TTCP Powder Research Articles

Novel artificial tricalcium phosphate and magnesium composite graft facilitates angiogenesis in bone healing

BackgroundBone grafting is the standard treatment for critical bone defects, but autologous grafts have limitations like donor site morbidity and limited availability, while commercial artificial grafts may have poor integration with surrounding bone tissue, leading to delayed healing. Magnesium deficiency negatively impacts angiogenesis and bone repair. Therefore, incorporating magnesium into a synthetic biomaterial could provide an excellent bone substitute. This study aims to evaluate the morphological, mechanical, and biological properties of a calcium phosphate cement (CPC) sponge composed of tetracalcium phosphate (TTCP) and monocalcium phosphate monohydrate (MCPM), which could serve as an excellent bone substitute by incorporating magnesium. MethodsThis study aims to develop biomedical materials composed mainly of TTCP and MCPM powder, magnesium powder, and collagen. The materials were prepared using a wet-stirred mill and freeze-dryer methods. The particle size, composition, and microstructure of the materials were investigated. Finally, the biological properties of these materials, including 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay for biocompatibility, effects on bone cell differentiation by alkaline phosphatase (ALP) activity assay and tartrate-resistant acid phosphatase (TRAP) activity assay, and endothelial cell tube formation assay for angiogenesis, were evaluated as well. ResultsThe data showed that the sub-micron CPC powder, composed of TTCP/MCPM in a 3.5:1 ratio, had a setting time shorter than 15 minutes and a compressive strength of 4.39±0.96 MPa. This reveals that the sub-micron CPC powder had an adequate setting time and mechanical strength. We found that the sub-micron CPC sponge containing magnesium had better biocompatibility, including increased proliferation and osteogenic induction effects without cytotoxicity. The CPC sponge containing magnesium also promoted angiogenesis. ConclusionIn summary, we introduced a novel CPC sponge, which had a similar property to human bone promoted the biological functions of bone cells, and could serve as a promising material used in bone regeneration for critical bone defects.

Synthesis and evaluation of bone morphogenetic protein (BMP)-loaded hydroxyapatite microspheres for enhanced bone regeneration

This study demonstrates a novel, simple way of synthesizing bone morphogenetic protein (BMP)-loaded hydroxyapatite (HA) microspheres for enhanced bone regeneration. To accomplish this, calcium phosphate cement (CPC) microspheres, consisting of α-TCP and TTCP powders, were prepared using water-in-oil (W/O) emulsion, followed by treatment at 37°C for 3 days to convert the CPC to HA. The BMP-loaded HA microspheres with a well-defined spherical shape had a highly porous structure, which was created by the entanglement of precipitated HA crystals with a needle-like morphology. The unique porous structure with the bone mineral-like HA phase resulted in a considerable improvement in in vitro biocompatibility. In addition, the bone regeneration ability of the HA microspheres was significantly enhanced by BMP loading, which was examined by in vivo animal testing using a rabbit vertical guided bone regeneration model.

Synthesis of Tetracalcium Phosphate at Reduced Temperatures

Tetracalcium phosphate (TTCP) requires the highest synthesis temperatures of all the calcium phosphates, but now a new process is available at 400 °C lower than previously, at 900 °C. Instead of ball-milling reactants for a homogeneous mix, the reactants were included in an amorphous phase. Heating produced hydroxyapatite, oxyapatite and then TTCP. Amorphous nanoparticles were synthesized and heated in air or in vacuum. The sequence of solid-state reactions were tracked with X-ray diffraction and Fourier transform infra-red spectroscopy. Heating in air stabilized the carbonate containing apatite, thereby requiring higher temperatures for decomposition, as per previous studies. Heating in vacuum promoted oxyapatite; a critical step for reaction with calcium oxide to generate TTCP. This faster process enables production at a lower temperature and reduces the use of ball milling for producing fine TTCP powders.

Synthesis of TTCP by using inverse micelle method

Tetracalcium phosphate (TTCP, Ca4(PO4)2O) is one of the major powder components of self-setting orthopedic and dental cements. Traditionally, TTCP powders are produced by a solid-state reaction of Ca- and P-containing precursors between 1350 and 1500 °C. Such procedures require expensive high-temperature furnaces and subsequent grinding of sintered particles. Grinding not only leads to the contamination but also alters the structure of TTCP, thereby reducing its bioactivity. The present paper offers an innovative approach to the inverse micelle method of TTCP synthesis, with a subsequent thermal treatment to obtain purer TTCP phase. The obtained powder was a mixture of TTCP and octacalcium phosphate (OCP) phases for thermally untreated sample, while for the sample thermally treated at 800 °C it was a mixture of TTCP and β-TCP phases. The TTCP phase was prevailing phase in both cases. A typical shape of TTCP nanoparticles was needle-like. Somewhere, needles are joined together forming platelet-like structures. Investigations of the obtained phases were made by XRD and TEM. The mechanism of chemical synthesis and structural arrangements of the obtained phases were particularly investigated.The specific surface area and mechanical properties (compressive and flexural strength) of cements based on needle like TTCP (n-TTCP) and irregular shape TTCP (i-TTCP), as the most frequent form of TTCP appeared in literature, were compared. It was shown, by corresponding calculations, that the specific surface area of n-TTCP (determined from TEM micrographs) was much higher than in the case of i-TTCP (determined from SEM micrographs). Beside, compressive and flexural strength of n-TTCP based cement were significantly improved, because n-TTCP whiskers considerably reinforced structure of hardened cement paste.

Surface modification of titanium by pack cementation treatment using calcium phosphate powders for biomedical applications

The surface modification of commercially pure titanium by pack cementation treatment using hydroxyapatite (HAp) or tetracalcium phosphate (TTCP) powder was investigated for temperatures ranging from 773 K to 1073 K. An HAp phase was detected on the surface of the titanium substrates after pack cementation treatment at temperatures of 973 K and 1073 K. After treatment using HAp and TTCP powders, a reaction layer with small HAp particles and pores containing small HAp particles, respectively, was observed. Apatite crystallites with a network pattern formed on the pack-cementation-treated titanium substrates after the substrates were immersed in Kokubo solution for 43.2 ks; such rapid apatite formation suggests that pack cementation treatment improves the biocompatibility of titanium.

Pack Cementation Treatment of Titanium Using Tetracalcium Phosphate Powder for Biomedical Applications

The surface modification of commercially pure titanium (CP Ti) by pack cementation treatment at 973 K using tetracalcium phosphate (Ca4(PO4)2O, TTCP) slurry was investigated. An HAp phase and a CaTiO3 phase were observed on the reaction layer of the CP Ti substrate after pack cementation treatment at 973 K for 86.4 ks. TTCP powder decomposed to HAp and CaO, and CaO reacted with TiO2 to form CaTiO3. The reaction layer on the CP Ti substrate consisted of inner and outer layers and the particles were in the outer reaction layer. The pores observed on the reaction layer were formed by the detachment of particles from the outer layer. The bonding strength of the reaction layer was 68.1 MPa. Apatite completely covered the surface of the pack-cementation-treated CP Ti after immersion in Kokubo solution for 21.6 ks; such rapid apatite formation suggests that pack cementation treatment improves the biocompatibility of titanium.

Synthesis of tetracalcium phosphate from mechanochemically activated reactants and assessment as a component of bone cements

The aim of this work was to gain a better understanding about the synthesis of tetracalcium phosphate (TTCP, Ca(4)(PO(4))(2)O) through a solid-state reaction from mechanochemically activated CaCO(3)-(NH(4))(2)HPO(4) mixtures. The evolution of the reaction was followed by DTA, XRD, FTIR and SEM techniques. An enhanced reactivity of the mixtures was detected as the mechanochemical treatment times increased. This effect was related to both the loss of crystallinity of the reactants and the production of defects on their surfaces. 6 h of mechanochemical processing at 1190 rpm, followed by 3 h of thermal treatment at 1500 degrees C, were enough to obtain pure TTCP. The crystallinity and purity of the obtained TTCP were checked by XRD and FTIR. The morphologic characteristics were analyzed by SEM and BET analysis. The behavior of synthesized TTCP powder in combination with commercial dicalcium phosphate anhydrous (DCPA, CaHPO(4)), as the solid phase of bone cements, was tested. Both the combination of different particle sizes of TTCP and DCPA and the effect of different kinds of accelerator agents (disodium hydrogen phosphate, tartaric acid, citric acid and oxalic acid) on setting time and degree of conversion to hydroxyapatite (HA, Ca(10)(PO(4))(6)(OH)(2)) were evaluated. The combination of TTCP (0.32 m(2)/g) with DCPA (1.52 m(2)/g), in a 1/1 molar ratio, showed the shortest setting times and high conversions to HA when an oxalic acid solution (5% volume fraction) was used as the liquid phase of the formulation. Results obtained from this work demonstrated that synthesized TTCP shows promising behavior as a component of bone cements, exhibiting not only a smaller particle size than that usually reported but also a low degree of crystallinity, all of which increases the reactivity of the obtained TTCP. This study provided a very efficient method for synthesizing pure TTCP through a modified solid-state reaction from mechanochemically activated reactants, employing very short times of thermal treatment in comparison with the conventional processes.

Morphology and mechanical behavior of TTCP-derived calcium phosphate cement subcutaneously implanted in rats

A pre-hardened, TTCP-derived CPC was immersed in Hanks' solution as well as subcutaneously implanted into abdomen of rats. The implant-soft tissue interfacial morphology was examined and properties of the CPC were evaluated and compared under in vitro and in vivo conditions. The results indicate that the surface of immersed samples appeared rougher and more porous than that of implanted samples and was covered with a layer of fine apatite crystals. The CPC samples implanted for 4 weeks or longer were surrounded by a layer of fibrous tissue, which was further surrounded by a soft tissue capsule comprising numerous fat cells. The soft tissue capsule had a non-uniform distribution in thickness, which increased most significantly between 4 weeks and 12 weeks after implantation. None of polymorphic cells, osteoblast cells or bone cells adjacent to the implant were observed. The majority of original TTCP powder was transformed into apatite after 1 day of either immersion in Hanks' solution or implantation. The average porosity values of samples immersed in Hanks' solution for 4 weeks or longer were significantly larger than those immersed for 1 day or 1 week. The porosity values of samples implanted for different times were not significantly different. The DTS values of Hanks' solution-immersed samples largely decreased after a few weeks of immersion. The implanted samples maintained their strengths throughout the study.

Synthesis of HA‐Seeded TTCP (Ca4(PO4)2O) Powders at 1230°C from Ca(CH3COO)2·H2O and NH4H2PO4

Tetracalcium phosphate (TTCP) Ca4(PO4)2O is one of the major powder components of self‐setting orthopedic and dental cements. Traditionally, TTCP powders are produced by a solid‐state process by soaking Ca‐ and P‐containing precursors between 1350° and 1500°C. Such procedures require expensive high‐temperature furnaces and subsequent grinding of sintered particulates. Grinding not only introduces contamination but alters the structure of TTCP, thereby reducing its bioactivity. The present paper offers a lower temperature synthesis process for TTCP with several interesting features. First, the synthesis procedure used Ca(CH3COO)2·H2O and NH4H2PO4 as separate sources for Ca and P, respectively. Second, the reactants underwent multiple melting and decomposition stages, thus increasing the reactivity of the synthesis process. NH4H2PO4 melted at 190°C and engulfed the calcium acetate particles. The Ca‐acetate component decomposed into CaCO3 at around 400°C while still surrounded by the molten phosphate liquid and an amorphous Ca‐metaphosphate phase. Hydroxyapatite, Ca10(PO4)6(OH)2 (HA), and β‐Ca3(PO4)2 crystallized upon heating the powder mixture to 700°C. Slightly above 1200°C, the TTCP phase was formed. This sequence of reactions led to a process temperature of 1230°C, the lowest temperature ever reported for the synthesis of TTCP. Third, the resulting powders required much less grinding, which itself is advantageous. Fourth, the resulting powders were in situ seeded with HA. HA‐seeded TTCP powders were tested for their apatite‐inducing ability by soaking them in synthetic body fluid at 37°C. TTCP powders of this study were readily covered with carbonated apatitic calcium phosphates within the first 72 h.

Effect of heat treatment on setting behavior and compressive strength of tetracalcium phosphate cement

Due to its superior biocompatibility and osteoconductivity, calcium phosphate cement (CPC) has been suggested for use as a filling material for dental and orthopedic applications [1–4]. The primary reaction product of most CPCs, after being implanted for a while, is various kinds of apatite [5], which is the main inorganic component of human bones and teeth [6, 7]. Tetracalcium phosphate (TTCP), a member of the calcium phosphate family, is known to have a high chemical activity and reacts with aqueous solution at room temperature [8]. The obtained hardened material has a high affinity for the living body and is therefore frequently used as a constituent in CPC powder [9–11]. Despite many advantages, there are problems for CPC during practical application, including working/setting time being too short or too long and dispersion upon early contact with blood or aqueous media. Appropriate working/setting times are critical for surgical applications. In the laboratory, a monolithic TTCP powder with nano-sized whiskers grown on surface was recently developed [12], which demonstrated excellent mechanical properties and biological responses with a relatively short working/setting time. The present work provides a simple heat treatment method for prolonging the working/setting time, while maintaining the strength, of the TTCP cement. The TTCP powder used for the study was fabricated in-house from the reaction of dicalcium pyrophosphate (Ca2P2O7) (Sigma Chem. Co., St. Louis, MO, USA) and calcium carbonate (CaCO3) (Katayama Chem. Co., Tokyo, Japan) by a weight ratio of 1:1.27. The powders were mixed uniformly in ethanol for 12 hr, followed by heating in an oven to let the powders dry. The dried powder mixture was then heated to 1400 ◦C and allowed to react and form TTCP [13]. Since a “whisker treatment” [12] has been very effective in enhancing the properties of TTCP cement, the same treatment was used to treat the TTCP powder for the present study. The whisker-treated TTCP powder was heat-treated in an air furnace (N 7/H, Nabertherm©R, Germany). Different heat-treatment temperatures (140–400 ◦C) and times (30 and 120 min) were used for the study. To form a CPC paste, the TTCP powder was mixed with a diammonium hydrogenphosphate ((NH4)2HPO4) hardening solution with a pH value of 8.6 and liquid/powder ratio of 0.3 ml/g. After mixing for 1 min, the cement

Transmission Electron Microscopic Study of Tetracalcium Phosphate Surface-Treated in Diammonium Hydrogen Phosphate Solution

The present study investigates the changes in microstructure and microchemistry (particularly Ca/P ratio) during whisker formation on the surface of a monolithic TTCP powder in a basic phosphoric acid solution. The XRD results indicate that when TTCP powder was treated for 10 min or less in (NH4)2HPO4 solution, the monolithic TTCP phase remains unchanged. When treated for 30 min, apatite whiskers appear, which continue to grow with treating time. When treated for 24 h, the apatite phase becomes dominant. TEM results show that on the surface of 1 min-treated TTCP are observed a large amount of globular-shaped fine particles which are substantially amorphous with an average Ca/P ratio of about 1.2. When treated for 10 min, whiskers primarily having a crystal structure of TTCP with an average Ca/P ratio of about 2.1 are observed to grow radially from TTCP surface. With time the whiskers continue to grow in length and width. The whiskers on 24 h-treated surface become Ca-rich apatite with an average Ca/P ratio of about 1.8. Both TTCP and apatite whiskers are essentially non-stoichiometric in chemistry.

Influence of cooling modes on purity of solid-state synthesized tetracalcium phosphate

Pure tetracalcium phosphate powder (TTCP) was prepared by a solid-state phase reaction at 1500 °C. Effects of cooling modes on the synthesizing process of TTCP powder and its thermal behavior at different heating temperatures were investigated by XRD and FTIR. The results show that cooling in dry air tends to promote formation of single phase TTCP, while in situ cooling in furnace results in a mixture of hydroxyapatite (HAP) and CaO. The examination of the thermal behavior of TTCP indicates that there exists a decomposing zone in the range of 500 °C–1200 °C, in which the high-temperature synthesized TTCP phase is transformed during the subsequent cooling process. This was confirmed by three additional cooling treatment routes that provide significant experimental evidence to the cooling modes effect on the purity of TTCP. The phase transformation course of starting materials heated to elevated temperatures was further studied by TGA–DSC analysis with the aid of XRD and FTIR, in order to fully understand the complicated synthesizing process of TTCP.

Hydrolysis of tetracalcium phosphate under a near-constant-composition condition—effects of pH and particle size

Tetracalcium phosphate (TTCP) is a component of a number of calcium phosphate cements used clinically for bone defect repairs. The strength, phase composition, and solubility of the set cement are highly dependent on the reactions of the cement components during setting. This study investigated hydrolysis reactions of TTCP under solution compositions chosen to mimic the compositions of the cement liquid during setting. The study utilized a pseudo-constant-composition technique that allowed both the rate and stoichiometry of the reaction to be determined while the reaction proceeded under a specific, constantly held solution pH, thereby keeping a constant calcium-to-phosphate ratio in solution. The hydrolysis experiments were conducted using either a fine (median particle size 3.5 μm) or coarse (median particle size 13.2 μm) TTCP powder at pH 7, 8 and 10. Low crystalline calcium (Ca)-deficient hydroxyapatite (HA) was the product in all experiments. Both the solution pH and TTCP particle size produced significant effects on all aspects of the hydrolysis reaction. At a given pH, the fine TTCP produced a HA product with a greater Ca deficiency than did the coarse TTCP. For a given particle size, the Ca deficiency generally decreased with increasing pH. Hydrolysis reaction rate generally decreased with increasing pH or TTCP particle size. At pH 7 and 8, the solution was undersaturated with respect to TTCP and supersaturated with respect to HA, suggesting that the reaction rate was limited by TTCP dissolution. In contrast, at pH 10, the solution was approximately saturated with respect to TTCP and highly supersaturated with respect to HA, suggesting that HA formation was the rate-determining step of the reaction. The findings provided useful insights into the setting reaction mechanisms of TTCP-containing calcium phosphate cements.

Transmission Electron Microscopic Study of Whisker Formation on Tetracalcium Phosphate in Hydrochloric Acid

The present study investigates the changes in microstructure and microchemistry (particularly Ca/P ratio) during whisker formation on the surface of TTCP powder in hydrochloric solution. XRD results indicate that the as-fabricated (non-treated) powder has a monolithic TTCP crystal structure. When the powder is treated for 10 min in HCl, a DCPD phase appears on TTCP surface, which increases in intensity with treating time. When treated longer than 4 h, the DCPD intensity decreases. When treated for 24 h, the DCPD phase disappears. TEM results indicate that, when TTCP is treated for 10 min, fine globular-shaped DCPD crystals are observed to precipitate on the surface of TTCP particles. When treated for 1 h, hexagonal Ca-deficient apatite whiskers with an average Ca/P ratio of 1.5 form. With treating time the apatite whiskers continue to grow in size and increase in Ca/P ratio. When treated for 24 h, the length and thickness of the apatite whiskers can reach >500 and >50 nm, respectively, with an average Ca/P ratio of 1.8. The microchemical data indicate that the apatite whiskers grown on the surface of TTCP particles are substantially non-stoichiometric in nature. Compressive strength data indicate that apatite whiskers grown on TTCP surface under a favorable condition can largely improve the properties of the resulting CPC.

Transmission electron microscopic study on setting mechanism of tetracalcium phosphate/dicalcium phosphate anhydrous-based calcium phosphate cement.

This work studied transmission electron microscopy on the setting mechanism of tetracalcium phosphate/dicalcium phosphate anhydrous (TTCP/DCPA)-based calcium phosphate cement. The results suggest the process for early-stage apatite formation as the follows: when TTCP and DCPA powders are mixed in the phosphate-containing solution, the TTCP powder is quickly dissolved because of its higher solubility in the acidic solution. The dissolved calcium and phosphate ions, along with those ions readily in the solution, are then precipitated predominantly on the surface of DCPA particles. Few apatite crystals were observed on the surface of TTCP powder. During the later stages of reaction, the extensive growth of apatite crystals/whiskers, with a calcium/phosphorous ratio very close to that of hydroxyapatite, effectively linked DCPA particles together and also bridged the larger TTCP particles. It is suggested that, when the large TTCP particles are locked in place by the bridging apatite crystals/whiskers, the CPC is set and would not dissolve when immersed in Hanks' solution after 20-40 min of reaction.

Variation in crystallinity of hydroxyapatite and the related calcium phosphates by mechanical grinding and subsequent heat treatment

The crystallinity of hydroxyapatite (HAp) and the related calcium phosphates for regenerating hard tissues was controlled by the mechanical grinding (MG) method and subsequent heat treatment. The HAp, carbonate-apatite (CO3Ap), fluorapatite (FAp), and α- and β-tricalcium phosphates (α-TCP and β-TCP, respectively) and tetracalcium diphosphate monoxide (TTCP) were used as initial materials. Variations in crystallinity and crystal structure were examined by the X-ray diffraction (XRD) method during MG and the following heat treatment. The crystallinity, based on crystallite size and crystal elastic strain, decreased with grinding time, and the decreasing rate depended on the type of calcium phosphate; crystallographic diffraction peaks disappeared more rapidly in CO3Ap than in FAp. The change in the morphology of powder during MG was influenced by the primary particle size of the first-stage product; α-TCP, β-TCP, and TTCP powders composed of large particles were predominantly shattered into small pieces and then gathered during MG, while the crystal strain in the HAp, CO3Ap, and FAp powders was mainly accumulated without significant refinement of crystallite size. The thermal-recovery process of crystallinity and crystal structure in the milled powders was investigated. The crystallinity of HAp, CO3Ap, and FAp powders recovered depended on annealing temperature. The novel phase of β’-TCP with higher ordering than β-TCP appeared during heat treatment from the amorphous state of α-TCP or β-TCP obtained during MG. The MG and subsequent heat treatment were, finally, concluded to be an effective process for controlling the crystallinity and changing crystal structure in calcium phosphate powders.