Na-doped β-tricalcium phosphate: physico-chemical and in vitro biological properties

Synthetic calcium phosphate ceramics as β-tricalcium phosphate (Ca₃(PO₄)₂; β-TCP) are currently successfully used in human bone surgery. The aim of this work was to evaluate the influence of the presence of sodium ion in β-TCP on its mechanical and biological properties. Five Na-doped-β-TCP [Ca_10.5−x/2Na _x (PO₄)₇, 0 ≤ x ≤ 1] microporous pellets were prepared via solid phase synthesis, and their physico-chemical data (lattice compacity, density, porosity, compressive strength, infrared spectra) denote an increase of the mechanical properties and a decrease of the solubility when the sodium content is raised. On the other hand, the in vitro study of MC3T3-E1 cell activity (morphology, MTS assay and ALP activity) shows that the incorporation of sodium does not modify the bioactivity of the β-TCP. These results strongly suggest that Na-doped-β-TCP appear to be good candidates for their use as bone substitutes.     

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.     

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.     

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.     

Electrical properties of α-zirconium phosphate and its alkali salts in a humid atmosphere

Humidity dependence of impedance was examined by using the complex impedance analysis for fine crystalline zirconium bis(monohydrogen phosphate) monohydrate and its alkali salts. The value of C ₀ in C _p()=C ₀(j/ ₀)^– was hardly dependent on the relative humidity and monovalent cation species. The resistive component and its activation energy decreased with increase in the relative humidity and the former was expressed as R=R ^* exp (–E/kT ^*) exp (E/kT) The pre-exponential factor R ^*, was hardly influenced by the humidity and decreased when the acidic protons were replaced with monovalent alkali cations. The activation energy of conduction was strongly affected by the degree of orientation and particle size of crystal with layered structure in a whole humidity region and increased by replacing the protons by alkali cations.     

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.     

Dual setting α-tricalcium phosphate cements

An extension of the application of calcium phosphate cements (CPC) to load-bearing defects, e.g. in vertebroplasty, would require less brittle cements with an increased fracture toughness. Here we report the modification of CPC made of alpha-tricalcium phosphate (α-TCP) with 2-hydroxyethylmethacrylate (HEMA), which is polymerised during setting to obtain a mechanically stable polymer-ceramic composite with interpenetrating organic and inorganic networks. The cement liquid was modified by the addition of 30–70 % HEMA and ammoniumpersulfate/tetramethylethylendiamine as initiator. Modification of α-TCP cement paste with HEMA decreased the setting time from 14 min to 3–8 min depending on the initiator concentration. The 4-point bending strength was increased from 9 MPa to more than 14 MPa when using 50 % HEMA, while the bending modulus decreased from 18 GPa to approx. 4 GPa. The addition of ≥50 % HEMA reduced the brittle fracture behaviour of the cements and resulted in an increase of the work of fracture by more than an order of magnitude. X-ray diffraction analyses revealed that the degree of transformation of α-TCP to calcium deficient hydroxyapatite was lower for polymer modified cements (82 % for polymer free cement and 55 % for 70 % HEMA) after 24 h setting, while the polymerisation of HEMA in the cement liquid was quantitative according to FT-IR spectroscopy. This work demonstrated the feasibility of producing fracture resistant dual-setting calcium phosphate cements by adding water soluble polymerisable monomers to the liquid cement phase, which may be suitable for an application in load-bearing bone defects.     

Long-term conversion of 45S5 bioactive glass–ceramic microspheres in aqueous phosphate solution

The conversion of 45S5 glass and glass–ceramics to a hydroxyapatite (HA)-like material in vitro has been studied extensively, but only for short reaction times (typically <3 months). In this paper, we report for the first time on the long-term conversion of 45S5 glass–ceramic microspheres (designated 45S5c) in an aqueous phosphate solution. Microspheres of 45S5c (75–150 μm) were immersed for 10 years at room temperature (~25 °C) in K₂HPO₄ solution with a concentration of 0.01 M or 1.0 M, and with a starting pH of 7.0 or 9.5. The reacted 45S5c microspheres and solutions were analyzed using structural and analytical techniques. Only 25–45 vol% of the 45S5c microspheres were converted to an HA-like material after the 10 year reaction. In solutions with a starting pH of 9.5, an increase in the K₂HPO₄ concentration from 0.01 to 1.0 M resulted in a doubling of the volume of the microspheres converted to an HA-like material but had little effect on the composition of the HA-like product. In comparison, reaction of the 45S5c microspheres in the solution with a starting pH of 7.0 resulted in an HA-like product in the 0.01 M K₂HPO₄ solution but a calcium pyrophosphate product, Ca₁₀K₄(P₂O₇)₆.9H₂O, in the 1.0 M solution. The consequences of these results for the long-term use of 45S5 glass–ceramics in biomedical applications are discussed.     

Calcium–phosphate–silicate composite bone cement: self-setting properties and in vitro bioactivity

In this study, a novel low temperature setting calcium phosphate–silicate cement was obtained by mixing CaHPO₄ · 2H₂O (DCPD) and Ca₃SiO₅ (C₃S) with 0.75 M sodium phosphate buffers (pH = 7.0) as liquid phase. The self-setting properties of the obtained DCPD/C₃S paste with liquid to powder ratio (L/P) of 0.6 ml/g, such as setting times, injectability, degradability and compressive strength were investigated and compared with that of DCPD/CaO cement system. The results indicated that, with the weight ratio of C₃S varied from 20% to 40%, the workable DCPD/C₃S pastes could set within 20 min, and the hydrated cement showed significantly higher compressive strength (around 34.0 MPa after 24 h) than that of the DCPD/CaO cement system (approximately 10.0 MPa). Furthermore, the in vitro pH value of the cements was investigated by soaking in simulated body fluid (SBF) for 12 h, and the result indicated that the DCPD/C₃S did not induce significant increase or decrease of pH value in SBF. Additionally, the composite cement possesses better ability to support and stimulate cell proliferation than the DCPD/CaO cement. With good hydraulic properties, improved biocompatibility and moderate degradability, the novel DCPD/C₃S bone cement may be a potential candidate as bone substitute.     

Subacute systemic toxicity assessment of β-tricalcium phosphate/carboxymethyl-chitin composite implanted in rat femur

The efficacy of a composite of β-tricalcium phosphate particles and carboxymethyl-chitin (β-TCP/CM-chitin) for bone repair has already been established in animal experiments. In the present study, subacute systemic toxicity was evaluated to further assess the biological safety of the implanted composite. β-TCP/CM-chitin (approximately 4 mg/kg and 7 mg/kg in male and female rats, respectively) was implanted for 28 days into penetrating defects (2 mm diameter) made artificially in the shaft of the right femur of rats. Sham operation groups with the defect only were prepared as controls. Haematology, blood chemistry, urinalysis, and the histopathology of 44 organs and tissues were investigated. Body weight measurements and clinical observations were performed daily throughout the study. No subacute systemic toxicity possibly caused by the implantation of β-TCP/CM-chitin was detected. These findings indicate that β-TCP/CM-chitin composite is a highly biocompatible bone substitute, at least with an implantation dosage of < 4–7 mg/kg.     

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.     

Characterization and distribution of mechanically competent mineralized tissue in micropores of β-tricalcium phosphate bone substitutes

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Osteoblast cell response to β-tricalcium phosphate scaffolds with controlled architecture in flow perfusion culture system

Porous β-tricalcium phosphate (β-TCP) scaffolds with controlled architecture and improved mechanical properties were fabricated by combining the gel-casting and rapid prototyping techniques. The pore morphology, size, and distribution of the β-TCP scaffolds were characterized using a scanning electron microscope. The porosity of the resulting scaffolds with pore size range from 300 to 500 μm was 46%. The average compressive strength was 16.1 MPa. X-ray diffraction was used to determine the crystal structure and chemical composition of scaffolds. The result indicated that the sintering process has not changed the composition of β-TCP. Flow perfusion culture system was developed in our lab to improve mass transfer for seeded cells. For scaffold/cell constructs cultured under flow perfusion for 4, 8, and 16 days, there was greater scaffold cellularity and alkaline phosphatase activity compared with static culture condition. These results indicated that flow perfusion culture system had evident effects on osteoblast viability and functions in vitro.     

In vitro corrosion behaviour of Mg alloys in a phosphate buffered solution for bone implant application

The corrosion behaviour of Mg–Mn and Mg–Mn–Zn magnesium alloy in a phosphate buffered simulated body fluid (SBF) has been investigated by electrochemical testing and weight loss experiment for bone implant application. Long passivation stage and noble breakdown potential in the polarization curves indicated that a passive layer could be rapidly formed on the surface of magnesium alloy in the phosphate buffered SBF, which in turn can protect magnesium from fast corrosion. Surfaces of the immersed magnesium alloy were characterized by SEM, EDS, SAXS and XPS. Results have shown that Mg–Mn and Mg–Mn–Zn alloy were covered completely by an amorphous Mg-containing phosphate reaction layer after 24 h immersion. The corrosion behaviour of magnesium alloys can be described by the dissolving of magnesium through the reaction between magnesium and solution and the precipitating of Mg-containing phosphate on the magnesium surface. Weight loss rate and weight gain rate results have indicated that magnesium alloys were corroded seriously at the first 48 h while Mg-containing phosphate precipitated fast on the surface of magnesium alloy. After 48–96 h immersion, the corrosion reaction and the precipitation reaction reach a stable stage, displaying that the phosphate layer on magnesium surface, especially Zn-containing phosphate layer could provide effective protection for magnesium alloy.     

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.     

A PLA/calcium phosphate degradable composite material for bone tissue engineering: an in vitro study

Biodegradable polymers reinforced with an inorganic phase such as calcium phosphate glasses may be a promising approach to fulfil the challenging requirements presented by 3D porous scaffolds for tissue engineering. Scaffolds’ success depends mainly on their biological behaviour. This work is aimed to the in vitro study of polylactic acid (PLA)/CaP glass 3D porous constructs for bone regeneration. The scaffolds were elaborated using two different techniques, namely solvent-casting and phase-separation. The effect of scaffolds’ micro and macrostructure on the biological response of these scaffolds was assayed. Cell proliferation, differentiation and morphology within the scaffolds were studied. Furthermore, polymer/glass scaffolds were seeded under dynamic conditions in a custom-made perfusion bioreactor. Results indicate that the final architecture of the solvent-cast or phase separated scaffolds have a significant effect on cells’ behaviour. Solvent-cast scaffolds seem to be the best candidates for bone tissue engineering. Besides, dynamic seeding yielded a higher seeding efficiency in comparison with the static method.     

Potentiostatic pulse-deposition of calcium phosphate on magnesium alloy for temporary implant applications — An in vitro corrosion study

In this study, a magnesium alloy (AZ91) was coated with calcium phosphate using potentiostatic pulse-potential and constant-potential methods and the in vitro corrosion behaviour of the coated samples was compared with the bare metal. In vitro corrosion studies were carried out using electrochemical impedance spectroscopy and potentiodynamic polarization in simulated body fluid (SBF) at 37 °C. Calcium phosphate coatings enhanced the corrosion resistance of the alloy, however, the pulse-potential coating performed better than the constant-potential coating. The pulse-potential coating exhibited ~ 3 times higher polarization resistance than that of the constant-potential coating. The corrosion current density obtained from the potentiodynamic polarization curves was significantly less (~ 60%) for the pulse-deposition coating as compared to the constant-potential coating. Post-corrosion analysis revealed only slight corrosion on the pulse-potential coating, whereas the constant-potential coating exhibited a large number of corrosion particles attached to the coating. The better in vitro corrosion performance of the pulse-potential coating can be attributed to the closely packed calcium phosphate particles.     

The copolymer of ε-caprolactone-lactide and tricalcium phosphate does not enhance bone growth in mandibular defect of sheep

In the field of craniomaxillofacial and orthopaedic surgery there is a constant need for bone or bone substitute. At the present, the most effective way to enhance bone healing clinically is to use autogenous bone grafts. The problems associated with the use of these autografts are donor site morbidity, limited supply and need for a second operative site. Currently there are several different synthetic products commercially available in the market; nevertheless, none of them is ideal for filling bone defects. Therefore, search for new synthetic materials for bone replacement is necessary. A mixture of tricalcium phosphate (TCP) and ε-caprolactone-lactide copolymer P(ε -CL/DL-LA) was prepared and implanted in critical size mandibular bone defects in twelve sheep. Contralateral side was used as a control. Follow-up times for histological and radiological studies were 9, 14, 24 and 52 weeks. We found that the implanted material did not enhance bone formation compared to control site. We also confirmed that defect size was of critical size, since there was no complete healing of the control site either. The results do not encourage us to continue our studies with the mixture of TCP and P(ε-CL/DL-LA) as a filling material for bone defects. Therefore the search for the ideal material is still ongoing.     

Microemulsion-based patch for transdermal delivery of huperzine A and ligustrazine phosphate in treatment of Alzheimer’s disease

The aim of the present study was to construct an innovative microemulsion-based patch for simultaneously transdermal delivery of huperzine A (HA) and ligustrazine phosphate (LP). The pseudo-ternary phase diagrams for microemulsion region were developed using oleic acid as oil, Cremophor RH40 as a surfactant, and ethanol as a cosurfactant. 1,8-cineole was added to the microemulsion as a penetration enhancer. The microemulsion-based transdermal patches were prepared by the lamination technique. The permeation studies were performed in vitro to evaluate the abilities of various microemulsions and transdermal patches to deliver HA and LP across the rat abdominal skin, showing that microemulsions increased the permeation rates of HA and LP compared with the control, and the penetration kinetics of the transdermal patch was in a zero order process. The results of the pharmacodynamic studies indicated that the transdermal combination therapy of HA and LP showed more benefits for fighting against amnesia in comparison with monotherapy. The anti-amnesic effects were also confirmed in scopolamine-induced amnesia rats after transdermal administration at multiple doses for 9 consecutive days, and the efficacy exhibited a dose-dependent manner. As a conclusion, the microemulsion-based transdermal patch containing HA and LP might provide a feasible strategy for the prevention of Alzheimer’s disease.