Quantitative histomorphometric evaluation of spinal arthrodesis after biphasic calcium phosphate ceramic implantation in sheep

The effectiveness of a macroporous biphasic calcium phosphate ceramic was studied after laterovertebral arthrodesis in sheep. A ceramic with a TCP/HAP ratio of 35/65 was compared with autogenous bone graft 1, 3, 6, 9 and 12 months after implantation. Surface, lengths, and relative pore parameters have been quantified by using light microscopy and image analysis. Quantitative analysis of the results indicated that the biphasic ceramic allows an arthrodesis after 12 months, although control graft is effective after six months. The implanted material is still present after 12 months. Degradation of the ceramic is initially fast, then slows down. The intra-implant new bone formation reaches 15% after three months and 19% after 12 months. In contrast, bone ingrowth increases from 9% after three months to 23% after 12 months in intertransverse zone. Bone formation was seen with a higher frequency in pores of 240–480 µm in average diameters. New bone fills only one part of the area of a pore. A spinal fusion can be obtained with this biphasic ceramic, but the use of this material cannot be currently recommended without further investigations.     

Synthesis of biphasic calcium phosphate containing nanostructured films by micro arc oxidation on magnesium alloy

The present research reports the synthesis of an innovative nanostructured composite film containing biphasic calcium phosphate (BCP) by the micro arc oxidation (MAO) method on AZ31 magnesium alloy. Nanometric structure of the used hydroxyapatite powder and the coatings were characterized by means of transmission and field-emission scanning electron microscope, respectively. Electrochemical behaviors of the pure MAO and nanocomposite films were also evaluated by electrochemical impedance spectroscopy and potentiodynamic polarization tests in simulated body fluid (SBF) environment. The results showed higher corrosion resistance of nanocomposite film compared to pure MAO coating, which was related to the blocking feature of the nanoparticles from the diffusing of the corrosive medium through the substrate. In addition, by immersing the specimens in simulated body fluid, greater apatite forming ability of the nanocomposite coating was proved.     

Macroporous biphasic calcium phosphate ceramics versus injectable bone substitute: a comparative study 3 and 8 weeks after implantation in rabbit bone

Macroporous biphasic calcium phosphate ceramics (MBCP) and a calcium phosphate injectable bone substitute (IBS), obtained by the association of biphasic calcium phosphate (BCP) ceramic granules and an aqueous solution of a cellulosic polymer, were compared in the same animal model. The two tested biomaterials were implanted in distal femoral osseous defects in rabbits. Qualitative and quantitative histological evaluation was performed three and eight weeks after implantation to investigate bone colonization and ceramic biodegradation associated with the two bone substitutes.Both biomaterials expressed osteoconduction properties and supported the apposition of a well-mineralized lamellar newly-formed bone. Bone colonization occurred much earlier and faster for IBS than for MBCP implants, although the respective rates of newly-formed bone after eight weeks of implantation did not differ significantly. For both biomaterials, ceramic resorption occurred regularly throughout the implantation period, though to a greater extent with IBS than with MBCP implants.The associated polymer in IBS produced intergranular spaces allowing body fluids to reach each BCP ceramic granule immediately after implantation, which may have favored osteoblastic activity, new bone formation and ceramic resorption. This completely interconnected open macroporosity could account for the earlier and more satisfactory bone substitution achieved with IBS. © 2001 Kluwer Academic Publishers     

The thermal stability of hydroxyapatite in biphasic calcium phosphate ceramics

Biphasic calcium phosphate ceramics (BCP) comprising a mix of non-resorbable hydroxyapatite (HA) and resorbable β-tricalcium phosphate (β-TCP) are particularly suitable materials for synthetic bone substitute applications. In this study, HA synthesised by solid state reaction was mechanically mixed with β-TCP, then sintered to form a suite of BCP materials with a wide range of HA/β-TCP phase content ratios. The influence of sintering temperature and composition on the HA thermal stability was quantified by X-ray diffraction (XRD). The pre-sinter β-TCP content was found to strongly affect the post-sinter HA/β-TCP ratio by promoting the thermal decomposition of HA to β-TCP, even at sintering temperatures as low as 850 °C. For BCP material with pre-sinter HA/β-TCP = 40/60 wt%, approximately 80% of the HA decomposed to β-TCP during sintering at 1000 °C. Furthermore, the HA content appeared to influence the reverse transformation of α-TCP to β-TCP expected upon gradual cooling from sintering temperatures greater than 1125 °C. Because the HA/β-TCP ratio dominantly determines the rate and extent of BCP resorption in vivo, the possible thermal decomposition of HA during BCP synthesis must be considered, particularly if high temperature treatments are involved.     

Current state of the art of biphasic calcium phosphate bioceramics

We have developed 15 years ago, with the collaboration of Lynch, Nery, and LeGeros in the USA, a bioactive concept based on biphasic calcium phosphate (BCP) ceramics. The concept is determined by an optimum balance of the more stable phase of HA and more soluble TCP. The material is soluble and gradually dissolves in the body, seeding new bone formation as it releases calcium and phosphate ions into the biological medium. The bioactive concept based on the dissolution/transformation processes of HA and TCP has been applied to both Bulk, Coating and Injectable Biomaterials. The events at the calcium phosphate (CaP) biomaterial/bone interface represent a dynamic process, including physico-chemical processes, crystal/proteins interactions, cells and tissue colonization, bone remodeling, finally contributing to the unique strength of such interfaces. An important literature and numerous techniques have been used for the evaluation of the fundamental physico chemical and biological performance of BCP concept. This type of artificial bone used from a long time in preclinical and in clinical trial, revealed the efficiency for bone filling, performance for bone reconstruction and efficacy for bone ingrowth at the expense of the micro macroporous BCP bioceramics.     

不同孔隙率的双相磷酸钙陶瓷降解性能研究

包含羟基磷灰石(HA)和磷酸三钙(TCP)的双相磷酸钙陶瓷(BCP)由于其具有良好的降解性能和良好的骨诱导性被看作是骨替代和修复的首选材料。BCP陶瓷的降解性能主要受相成份、孔隙率、材料微观形貌的影响。通过化学共沉淀法制得了成份为60/40(HA/TCP)的双相BCP粉体,通过双氧水发泡法制得了孔隙率分别为40%、60%和80%的多孔双相BCP陶瓷。选用Tris缓冲液浸泡的方法测试材料的体外降解行为。结果显示,孔隙率的改变有效地调控了BCP陶瓷的降解性能。随孔隙率的增加材料的溶出显著加快。高孔隙率材料的快速降解,在体系中释放出相对较高的钙、磷浓度,这可能是其高生物活性的重要影响因素。     

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.     

Preparation and characterization of biphasic calcium phosphate ceramics of desired composition

A modified processing route for fabricating dense and porous biphasic calcium phosphate (BCP) ceramics of desired and reproducible phase composition (hydroxyapatite (HA)/beta-tricalcium phosphate (beta-TCP) ratio) has been developed. The principal idea of the route was combining a precipitation and a solid phase methods. First, a nonstoichiometric (slightly carbonated calcium-deficient) HA (CdHA) precipitate was synthesized by mixing a calcium carbonate (CaCO(3)) water suspension with an orthophosphoric acid (H(3)PO(4)) solution in abundance (related to the amount resulting in a stoichiometric HA) under definite conditions, and a powder of the precipitate was prepared and calcinated in air (860 degrees C, 1.5 h). In the second stage, a BCP ceramics of the composition determined by the calcium-deficiency in a calcinated powder (the acid abundance in a mixture) was processed by sintering powder compacts with or without a porosizer under appropriate conditions (1,200 degrees C, 2h). A calibrating dependence of the HA/beta-TCP ratio in the ceramics on the acid abundance has been plotted which enabled a controlled preparation of BCP ceramics. A correlation based on unresolved bands in nu(4)-PO (4) (3-) domain in IR-spectra of nanostructured BCP materials was found. Using the correlation, the process of CdHA --> beta-TCP transformation could be easily monitored. The density and microhardness of the BCP ceramics neglectly depended on the composition, however, the compressive strength did: the lower the HA/beta-TCP ratio, the higher the strength in the dense materials.     

Physicochemical properties and cytotoxicities of Sr-containing biphasic calcium phosphate bone scaffolds

This study demonstrates a new biomaterial system composed of Sr-containing hydroxyapatite (Sr-HA) and Sr-containing tricalcium phosphate (Sr-TCP), termed herein Sr-containing biphasic calcium phosphate (Sr-BCP). Furthermore, a series of new Sr-BCP porous scaffolds with tunable structure and properties has also been developed. These Sr-BCP scaffolds were obtained by in situ sintering of a series of composites formed by casting various Sr-containing calcium phosphate cement (Sr-CPC) into different rapid prototyping (RP) porous phenol formaldehyde resins, which acted as the negative moulds for controlling pore structures of the final scaffolds. Results show that the porous Sr-BCP scaffolds are composed of Sr-HA and Sr-TCP. The phase composition and the macro-structure of the Sr-BCP scaffold could be adjusted by controlling the processing parameters of the Sr-CPC pastes and the structure parameters of the RP negative mould, respectively. It is also found that both the compressive strength (CS) and the dissolving rate of the Sr-BCP scaffold significantly vary with their phase composition and macropore percentage. In particular, the compressive strength achieves a maximum CS level of 9.20 ± 1.30 MPa for the Sr-BCP scaffold with a Sr-HA/Sr-TCP weight ratio of 78:22, a macropore percentage of 30% (400–550 μm in size) and a total-porosity of 63.70%, significantly higher than that of the Sr-free BCP scaffold with similar porosity. All the extracts of the Sr-BCP scaffold exhibit no cytotoxicity. The current study shows that the incorporation of Sr plays an important role in positively improving the physicochemical properties of the BCP scaffold without introducing obvious cytotoxicity. It also reveals a potential clinical application for this material system as bone tissue engineering (BTE) scaffold.     

Ultrasonic implantation of calcium metasilicate glass particles into PMMA

Polymer materials for clinical applications should be bioactive and have a bone-bonding ability. In order to provide poly(methyl methacrylate) (PMMA) with bioactivity, granules (<45 m) of a bioactive glass 50CaO·50SiO2 (mol %) were implanted into PMMA: they were suspended together with a piece of PMMA in a 40 tetrahydrofuran-60 ethanol (vol %) solution and ultrasonically agitated. The granules of <10 m in size were impregnated at 40–20 m depth below the substrate surface. Two types were detected on the PMMA surface: (a) a glass-granule layer on PMMA, and (b) an inner granule layer, a PMMA layer, and an outer granule layer on the PMMA. The bioactivity of the implanted PMMA substrates was examined in vitro with a simulated body fluid (Kokubo solution). Apatite was precipitated on all glass granules and the whole substrate surfaces within 1 d. After 4 h soaking in the Kokubo solution, aggregates of apatite particles appeared on the substrate surface, independently of those on the glass granules, and they grew and proliferated on the whole subtrate surface in 7 d. Silica gel islands on PMMA due to the silicate anions from the glass were considered to induce nucleation of the apatite particles.     

Strontium substituted calcium phosphate biphasic ceramics obtained by a powder precipitation method

Strontium (Sr) substituted calcium phosphate ceramics were fabricated using a powder precipitation method. The Sr ions were added up to 8 mol % to replace the Ca ions during the powder preparation. Composition analysis showed that the added Sr was not fully incorporated within the as-precipitated apatite structure, presumably being washed out during the powder preparation. After calcination, the Sr containing powders were crystallized into apatite and tricalcium phosphate (TCP), that is, biphasic calcium phosphates were formed. The amount of TCP increased with increasing the Sr addition. The lattice parameters of the calcined powders increased gradually with Sr substitution in both the a- and c-axis. However, the obtained values deviated slightly from the calculated ones at higher Sr additions (>4%) due to the partial substitution of Sr ions. The microstructure of the sintered bodies changed with the Sr addition due to the formation of TCP. The Vickers hardness increased slightly from 5.2 to 5.5 MPa with increasing Sr addition, which was driven by the HA+TCP biphasic formation. The osteoblast-like cells cultured on the Sr-substituted biphasic sample spread and grew actively. The proliferation rate of the cells was higher in the samples containing more Sr. The alkaline phosphate activity of the cells was expressed to a higher degree with increasing Sr addition. These observations confirmed the enhanced cell viability and differentiation of the Sr-substituted biphasic calcium phosphate ceramics.     

Mesenchymal stem cells combined with biphasic calcium phosphate ceramics promote bone regeneration

The reconstruction and repair of large bone defects, resulting from trauma, cancer or metabolic disorders, is a major clinical challenge in orthopaedics. Clinically available biological and synthetic grafts have clear limitations that necessitate the development of new graft materials and/or strategies. Human mesenchymal stem cells (MSCs), obtained from the adult bone marrow, are multipotent cells capable of differentiating into various mesenchymal tissues. Of particular interest is the ability of these cells to differentiate into osteoblasts, or bone-forming cells. At Osiris, we have extensively characterized MSCs and have demonstrated MSCs can induce bone repair when implanted in vivo in combination with a biphasic calcium phosphate, specifically hydroxyapatite/tricalcium phosphate. This article reviews previous and current studies utilizing mesenchymal stem cells and biphasic calcium phosphates in bone repair.     

Cytocompatibility evaluation of microwave sintered biphasic calcium phosphate scaffolds synthesized using pH control

Compounds belonging to the calcium phosphate (CaP) system are known to be major constituents of bone and are bioactive to different extents in vitro and in vivo. Their chemical similarity makes them prime candidates for implants and bone tissue engineering scaffolds. CaP nanoparticles of amorphous hydroxyapatite (aHA) and dicalcium phosphate dihydrate (DCPD) were synthesized using chemical precipitation. Uniaxially pressed aHA and DCPD powders were subjected to microwave radiation to promote solid state phase transformations resulting in crystalline hydroxyapatite (HA), tricalcium phosphate (TCP) and biphasic compositions: HA/TCP and TCP/calcium pyrophosphate (CPP) and their subsequent densification. Phase composition of microwave sintered compacts was confirmed via X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). Solution pH during crystal growth was found to have a profound effect on particle morphology and post-sintered phases, despite constant sintering temperature.Cytocompatibility assessment using 7F2 cells, corresponding to adult mouse osteoblasts, on microwave and conventional, furnace sintered samples demonstrated that manufacturing method does not impact cellular viability after 24 h or proliferation over 7 days. New CaP deposition and extracellular matrix components were observed in vitro via scanning electron microscopy (SEM).     

Treatment of periodontal defects in dogs using an injectable composite hydrogel/biphasic calcium phosphate

An injectable composite silanized hydroxypropyl methyl cellulose/biphasic calcium phosphate (Si-HPMC/BCP) has been investigated in humans with promising results. The aim of this study was to evaluate his efficacy for treating periodontal defects (canine fenestration and premolar furcation) in dog models. At 3?months, we observed that bone formation around BCP particles in furcation model is more discernible but not statistically significant in defects filled with Si-HPMC/BCP compared to healing in control. We suggest that BCP particles sustain the bone healing process by osteoconduction, while the Si-HPMC hydrogel enhances intergranular cohesion and acts as an exclusion barrier. Furthermore, bone ingrowth is not so distinctive in superficial defects where the biomaterial appears unstable. These results with Si-HPMC/BCP are encouraging. In addition, this biomaterial is easy to use and simplifies the process of filling periodontal lesions. However, more researches are needed to improve the viscosity and hardness to adjust the material to the specificities of periodontal defects.     

Elaboration conditions influence physicochemical properties and in vivo bioactivity of macroporous biphasic calcium phosphate ceramics

Two different preparations of biphasic calcium phosphate (BCP) were characterized in vitro: BCP₁ from a mechanical mixture of hydroxyapatite (HA) and -tricalcium phosphate (-TCP) powders, and BCP₂ from calcination of a calcium-deficient apatite (CDA). The structural, physicochemical and mechanical parameters of these two preparations were investigated, and two different macroporous BCP₁ (MBCP₁) and BCP₂ MBCP₂) implants were manufactured and implanted in rabbit bone for in vivo bioactivity studies. Scanning electron microscopy observations showed that MBCP₁ implants had a significantly higher degradation rate (P<0.0001) than MBCP₂ implants. This was probably caused by the presence of calcium oxide impurities in BCP₁ and the more intimate mixture and stable ultrastructure of BCP₂. No significant difference about the newly formed bone rate in these two BCP preparations was observed. Very slight variations in sintering conditions appeared to influence the biodegradation behavior of the two MBCP implants despite their identical HA/-TCP ratios and similar porosity. Precise and complete in vitro characterization enabled us to understand and predict in vivo degradation behavior. © 1999 Kluwer Academic Publishers     

Laser engineered multilayer coating of biphasic calcium phosphate/titanium nanocomposite on metal substrates

In this work, laser coating of biphasic calcium phosphate/titanium (BCP/Ti) nanocomposite on Ti-6Al-4 V substrates was developed. A continuous wave neodymium-doped yttrium aluminium garnet (Nd:YAG) laser was used to form a robust multilayer of BCP/Ti nanocomposite starting from hydroxyapatite and titanium nanoparticles. In this process, low power coating is realized because of the strong laser-nanoparticle interaction and good sinterability of nanosized titanium. To guide the optimization of laser processing conditions for the coating process, a multiphysics model coupling electromagnetic module with heat transfer module was developed. This model was validated by laser coating experiments. Important features of the coated samples, including microstructures, chemical compositions, and interfacial bonding strength, were characterized. We found that a multilayer of BCP, consisting of 72% hydroxyapatite (HA) and 28% beta-tricalcium phosphate (β-TCP), and titanium nanocomposite was formed on Ti-6Al-4 V substrates. Significantly, the coating/substrate interfacial bonding strength was found to be two times higher than that of the commercial plasma sprayed coatings. Preliminary cell culture studies showed that the resultant BCP/Ti nanocomposite coating supported the adhesion and proliferation of osteoblast-like UMR-106 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.     

Synthesis of nano-sized biphasic calcium phosphate ceramics with spherical shape by flame spray pyrolysis

Nanometer size biphasic calcium phosphate (BCP) powders with various Ca/P molar ratios satisfied with appropriate phase ratios of HA/β-TCP were prepared by high temperature flame spray pyrolysis process. The BCP powders had spherical shapes and narrow size distributions irrespective of the ratios of Ca/P. The mean size of the BCP powders measured from the TEM image was 38 nm. The composition ratio of Ca/P was controlled from 1.500 to 1.723 in the spray solution, and required phase ratios of HA/TCP are controlled systematically. The calcium dissolution of the pellets obtained from the BCP powders directly prepared by flame spray pyrolysis in buffer solution increased with the decrease of Ca/P ratios except with the Ca/P ratio of 1.713. The pellet surface with Ca/P ratio of 1.500, which consisted of β-TCP, was eroded dramatically for 7 days. On the other hand, the pellet surface with Ca/P ratio of 1.667 was stable and did not disintegrate after immersion in Tris–HCl buffer solution based on the SEM observation.     

Developments in injectable multiphasic biomaterials. The performance of microporous biphasic calcium phosphate granules and hydrogels

Calcium phosphate bioceramic granules associated with hydrosoluble polymers were developed as bone substitutes for various maxillofacial and orthopaedic applications. These injectable bone substitutes, support and regenerate bone tissue and resorb after implantation. The efficiency of these multiphasic materials is due to the osteogenic and osteoconductive properties of the microporous biphasic calcium phosphate. The associated hydrosoluble polymers are considered as carriers in order to achieve the rheological properties of injectable bone substitutes (IBS). In this study, we used 2 semi synthetic hydrosoluble polymers of polysaccharidic origin. The hydroxy propyl methyl cellulose (HPMC), with and without silane, was combined with microporous BCP granules. The presence of silane induced considerable gelation of the suspension. The 2 IBS used (without gelation, IBS1, with gelation, IBS2) were implanted in critical size femoral epiphysis defects in rabbits. No foreign body reactions were observed in either sample. However, because of the higher density from gelation, cell colonisation followed by bone tissue ingrowth was delayed over time with IBS2 compared to the IBS1 without gelation. The results showed resorption of the BCP granule and bone ingrowth at the expense of both IBS with different kinetics. This study demonstrates that the hydrogel cannot be considered merely as a carrier. The gelation process delayed cell and tissue colonisation by slow degradation of the HPMC Si, compared to the faster release of HPMC with IBS1, in turn inducing faster permeability and spaces for tissue ingrowth between the BCP granules.