Multiphasic Calcium Phosphate Research Articles

Direct 3D Printing of Seashell Precursor toward Engineering a Multiphasic Calcium Phosphate Bone Graft

Multiphasic calcium phosphate (Ca-P) has widely been explored for bone graft replacement. This study represents a simple method of developing osteoinductive scaffolds by direct printing of seashell resources. The process demonstrates a coagulation-assisted extrusion-based three-dimensional (3D) printing process for rapid fabrication of multiphasic calcium phosphate-incorporated 3D scaffolds. These scaffolds demonstrated an interconnected open porous architecture with improved compressive strength and higher surface area. Multiphasic calcium phosphate (Ca-P) and hydroxyapatite present in the multi-scalar naturally resourced scaffold displayed differential protein adsorption, thus facilitating cell adhesion, migration, and differentiation, resulting in enhanced deposition of the extracellular matrix. The microstructural and physicochemical attributes of the scaffolds also lead to enhanced stem cell differentiation as witnessed from gene and protein expression analysis. Furthermore, the histological study of subcutaneous implantation evidently portrays promising biocompatibility without foreign body reaction. Neo-tissue in-growth was manifested with abundant blood vessels, thus indicative of excellent vascularization. Notably, cartilaginous and proteoglycan-rich tissue deposition indicated ectopic bone formation via an endochondral ossification pathway. The hierarchical interconnected porous architectural tribology accompanied with multiphasic calcium phosphate composition manifests its successful implication in enhancing stem cell differentiation and promoting excellent tissue in-growth, thus making it a plausible alternative in bone tissue engineering applications.

Effect of pH level and calcination on the production of calcium phosphates by acidic route of wet precipitation

Multiphasic calcium phosphate powders were synthesized by the acidic route, using lactic acid as a chelating agent that allows the production of a stable and rich solution of calcium and phosphate ions at room temperature. After pH adjusting, without varying the concentrations of precursor solution, XRD and FTIR analyses showed different CaP phases before and after heat treatment at 1000 °C. At pH 5, brushite plate-like particles were produced, while at higher pH levels (7 to 12) nano-hydroxyapatite particles were formed. After the calcination process, partial and total decomposition of hydroxyapatite in the β-TCP phase was explained by XRF analysis due to the calcium-deficient hydroxyapatite of synthesized samples at pH 7 and 10. This work presents an important method to synthesize any desired CaP phase compositions by varying the pH level and subsequently performing heat treatment, which has a direct effect on morphology, crystallinity, and formation of different CaP powders.

Effect of calcium phosphate synthesis conditions on its physico-chemical properties and evaluation of its antibacterial activity

The antibacterial activity of non-stoichiometric calcium phosphate particles prepared by precipitation under controlled experimental conditions at pH ∼ 9 and sintered at high temperature was studied against Staphylococcus aureus bacteria. The effects of operating parameters developed according to an experimental design of Plackett-Burman type on the physicochemical characteristics and the capacity to inhibit bacterial growth were identified using a thermal analysis (TGA-DTA-DSC), x-ray Diffraction (XR), Raman Spectroscopy, Scanning Electron Microscope (SEM) and the Kirby Bauer Method. The XRD spectrum shows that the synthetic crystalline nanoparticles powders consist of multiphasic calcium phosphate β-TCP/β-CPP/OCP/HA and that the average particle size is between 56 and 123 nm calculated by the Debay-Shearer equation. The Raman spectrum of sintered powder shows the main absorption bands that are assigned to the asymmetric / symmetric P-O stretching vibrations in PO4−3 and the symmetric O-H stretching mode of the hydroxyl group in addition of Ca-PO4 and Ca-OH modes. The samples were found to possess different morphologies consisting of nano-rods of different lengths, semi / spherical structures and fine granules, in addition to irregular clusters. The antibacterial tests results showed that the high concentration calcium phosphate powder exhibited better antibacterial activity against Staphylococcus aureus bacteria with inhibition zones ranging from 0.2–0.7 cm.

Characterization of microstructure and phase distribution of sintered multiphasic calcium phosphate bioceramics

The microstructures of sintered multiphasic calcium phosphate (CaP) ceramics with three initial hydroxyapatite to tricalcium phosphate ratios ranging from 1:4 to 4:1 were characterized at the grain scale. The grain structure, phase composition and spatial phase distribution in sintered calcium phosphate bioceramics were examined by the electron backscatter diffraction (EBSD) supplemented with X-ray diffraction, scanning electron microscopy, and transmission electron microscopy. EBSD proved to be a powerful analytical tool for characterizing grain and phase distribution of CaP bioceramics. The EBSD grain maps connected with the EBSD phase analysis successfully described the phase distribution and grain microstructure of the sintered ceramics in detail. The change in the overall phase composition after sintering was explained by a chemical reaction between a phase impurity of TCP powder and HA. These results can lead to a better understanding of the mechanical and biodegradation properties of CaP bioceramics in both in-vivo and in-vitro conditions.

Evaluation of physicochemical, mechanical and biological properties of chitosan/carboxymethyl cellulose reinforced with multiphasic calcium phosphate whisker-like fibers for bone tissue engineering

In this study porous scaffolds of chitosan (CS) and carboxymethyl cellulose (CMC) reinforced with whisker-like biphasic and triphasic calcium phosphate fibers were fabricated by freeze drying method. The effect of addition of CMC, fiber type and content on the mechanical, physicochemical and biological properties of the composite scaffolds was evaluated. The fibers were synthesized by homogenous precipitation method and were characterized. Biphasic fibers contained two phases of hydroxyapatite (HA) and monetite, and triphasic fibers consisted of HA, β-tricalcium phosphate and calcium pyrophosphate and were 20–270 μm and 20–145 μm in length, respectively. The composite scaffolds exhibited desirable microstructures with high porosity (61–75%) and interconnected pores in range of 35–200 μm. Addition of CMC to CS led to a significant improvement in the mechanical properties (up to 150%) but did not affect the water uptake ability and biocompatibility. Both fibers improved the in vitro proliferation, attachment and mineralization of MG63 cells on scaffolds as evidenced by MTT assay, DAPI staining, SEM and Alizarin red staining. Triphasic fibers were more effective in reinforcing the scaffolds and resulted in higher cell viability. Composite scaffolds of CS and CMC reinforced with 50 wt% triphasic fibers were superior in terms of mechanical and biological properties and showed compressive strength and modulus of 150 kPa and 3.08 MPa, respectively, which is up to 300% greater than pure CS scaffolds. The findings indicate that the developed composite scaffolds are potential candidates for bone tissue engineering although they need further enhancement in mechanical properties.

A Simple Approach for an Eggshell-Based 3D-Printed Osteoinductive Multiphasic Calcium Phosphate Scaffold

Natural origin bioceramics are widely used for bone grafts. In the present study, an eggshell-derived bioceramic scaffold is fabricated by 3D printing as a potential bone-graft analogue. The eggshell, a biological waste material, was mixed with a specific ratio of phosphoric acid and chitosan to form a precursor toward the fabrication of an osteoinductive multiphasic calcium phosphate scaffold via a coagulation-assisted extrusion and sintering for a multiscalar hierarchical porous structure with improved mechanical properties. Physicochemical characterization of the formed scaffolds was carried out for phase analysis, surface morphology, and mechanical properties. A similar scaffold was prepared using a chemically synthesized calcium phosphate powder that was compared with the natural origin one. The higher surface area associated with the interconnected porosity along with multiple phases of the natural origin scaffold facilitated higher cell adhesion and proliferation compared to the chemically synthesized one. Further, the natural origin scaffold displayed relatively higher cell differentiation activity, as is evident by protein and gene expression studies. On subcutaneous implantation for 30 days, promising vascular tissue in-growth was observed, circumventing a major foreign body response. Collagen-rich vascular extracellular matrix deposition and osteocalcin secretion indicated bonelike tissue formation. Finally, the eggshell-derived multiphasic calcium phosphate scaffold displayed improvement in the mechanical properties with higher porosity and osteoinductivity compared to the chemically derived apatite and unveiled a new paradigm for utilization of biological wastes in bone-graft application.

Reinforcement of freeze-dried chitosan scaffolds with multiphasic calcium phosphate short fibers

The composite scaffolds of the chitosan and multiphasic calcium phosphate (HW) short fibers were prepared by freeze drying and characterized by X-ray diffractometry (XRD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM and FE-SEM). The mechanical properties of the scaffolds were assessed by compression test. The incorporation of HW fibers consisting three phases of hydroxyapatite (HA), beta-tricalcium phosphate (β-TCP) and calcium pyrophosphate (CPP) into the chitosan matrices was associated with an increase in pore size, density and compressive strength and modulus, and a decrease in porosity and swelling ratio of the scaffolds. The strongest composite scaffolds in this study with a chitosan: HW fibers weight ratio of 1:1 showed a mean porosity of 69% and a mean strength and modulus of 420kPa and 3.87MPa, respectively. The in vitro bioactivity of the composites was confirmed by the formation of a calcium phosphate rich layer on the surface of soaked scaffolds in simulated body fluid. The findings of this initial work indicate that the chitosan–multiphasic calcium phosphate short fibers may be a suitable material for bone scaffolding.

Biocompatibility and Biodegradation of Multiphasic Calcium Phosphate Ceramic Bone Substitute Transformed by Ostrich Cancellous Bone for Bone Tissue Engineering

A kind of novel multiphasic calcium phosphate ceramic bone substitute transformed by ostrich cancellous bone (OCB) was explored in this article. In this study we investigated the characterization, biocompatibility, biodegradability and in vivo osteoconductive property of the material. Scanning electron microscope (SEM) and X-ray diffraction (XRD) were used to characterize the material. Biocompatibility and biodegradation were confirmed by testing the hemolysis and intramuscular implantation. Bone marrow stromal cells (BMSCs) were also used to perform biocompatibility. Besides, material/BMSCs composites were implanted into the subcutaneous of back of nude mice to evaluate the osteoconductive property of the material. SEM analysis showed that the presence of a large amount of micropores within macropore walls and the interporous connections were abundant which indicated that the material possessed enough surface area. XRD pattern showed the peaks were corresponding to the characteristic for hydroxyapatite (HA), β-tricalcium phosphate (β-TCP), and sodium calcium phosphate (NaCaPO4). Culturing of BMSCs on the scaffolds revealed the cells were able to attach and proliferate easily on the surface and the pores of the scaffolds. Moreover, this scaffolds had a good performance of biodegradation in vivo. Finally, histological observation of heterotopic bone formation revealed newly formed bone at 8 weeks after transplantation. The results of this study demonstrated that the multiphasic calcium phosphate ceramic had good biocompatibility and its surface configuration and pore structure were suitable for BMSCs to attach and proliferate and also had a good osteoconductive property, which suggested that the material could be used as a bone tissue engineering scaffold in the future.

Novel multiphasic calcium phosphate cements with enhanced degradative properties

Novel multiphasic calcium phosphate cements with enhanced degradative properties

Effects of Calcium Carbonate Synthesized from Cockle Shell on Characteristics of Calcium Phosphate Biphasic

Calcium phosphate is a biomaterial which exhibits excellent biocompatibility, bioactivity and biodegradability for bone replacement and other medical applications. In the present work, multiphasic calcium phosphate compounds consisting of hydroxyapatite (HAp) and tri-calcium phosphates (TCP) were prepared by a solid state reaction method. Effects of calcium carbonate (CaCO3: ±3-5 wt%) on the properties of the studied powders were investigated where calcium carbonate powder was synthesized from a natural product (cockle shell). The phase formation and morphology of the powders were characterization by many techniques such as XRD, SEM and dynamic light scattering techniques. The results showed that amount of calcium carbonate additive affected on the properties of the studied samples.

Decomposition combustion synthesis of calcium phosphate powders for bone tissue engineering

The aim of this research was to study a potential processing mechanism for the synthesis of biologically active calcium phosphate (CaP) powders. Decomposition combustion synthesis (DCS) is a modified combustion synthesis technique that can be used to produce multiphasic calcium phosphate powders for use in bone tissue engineering applications. During DCS, the stoichiometric ratio of reactant salt to fuel was adjusted to alter product chemistry and morphology, and the resultant CaP powders were analytically characterized. Novel CaP powders produced via DCS represent a potential low-cost/high-efficiency source for mass-produced biomaterials that can be customized for specific biologic manufacturing requirements.

Processing and in vivo evaluation of multiphasic calcium phosphate cements with dual tricalcium phosphate phases

Calcium phosphate cements (CPCs) use the simultaneous presence of several calcium phosphates phases. This is done to generate specific bulk and in vivo properties. This work has processed and evaluated novel multiphasic CPCs containing dual tricalcium phosphate (TCPs) phases. Dual TCPs containing α- and β-TCP phases were obtained by thermal treatment. Standard CPC (S-CPC) was composed of α-TCP, anhydrous dicalcium phosphate and precipitated hydroxyapatite, while modified CPC (DT-CPC) included both α- and β-TCP. Physicochemical characterization of these CPCs was based on scanning electron microscopy, X-ray diffraction, specific surface area (SSA) and particle size (PS) analysis and mechanical properties. This characterization allowed the selection of one DT-CPC for setting time, cohesion and biological assessment compared with S-CPC. Biological assessment was carried out using a tibial intramedullary cavity model and subcutaneous pouches in guinea pigs. Differences in the surface morphology and crystalline phases of the treated TCPs were detected, although PS analysis of the milled CPC powders produced similar results. SSA analysis was significantly higher for DT-CPC with α-TCP treated at 1100°C for 5h. Poorer mechanical properties were found for DT-CPC with α-TCP treated at 1000°C. Setting time and cohesion, as well as the in vivo performance, were similar in the selected DT-CPC and the S-CPC. Both CPCs created the desired host reactions in vivo.

PL DLLA Calcium Phosphate Composite Combined with Macroporous Calcium Phosphate Cement MCPC® for New Surgical Technologies Combining Resorbable Osteosynthesis and Injectable Bone Substitute

Resorbable osteosynthesis based on PLLA and derivatives will be associated to bone substitute for bone reconstruction. We have performed rand evaluated a composite combining PL DLLA and Biphasic calcium phosphate able to have a), a better controlled hydrolysis in the purpose to preserve on time the mechanical property, and b), for long term efficiency, bone ingrowth at the expense of the osteosynthesis and the associated bone substitute. A new calcium phosphate cement MCPC® was tested with such composite. The novel macroporous calcium phosphate cement MCPC sets to poorly crystalline apatite after mixing the powder component and an aqueous solution. Interconnective macroporosity was induced on time by resorption of one part of the MCPC®. The multiphasic calcium phosphate components in the cement, are resorbed at different rates allowing the replacement by newly formed bone. This study reports the biocompatibility and the interactions of a composite using PL DLLA (Poly [L-Lactide-co-D,L-Lactide] acid) charged with biphasic calcium phosphate granules and a self setting calcium phosphate cement of new generation.

Effects of Changes in Structural Hydration of Multiphasic Heterogeneous Calcium Phosphate Powders Created via Auto-Ignition Combustion Synthesis

ABSTRACTCalcium phosphate (CaP) materials are commonly used in bone tissue engineering applications since they closely resemble the chemistry of bone and teeth. The inorganic component of mineralized tissue is multiphasic in nature-thus to better replicate those tissues, CaP materials should also be multiphasic. Combustion synthesis is a process that creates multiphasic CaP (HCaP) with low energy input over a relatively short time. The structure and chemistry of HCaP synthesized via auto-ignition combustion synthesis (AICS) varies greatly with respect to structural hydration. Product hydration was accomplished by modifying hydrogen and oxygen content in the combustion reaction by changing the amount of fuel, urea [ ] pre-reaction, and heating or sintering products post-reaction. The reaction equation for this specific system is given below. Calcium nitrate [ ], and ammonium nitrate [ ] are the components that form HCaP. Urea acts as an ignition source and fuel. Changes in the amount of urea dictate the amount of excess hydrogen to form water within the reaction. Excess products formed include water, carbon dioxide, and nitrogen. Salts of the reactants were mixed with 10 milliliters of de-ionized water in a Pyrex beaker, heated on a hot plate for 20 minutes or until the reactants began to foam, and then placed in a muffle furnace at 1000°C until the foam ignited in a combustion reaction. This was noted by the progression of a combustion wave throughout the foam. Post-AICS, products were heated at 105°C for 8 hours and 24 hours and massed to determine water content of the product. Subsequently, the products were sintered at 1000°C for 8 hours and massed again. The primary products formed using AICS are hydroxyapatite (HA), á-tricalcium calcium phosphate (TCP) and hydrated forms of tricalcium phosphate (HTCP). During low temperature heating, 105°C, water content decreases as time increases and the products began to densify. Initial results indicate that surface porosity decreases during the powder densification. XRD shows that peak intensity increases after low temperature heating, indicating an increase in crystallinity and grain orientation. XRD confirms that both crystalline and amorphous phases occur in the hydroxyapatite (HA), á-TCP and HTCP products. The amount of structural hydration has an effect on CaP, and these effects are noted by an increase in density and decrease in porosity as structurally bound water is removed from the system. Future research will be dedicated to determining hydration ratio (amount of urea in the reaction to the amount of water within the products) and a Ca:P ratio that result in optimal powder porosity, ductility and grain size generating a multiphasic HCaP implant biomaterial that accurately replicates natural bony tissue.

Osteoblast-like cell mineralization induced by multiphasic calcium phosphate ceramic

The work presented here examines the effect of multiphasic CaP materials on the activity of SaOS-2 osteoblast like cells. Heterogeneous calcium phosphate (HCaP) was synthesized using self-propagating high temperature combustion synthesis (SHS). All samples were characterized using XRD, SEM, and FTIR. Cell culture procedures of HCaP with SaOS-2 cells followed ASTM 813-01. Samples were examined by Environmental SEM (ESEM) and EDS to characterize the mineral content of the cultures. Gold-coated samples were used for high-resolution imaging. Biomineralization was noted in unconditioned cultures exposed to HCaP while the controls (cells only in media and HCaP only in media) showed no mineralization. Calcium phosphate plate-like structures were identified adjacent to cells expressing calcium phosphate containing vesicles. These structures are similar to the organophosphate crystals seen in other published works. Plate-like crystals were noted in the larger membrane vesicles of SaOS-2 cells indicating the occurrence of cellularly mediated biomineralization.