Bone Tissue Engineering. In this article, we report on the effect of iron-modified α -tricalcium phosphate, which is the main reactant of commercial calcium phosphate bone cements, on their new setting and hardening properties.     

Effect of β-TCP granularity on setting time and strength of calcium phosphate hydraulic cements

The effects of -tricalcium phosphate (-TCP) granularity on the properties of calcium phosphate hydraulic cements (CPHC) have been investigated. A model system based on mixtures of (-TCP) and aqueous solution of orthophosphoric acid has been used. Powders with different shapes (irregular agglomerates or spheres) and sizes (d ₅₀=7 to 130 m) were prepared from two different calcium phosphate sources: Ca-deficient hydroxyapatite (DAP) or hydroxyapatite dicalcium phosphate mixtures (HAP-DCP). The cements exhibited setting times (ST) ranging from 65 to 510 s; they are mainly affected by the specific surface area (S _BET) of the -TCP powders, longer ST corresponding to lower S _BET. In general, lower S _BET and shorter ST values were obtained with HAP-DCP powders. Diametral tensile strengths (DTS) ranging from 3.5 to 10.4 MPa were obtained. The results show that DTS is affected in a complex way by the experimental variables. In general, DTS is higher with irregular agglomerates compared to spheres (+1.2 MPa), while better results are obtained with HAP-DCP powders (+0.9 MPa). The dependence of DTS on particle size is variable according to powder source and shape. The highest DTS (10.4 MPa) was obtained with irregular agglomerates prepared from HAP-DCP mixtures (d ₅₀=16 m), and the lowest (3.5 MPa), with irregular agglomerates prepared with DAP (d ₅₀=123 m). It can be concluded from this work that specific CPHC formulations can give quite different cement properties, such as setting time and ultimate mechanical strength, depending on the characteristics of the raw materials used. In the case of -TCP based cements, the granularity of the starting cement powder, including particle size, shape and specific surface area, is of crucial importance and should be specified when the performances of different formulations are to be compared.     

The controlled resorption of porous α-tricalcium phosphate using a hydroxypropylcellulose coating

Tricalcium phosphate (TCP) ceramic is known in orthopedics to be a bioresorbable bone substitute. A porous TCP ceramic body also has high potential as a drug delivery system in bony defects. Porous alpha-TCP ceramic can be easily fabricated using conventional sintering of beta-TCP, since alpha-TCP is the thermodynamically stable phase at temperatures above 1 100 degrees C. However, the solubility of alpha-TCP is much higher than that of beta-TCP. Therefore, the dissolution of porous alpha-TCP progresses at a higher rate than bone repair. In the present study, we attempted to reduce the dissolution rate of porous alpha-TCP by employing an organic polymer coating. We fabricated porous alpha-TCP ceramic with a continuous 10-50 microm diameter pore structure by sintering a body made from a beta-TCP and potato starch slurry. The porous body obtained was coated with hydroxypropylcellulose (HPC), and then subjected to heat treatment. The chemical durability and mechanical properties of the body were examined before and after coating with the HPC. The dissolution of porous alpha-TCP in buffered solutions was reduced by coating with HPC and drying at 60 degrees C. The compressive strength of the porous alpha-TCP was also improved by coating with HPC. The results of in vivo experiments showed that some parts of the porous alpha-TCP ceramic coated with HPC remained in the canal of the tibia of a rabbit four weeks after implantation, whereas no residual was observed in a non-coated alpha-TCP ceramic. Coating with HPC was found to be effective for controlling bioresorption and improving the workability of porous alpha-TCP ceramic. The prepared porous alpha-TCP ceramic is expected to be useful as a novel material for bone fillers by incorporating it with drugs or osteoinductive factors.     

Calcium phosphate cement: in vitro and in vivo studies of the α-tricalcium phosphate-dicalcium phosphate dibasic-tetracalcium phosphate monoxide system

In this paper, calcium phosphate cement consisting of -tricalcium phosphate (-TCP), dicalcium phosphate dibasic (DCPD) and tetracalcium phosphate monoxide (TeCP) was investigated in vitro and in vivo. Measurements of compressive strength against soaking time in simulated body fluid (SBF) showed a rapid increase of the hardness for the first 7 days. The gained strength was retained up to 1 year and the maximal mean value was 94.7 (±14.4) MPa. X-ray diffraction (XRD) and scanning electron microscopy (SEM) presented precipitates of hydroxyapatite (HA) after mixing, also after soaking in SBF and after implantation in rat subcutaneous tissues. However, the conversion to HA happened in different ways between in vitro and in vivo exposures. Histologic examinations showed that the cement causes the same reactions at the interface with surrounding soft tissues as HA. The authors consider the cement to be a promising material as a bone substitute, bone cement or dental material, however, further studies in a paste form and in bone tissue environments are necessary.     

Effect of silicate incorporation on in vivo responses of α-tricalcium phosphate ceramics

In addition to calcium phosphate-based ceramics, glass-based materials have been utilized as bone substitutes, and silicate in these materials has been suggested to contribute to their ability to stimulate bone repair. In this study, a silicate-containing α-tricalcium phosphate (α-TCP) ceramic was prepared using a wet chemical process. Porous granules composed of silicate-containing α-TCP, for which the starting composition had a molar ratio of 0.05 for Si/(P + Si), and silicate-free α-TCP were prepared and evaluated in vivo. When implanted into bone defects that were created in rat femurs, α-TCP ceramics either with or without silicate were biodegraded, generating a hybrid tissue composed of residual ceramic granules and newly formed bone, which had a tissue architecture similar to physiological trabecular structures, and aided regeneration of the bone defects. Supplementation with silicate significantly promoted osteogenesis and delayed biodegradation of α-TCP. These results suggest that silicate-containing α-TCP is advantageous for initial skeletal fixation and wound regeneration in bone repair.     

Tubular hydroxyapatite formation through a hydrothermal process from α-tricalcium phosphate with anatase

Hydroxyapatite (HA) is a useful substance because of its biocompatibility and adsorption capability. Tubular HA particles are highly attractive as novel tissue scaffolds and as drug carriers because of their hollow structures. We previously found that tubular HA particles were formed when a mixture of α-tricalcium phosphate (α-TCP) and anatase was hydrothermally treated. However, the formation mechanism is still unclear. In this study, we investigated the effect of anatase on the formation of tubular HA particles. The formation of tubular HA particles was enhanced when a pellet of a mixture of α-TCP and anatase was UV-irradiated before hydrothermal treatment. The tubular HA formation was observed only when anatase particles with adequate size were added in adequate quantities. We found that the surface properties, size, and quantity of the anatase particles were important for the formation of tubular HA particles. The tubular HA particles were not formed in the early stages of the reaction, they were formed only after some crystal growth had occurred. The anatase particles controlled the nucleation and crystal growth of HA.     

Development of some calcium phosphate cements from combinations of α-TCP,MCPM and CaO

From previous studies it is known that alpha-tertiary calcium phosphate (-TCP), monocalcium phosphate monohydrate (MCPM) and calcium oxide form cements upon mixing combinations of them with water. In this study some formulations were optimized with respect to the particle size of the constituents, their molar ratio, amounts of hydroxyapatite or beta-tertiary calcium phosphate (-TCP) added and the water/powder ratio. Three suitable products were obtained. Product 1 had a relatively short setting time and might be suitable as a dental cavity liner or for filling parodontological pockets or for filling alveolar cavities to prevent alveolar ridge resorption. Products 2 and 3 may be more suitable for orthopaedic purposes. Their compressive strength being 35 and 12 MPa, respectively, after soaking for 1 day in Ringer's solution at 37°C. Product 1 reaches its full strength within 4 h, whereas products 2 and 3 take about 12 h and 10 h, respectively.     

Dense polycrystalline β-tricalcium phosphate for prosthetic applications

A method is described for preparing dense, polycrystalline-tricalcium phosphate. The compressive and flexural strengths of the polycrystalline bodies sintered at a temperature of 1150° C for 1 h were found to be 459 and 138 MPa, respectively. Observation of the fracture surfaces by scanning electron microscopy indicates a predomination of transgranular failures. The polycrystalline tricalcium phosphate is non-toxic in a cell culture system.     

Doped calcium–aluminium–phosphate cements for biomedical applications

Calcium–aluminium–phosphate cements (CAPCs) for biomedical applications, mainly intended for applications in the dental field as non-resorbable fillers, were obtained by reacting Ca-aluminates compounds, i.e. CaO·Al₂O₃ (CA) and CaO·2 Al₂O₃ (CA₂), with Al(H₂PO₄)₃ aqueous solution. Hydroxyapatite was also introduced as a bioactive dispersed phase. Suitable elements like Sr and La were used to increase the radiopacity of the set yielded pastes towards X-ray wavelength used in clinical diagnostic radiographic equipments. La and Sr doped Ca-aluminates powders have been synthesized by solid state reaction at 1,400°C from a mixture of CaCO₃, Al₂O₃, La₂O₃ and SrCO₃. The characteristics of the obtained powders were analyzed and related to the starting compositions and synthesis procedures. The microstructure, setting time, radiopacity and compressive strength of the CAPCs have been investigated and discussed.     

Biodegradable β-tricalcium phosphate cement with anti-washout property based on chelate-setting mechanism of inositol phosphate

Novel biodegradable β-tricalcium phosphate (β-TCP) cements with anti-washout properties were created on the basis of chelate-setting mechanism of inositol phosphate (IP6) using β-TCP powders. The β-TCP powders were ball-milled using ZrO₂ beads for 0–6 h in the IP6 solutions with concentrations from 0 to 10,000 ppm. The chelate-setting β-TCP cement with anti-washout property was successfully fabricated by mixing the β-TCP powder ball-milled in 3,000 ppm IP6 solution for 3 h and 2.5 mass% Na₂HPO₄ solution, and compressive strength of the cement was 13.4 ± 0.8 MPa. An in vivo study revealed that the above cement was directly in contact with host and newly formed bones without fibrous tissue layers, and was resorbed by osteoclast-like cells on the surface of the cement. The chelate-setting β-TCP cement with anti-washout property is promising for application as a novel injectable artificial bone with both biodegradability and osteoconductivity.     

Preparation of nanosized β-tricalcium phosphate particles with Zn substitution

Nanosized β-ZnTCP powders with different Zn contents were prepared through coprecipitation of ACP out of the CaCl₂-ZnCl₂-Na₃PO₄-PEG system, and calcination of the ACP precursor at 800 °C for 3 h. The characterizations of the products showed that the products belong to β-TCP phase, and the particles sizes of them are about 300 nm, smaller than that of β-TCP (500 nm). Both the Zn_2p binding energy and lattice parameter variations of β-TCP evidenced that Zn had substituted for Ca in the lattice. Such nanosized β-ZnTCP powders could be used as bone repair materials with desired and sustained release of Zn.     

Phase transformation and structural characteristics of zinc-incorporated β-tricalcium phosphate

Biological performance of bioceramics such as calcium phosphate has been proved to be improved by substitution of different ions like Mg, Sr and Si. In this study, different amounts of Zn ions in nitrate form were incorporated into β-tricalcium phosphate in which various molar ratios of Ca:Zn were achieved: 3:0, 2.8:0.2, 2.6:0.4, 2.4:0.6, and 2.2:0.8. The mixtures were heated at different temperatures ranging from 800–1100 °C. The phase composition, amount of each phase and lattice parameters of β-tricalcium phosphate were determined by means of X-ray diffractometry and coupled software. Also, solubility of the heated mixtures was investigated by determining the amount of Ca and Zn released into a simulated body fluid during 120 h. The results revealed that only limited amount of Zn ions could be incorporated into β-tricalcium phosphate lattice and ZnO phase was formed when high content of zinc nitrate was introduced in initial mixture. Both a and c lattice parameters of β-tricalcium phosphate were reduced by adding Zn. The release rate of calcium ions into the simulated body fluid was approximately constant during 120 h while for Zn minor release was observed.     

Fabrication and biological characteristics of β-tricalcium phosphate porous ceramic scaffolds reinforced with calcium phosphate glass

The fabrication process, compressive strength and biocompatibility of porous β-tricalcium phosphate (β-TCP) ceramic scaffolds reinforced with 45P₂O₅–22CaO–25Na₂O–8MgO bioglass (β-TCP/BG) were investigated for their suitability as bone engineering materials. Porous β-TCP/BG scaffolds with macropore sizes of 200–500 μm were prepared by coating porous polyurethane template with β-TCP/BG slurry. The β-TCP/BG scaffolds showed interconnected porous structures and exhibited enhanced mechanical properties to those pure β-TCP scaffolds. In order to assess the effects of chemical composition of this bioglass on the behavior of osteoblasts cultured in vitro, porous scaffolds were immersed in simulated body fluid (SBF) for 2 weeks, and original specimens (without soaked in SBF) seeded with MC3T3-E1 were cultured for the same period. The ability of inducing apatite crystals in simulated body fluid and the attachment of osteoblasts were examined. Results suggest that apatite agglomerates are formed on the surface of the β-TCP/BG scaffolds and its Ca/P molar ratio is ~1.42. Controlling the crystallization from the β-TCP/BG matrix could influence the releasing speed of inorganic ions and further adjust the microenvironment of the solution around the β-TCP/BG, which could improve the interaction between osteoblasts and the scaffolds.     

Pulse electric current sintering of hydroxyapatite/β-tricalcium phosphate composites

Hydroxyapatite (HA)/β-tricalcium phosphate (β-TCP) composites attract attentions as bone implant materials. As one of the fabrication method of HA/β-TCP is mixing of HA and β-TCP powder in advance of sintering. This method enables to control the ratio of content of β-TCP easier. However, it is difficult to obtain dense composites. In this study, we focused on pulse electric current sintering (PECS) to obtain dense HA/β-TCP composites. The sinterability is evaluated with relative density and grain size measurements. Composition of sintered body was also characterized by X-ray diffraction. In comparison with pressureless sintering, PECS increased relative density of the composites without grain growth. In HA/β-TCP sintered by PECS, the phase transformation from β-TCP to α-TCP was promoted. This is due to higher thermal energy by spark discharge during PECS. On the other hand, sintering additives (MgO) inhibited phase transformation. It was suggested that sinterability of HA/β-TCP composites was improved by PECS.     

Microwave-assisted co-precipitation synthesis of high purity β-tricalcium phosphate crystalline powders

A new fabrication technique of β-tricalcium phosphate (β-TCP) has been investigated by the microwave-assisted co-precipitation method and subsequent calcinations. The synthesized powders were characterized by X-ray diffraction (XRD), Fourier transform-infrared spectroscopy (FT-IR), thermogravimetry-differential thermal analysis (TG-DTA), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The calcium/phosphorous atomic ratio of the as-dried sample was determined by energy dispersive analysis of X-rays (EDAX). The results showed that high purity and well-crystallized β-TCP powders could be obtained in a rapid way by microwave processing. The formation process of β-TCP was explained based on the TG-DTA analysis. The effects of microwave irradiation on the reaction mechanism and reaction rate were preliminarily analyzed in the paper.