Inflammatory reaction in rats muscle after implantation of biphasic calcium phosphate micro particles

Several studies have shown that macro micro porous bioceramics ectopically implanted promote bone tissue formation. This study aims at investigating the inflammatory response towards biphasic calcium phosphate (BCP) ceramic micro particles. BCP composed of hydroxyapatite (HA) and beta-tricalcium phosphate, HA/β -TCP ratio of 50/50, were prepared by sintering at 1200°C for 5 h. After crushing, 3 fractions of BCP micro particles < 20, 40–80 and 80–200 μ m were sieved. The micro particles were carefully characterized by using X-ray diffraction (XRD), scanning electron microscopy (SEM) and laser scattering. The inflammatory reactions induced by BCP micro particles implanted in quadriceps muscles of rats for 7, 14 and 21 days were studied by histology (n = 8/group). A fibrous tissue encapsulation of the BCP micro particles implanted in muscle tissue was observed and fibrosis was similar for the 3 groups of micro particles. The comparison of the cellular response indicated that the total number of cells was significantly higher for BCP < 20 μ m than for 40–80 and 80–200 μ m (p < 0.0001). The number of macrophages was relatively higher for the smallest than for the intermediate and largest fractions (p < 0.0001). The relative percentage of giant cells was higher for the intermediate and largest size of particles than for the smallest. The number of lymphocytes was comparable for the 3 fractions and after the 3 delays. Therefore, the BCP micro particles < 20 μ m initiated an inflammatory response which might play an important role in osteogenesis.     

Bone tissue formation in sheep muscles induced by a biphasic calcium phosphate ceramic and fibrin glue composite

Some biomaterials are able to induce ectopic bone formation in muscles of large animals. The osteoinductive potential of macro- micro-porous biphasic calcium phosphate (MBCP) ceramic granules with fibrin glue was evaluated by intramuscular implantation for 6 months in six adult female sheep. The MBCP granules were 1–2 mm in size and were composed of hydroxyapatite (HA) and beta-tricalcium phosphate (β-TCP) in a 60/40 ratio. The fibrin glue was composed of fibrinogen, thrombin and other biological factors. After 6 months of implantation in the dorsal muscles of sheep, the explants were rigid. Histology, back-scattered electron microscopy and micro-computed tomography of the implants indicated that approximately 12% of mineralized bone had formed in between the MBCP granules. The ectopic bone appeared well-mineralized with mature osteocytes and Haversian structures. In addition, the number and thickness of bone trabeculae formed in between the MBCP particles were similar to those measured in trabecular bone in sheep. The overall results therefore confirmed the formation of well-mineralized ectopic bone tissue after intramuscular implantation of MBCP/fibrin glue composites. These bone substitutes exhibiting osteoinductive properties could be used for the reconstruction of large bone defects.     

Immunohistochemical evaluation after Sr-enriched biphasic ceramic implantation in rabbits femoral neck: comparison of seven different bone conditions

Strontium (Sr) has shown effectiveness for stimulating bone remodeling. Nevertheless, the exact therapeutic values are not established yet. Authors hypothesized that local application of Sr-enriched ceramics would enhance bone remodeling in constant osteoporosis of rabbits’ femoral neck bone. Seven different bone conditions were analyzed: ten healthy rabbits composed a control group, while other twenty underwent ovariectomy and were divided into three groups. Bone defect was filled with hydroxyapatite 30% (HAP) and tricalcium phosphate 70% (TCP) granules in 7 rabbits, 5% of Sr-enriched HAP/TCP granules in 7, but sham defect was left unfilled in 6 rabbits. Bone samples were obtained from operated and non-operated legs 12 weeks after surgery and analyzed by histomorphometry and immunohistochemistry (IMH). Mean trabecular bone area in control group was 0.393 mm2, in HAP/TCP – 0.226 mm2, in HAP/TCP/Sr – 0.234 mm2 and after sham surgery – 0.242 mm2. IMH revealed that HAP/TCP/Sr induced most noticeable increase of nuclear factor kappa beta 105 (NFkB 105), osteoprotegerin (OPG), osteocalcin (OC), bone morphogenetic protein 2/4 (BMP 2/4), collagen type 1α (COL-1α), interleukin 1 (IL-1) with comparison to intact leg; NFkB 105 and OPG rather than pure HAP/TCP or sham bone. We concluded that Sr-enriched biomaterials induce higher potential to improve bone regeneration than pure bioceramics in constant osteoporosis of femoral neck bone. Further studies on bigger osteoporotic animals using Sr-substituted orthopedic implants for femoral neck fixation should be performed to confirm valuable role in local treatment of osteoporotic femoral neck fractures in humans.

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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).     

Periodontal regeneration in experimentally-induced alveolar bone dehiscence by an improved porous biphasic calcium phosphate ceramic in beagle dogs

Regeneration of lost periodontium is the focus of periodontal therapy. To achieve the effective regeneration, a number of bone graft substitute materials have been developed. This study aimed to investigate the histological response in alveolar bone dehiscences which were filled with an improved biphasic calcium phosphate (BCP) ceramic with more reasonable pore diameter, pore wall thickness and porosity. Twenty-four alveolar bone dehiscences were made surgically in twelve beagle dogs by reflecting mucoperiosteal flaps on the buccal aspect of bilateral lower second premolars and removing alveolar bone. The left dehiscences were treated with BCP ceramic and the contralaterals were cured with the open flap debridement (OFD) as controls. Three dogs were used at week 4, 12, and 24 respectively. Histological observations were processed through three-dimensional micro-computed tomographic imaging, fluorescence and light microscopy. The histological study indicated that the biphasic ceramic was biocompatible, and regeneration was achieved more effectively through the BCP treatment. There were also arrest of epithelial migration apically and formation of new bone and cementum, as well as proliferation of fibrous connective tissues that became attached to the newly formed cementum at week 24, while there was no significant periodontal regeneration in the OFD group only with epithelial tissue migrating into the dehiscence regions. Clinically speaking, though the surgical location formed a limitation to the application of the improved BCP on the periodontal regeneration, the actual result was positive. It proved that the BCP had biocompatibility and was able to act as a stable scaffold to induce periodontal regeneration effectively.     

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。     

The growth of calcium phosphate on ceramic surfaces

Calcium phosphate ceramics are very important materials for prosthetic applications because of their excellent biocompatibility. The chemical composition of the ceramics is determining, both with respect to their capability of inducing calcium phosphate formation and regarding the crystalline phase formed. From a series of porous ceramics based on hydroxyapatite (HAP) containing metal oxides, it was found that only those containing ZrO₂ stabilized with 8% Y₂O₃ were able to induce calcium phosphate formation upon introduction into calcium phosphate supersaturated solutions. The overgrowing phase was increasingly crystalline, did not show any other characteristic X-ray peaks and infrared bands than those pertinent to a-tricalcium phosphate and had a molar CaP ratio of 32. Kinetics analysis with respect to Ca(PO₄)₂ yielded an apparent order of reaction of 5.0±0.5, suggesting polynuclear nuclei above nuclei growth. The surface energy calculated from the kinetics data for the crystalline overgrowth was found to be 88 mJ m^–2.     

Preparation and evaluation of osteoinductive porous biphasic calcium phosphate granules obtained from eggshell for bone tissue engineering

The synthetic bone graft substitutes currently used clinically are osteoconductive but not osteoinductive; their low success rate is thus their biggest disadvantage. The use of biomass raw materials for synthesizing calcium phosphate has gradually attracted increased research attention. In this study, hydroxyapatite powder was prepared through liquid-phase precipitation from eggshell, and porous biphasic calcium phosphate granules (EBGs) were then obtained using a pore former and sintering procedure. The EBGs were discovered to have high biocompatibility and no cytotoxicity. The results of animal experiments showed that the area of new bone growth was high, numerous Haversian canals could be observed, and almost all EBGs were surrounded by new bone tissue, which proved that the EBGs had excellent osteoinductivity. By contrast, numerous fat cells were found in the femoral defect area when a commercial bone graft was employed. Various biological inorganic ions (Mg, Sr, Na, and Fe) originally in the eggshell raw materials were incorporated into the EBGs, and the EBGs exhibited excellent osteogenic abilities. The developed approach provides an economical and feasible solution for the treatment of bone defects.     

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.     

The stability mechanisms of an injectable calcium phosphate ceramic suspension

Calcium phosphate ceramics are widely used as bone substitutes in dentistry and orthopedic applications. For minimally invasive surgery an injectable calcium phosphate ceramic suspension (ICPCS) was developed. It consists in a biopolymer (hydroxypropylmethylcellulose: HPMC) as matrix and bioactive calcium phosphate ceramics (biphasic calcium phosphate: BCP) as fillers. The stability of the suspension is essential to this generation of “ready to use” injectable biomaterial. But, during storage, the particles settle down. The engineering sciences have long been interested in models describing the settling (or sedimentation) of particles in viscous fluids. Our work is dedicated to the comprehension of the effect of the formulation on the stability of calcium phosphate suspension before and after steam sterilization. The rheological characterization revealed the macromolecular behavior of the suspending medium. The investigations of settling kinetics showed the influence of the BCP particle size and the HPMC concentration on the settling velocity and sediment compactness before and after sterilization. To decrease the sedimentation process, the granule size has to be smaller and the polymer concentration has to increase. A much lower sedimentation velocity, as compared to Stokes law, is observed and interpreted in terms of interactions between the polymer network in solution and the particles. This experimentation highlights the granules spacer property of hydrophilic macromolecules that is a key issue for interconnection control, one of the better ways to improve osteoconduction and bioactivity.     

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     

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.     

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.