Soluble phosphate salts as setting aids for premixed calcium phosphate bone cement pastes

Recently, premixed calcium phosphate cement pastes have been proposed as biomaterials for bone tissue repair and regeneration. Use of premixed pastes saves the time and removes an extra step during a medical operation. α-Tricalcium phosphate (α-TCP) based cements set to form calcium deficient hydroxyapatite which has a moderate bioresorbtion speed. α-TCP cements require a setting aid, usually a sodium or potassium phosphate salt, to speed up the setting process. Within the current research we investigated which setting aid has significant advantage, if α-TCP is used in form of non-aqueous premixed paste. This approach offers the application of simple ingredients to produce a premixed calcium phosphate cement. The following properties of cement formulations were evaluated: cohesion, phase composition, microstructure, pH value of the liquid surrounding the cement, and compressive strength.Compositions using mixture of basic and acidic potassium phosphate salts (KH₂PO₄ and K₂HPO₄) in sufficient amounts give the best overall results (adequate cohesion and pH of the surrounding liquid, hydrolysis of starting materials within 48 h, and compressive strength of 12 ± 3 MPa). Cement prepared with basic sodium phosphate salt (Na₂HPO₄) as setting aid had considerably higher compressive strength 22 ± 1 MPa, but the pH of the surrounding liquid was basic (9.0).     

Investigation of biocompatible nanosized materials for development of strong calcium phosphate bone cement: Comparison of nano-titania,nano-silicon carbide and amorphous nano-silica

The present paper deals with the effect of adding SiC, TiO₂ and SiO₂ nanoparticles on setting time, mechanical strength and hydraulic reactions of calcium phosphate cements (CPCs). The initial and final setting times of CPC increased by adding both nano-SiC and nano-TiO₂ additives but decreased by using nano-silica. Nano-titania and nano-silica had great effect on compressive strength of as-set CPC whereas slight changes were found by using nano-SiC. Although a sharp increase in compressive strength of all cements was observed by soaking them in physiological solution, the soaked additive-free cements and nano-SiO₂-added ones exhibited the greatest strength values. The results showed that adding these nano-additives did not influence on conversion rate of cement reactants to apatite phase during soaking in physiological solution period but the morphology of the formed phase was almost different. Overall, the results determined that nano-SiO₂ and nano-TiO₂ particles were appropriate additives to improve short-term mechanical strength of CPCs a(s-set CPCs), though nano-SiO₂ was found more effective because it improves the long-term mechanical strength of CPC (after soaking) too.     

In vitro characterization of porous calcium phosphate scaffolds capped with crosslinked hydrogels to avoid inherent brittleness

Ductile composite scaffolds can avoid being crushed during filling processes in clinical applications. Thus, this study aimed to modify brittle porous ceramic scaffolds into ductile scaffolds through hydrogel capping. The surfaces of calcium phosphate (CaP) ceramic scaffolds were effectively capped with alginate/gelatin hydrogels. The composite scaffolds were then crosslinked, subjected to vacuum drainage, and washed prior to lyophilization. The detailed controlled approach in this study was proposed with the aim to develop biocompatible composites with anisotropic open pores through the formation of a thin, homogenous, hydrogel film coating on porous ceramic scaffold surfaces. The performances of the hydrogel/CaP composite scaffolds were evaluated on the basis of their morphological characteristics, compressive strengths, and cell viabilities. Results showed that strength, toughness, and specimen integrity after cracking are strongly related to the concentration of the hydrogel cap. Strength testing results showed that the use of 50 vol% alcohol as the crosslinker removal solution yielded scaffolds with high toughness and ductility. Moreover, the cracked specimen dipped in 50 vol% alcohol possessed better integrity than that dipped in water only. This study successfully identified the optimal hydrogel quantity for the fabrication of biocompatible scaffolds with open connective pores. The advantages of the fabricated scaffolds indicate the relevance of the proposed method to clinical applications, such the production of fillers for successful alveolar bone augmentation.     

Investigation of calcium phosphate formation from calcium propionate and triethyl phosphate

Synthetic calcium phosphates are used in for example bone cements and implant coatings to increase biocompatibility. The common method to produce tricalcium phosphate (TCP) uses high temperatures, which creates large crystals with low specific surface areas. In order to investigate new methods to produce TCP at lower temperatures, the reaction between calcium propionate and triethyl phosphate conducted at 220 °C was studied. The method had a near 100% conversion rate, the main synthesis products were calcium phosphate and ethyl propionate. The formed calcium phosphate polymorph could be controlled depending on the water content of the precursor mixture. Anhydrous conditions created amorphous calcium phosphate. As the concentration of water increased, β-TCP was formed, followed by calcium deficient hydroxyapatite and monetite. The particle size increased with the water content, from 20 to 40 nm for amorphous calcium phosphate to tenths of micrometers for monetite. The specific surface areas varied between 209 m²/g for the amorphous product to 3.6 m²/g for the monetite product.     

Microwave-assisted preparation of Nano-hydroxyapatite for bone substitutes

Calcium phosphate-based biomaterials have been frequently used as bone substitutes and osteoconductive scaffolds due to their chemical similarity to the inorganic phase of bone. Hydroxyapatite (HA) is the most frequently used calcium phosphate-based biomaterials and it could be prepared from natural or synthesized sources via several processes. Nano-HA (nHA) particles were suggested to have superior bioresorbtion and close chemical and crystallographic structure to natural bone apatite.Currently, microwave-assisted (MW) processing of biomaterials, particularly bioceramics, has more appealing advantages than conventional heating methods. It works through an internally generated heat inside the materials molecules instead of originating external heating source and subsequent radiative transfer as in the conventional heating. Thus, MW synthesis of HA offers several benefits including rapid heating, shorter synthesis time, efficient energy transformation and throughout volume heating.The purpose of this review is to highlight the various methods and the paradigm shift of MW techniques to synthesize nHA in the past 25 years. Peer-reviewed journal publications (Scopus and Google Scholar indexed articles) in which titles and keywords combining “Microwave AND Nano-Hydroxyapatite” were collected from 1990 till May 2015. The discussions include the state-of-the-art and the role of reaction parameters on the HA structures, size, morphology and biocompatibility to be further used for bone regeneration applications.     

Investigation on structural properties and bioactivity of nanosized biphasic calcium phosphate

Synthetic biphasic bioceramics composed of hydroxyapatite (HA) and other calcium phosphates (CPs) can provide promising efficiency in the treatment of bone defects based on the rapid dissolution of the CPs, to allow its replacement by freshly formed bone, along with the slow resorption of the HA, to preserve the volume of the grafted area. For this purpose, the present study was conducted to prepare nanosized biphasic calcium phosphate (n-BCP) using a facile mechanochemical process. The experimental outputs were also compared to the commercial-grade HA in terms of physicochemical features. The Rietveld refinement was utilized to calculate the phase contents and crystal structures of the composites, including crystallite size, lattice parameters, and unit cell volume. Besides, the atomic arrangement of Ca1, Ca2, PO₄³⁻, and OH⁻ groups in the hexagonal crystal structure of HA and triclinic structure of anhydrous dicalcium phosphate (DCPA) was determined. The phase fraction, crystallite size, and the powder density of the biphasic structures, which were derived by Rietveld refinement, were found to be affected by ball-milling. In addition to biphasic structures, a monolithic hexagonal HA with an isotropic crystal growth was formed after 7 h of ball-milling under an argon atmosphere. The in vitro test in a simulated body fluid (SBF) confirmed the bioactivity of the biphasic structure through the formation of a bone-like apatite layer after one week.     

3D pore-interconnected calcium phosphate bone blocks for bone tissue engineering

Pore size and connectivity of artificial bone scaffolds play key role in regulating cell ingrowth and vascularization during healing. The objective of this study was to develop a novel process for preparing 3D pore-interconnected open-cell bone substitutes with varying pore sizes. This was achieved by thermal-induced expansion, drying, then sintering the mixture of biphasic calcium phosphate (BCP) and a thermal responsive porogen comprising chitosan (CS) and hydroxypropyl methyl cellulose (HPMC). The interpolymer complexes (IPCs) of CS/HPMC were prepared and investigated by FT-IR. The mixtures of IPCs/BCP were heated up to 100 °C for analyzing their thermal expansion properties. This resulted in ~13% and ~42% volume increment for IPC-1/BCP and IPC-2/BCP, respectively, while ~230% volume increased in the case of IPC-3/BCP (therefore chosen for sintering bone blocks). Heating rate-dependent (0.20–0.25 °C/min range) sintering profiles for IPC-3/BCP were utilized to produce BCP bone blocks. Gasification of IPC during sintering resulted in the formation of interconnected porous structures, and the morphology was investigated by SEM, revealing varying sizes ranging from 106 ± 13 μm to 1123 ± 75 μm. The pore size range of bone blocks from 235 ± 46 μm to 459 ± 76 μm portrayed significantly high MC3T3-E1 cell viability with prominent filopodial extensions, and elongated cells, depicting efficient biocompatibility. Therefore, the process for preparing porous interconnected 3D bone blocks were feasible, thereby serving as an alternative for potential bone tissue engineering applications.     

Synthesis of weakly crystallized hydroxyapatite with Zn substitution and its effect on the calcium phosphate and calcium sulfate cements

Zn is an essential trace element in the normal growth and loading Zn into biomaterials for biomedical applications has always been a hot topic due to its immune regulation. The preparation and characterization of Zn-substituted weakly crystallized hydroxyapatite (WCH) are studied in this work, and Zn-substituted WCH was added to calcium phosphate and calcium sulfate cements (CPC and CSC) to address the effect of Zn²⁺ on the hydration crystallization behavior of calcium phosphate and calcium sulfate. Our results demonstrate that Zn²⁺ will inhibit the transformation of α-TCP to HA during the hydration reaction of CPC. And the adding of Zn²⁺ in CSC changed the crystallization morphology of calcium sulfate. The regulation of Zn on the crystallization behavior of calcium phosphate and calcium sulfate resulted in the different in vitro degradation behaviors of CPC and CSC. With the purpose of improving the biological effects of materials, the polarization of Zn²⁺ released from cements on macrophages was also characterized in this work, and the results showed that appropriate concentrations of Zn²⁺ can inhibit inflammation after stimulating RAW264.7 cells for an appropriate period of time. The presented results may be useful guidelines for the preparation and design of composite bone cement with specific Zn content.     

In-vitro behavior of different fully dense calcium phosphate materials fabricated by Spark Plasma Sintering

Results obtained during in-vitro experiments concerning human osteoblasts cultivated on the surface of dense samples produced by Spark Plasma Sintering from three types of hydroxyapatite powders are described and discussed. The sintered products display diverse composition and microstructures which are found to significantly influence the biological response of the cells. Osteoblasts adhesion, viability and proliferation are quantitatively comparable for the three classes of bioceramics, whereas matrix mineralization occurs only in products exclusively consisting of hydroxyapatite. Correspondingly, a calcium-phosphate layer exhibiting a trabecular-like microstructure is deposited on the materials surface. Matrix mineralization is favored when the substrate is composed of submicrometer-sized apatite grains. On the other hand, the latter phenomena is markedly suppressed, and so does the formation of the new apatite phase, when cells are seeded on sintered disks composed of β-Tri-Calcium Phosphate, which was formed during the sintering process from the decomposition of initial apatite.     

Tailoring the pore structure and property of porous biphasic calcium phosphate ceramics by NaCl additive

This study investigated the effects of NaCl additive on the phase composition, pore structure and mechanical property of porous biphasic calcium phosphate (BCP) ceramics, which were prepared by freeze-casting. The results indicated that the addition of NaCl promoted transformation of β-tricalcium phosphate to hydroxyapatite in the BCP ceramics; the OH group in HA phase of BCP ceramic was partially replaced by chloride ion. As the mass fraction of NaCl in the slurry increased from 0 to 3%, the porosity of obtained porous BCP ceramics decreased from 77.76% to 60.22%; the average width of dendritic pores increased from 74.37 µm to 111.27 µm; the compressive strength achieved threefold increase. As the amount of NaCl additive reached 4.5%, the porosity, pore width, and compressive strength of the porous BCP ceramics were comparable with those modified by 3% NaCl. NaCl is regarded as an effective additive to tailor the pore structure and property of freeze-cast porous ceramics.     

Evaluation of ascorbic acid-loaded calcium phosphate bone cements: Physical properties and in vitro release behavior

In this study, different concentrations of ascorbic acid (50, 100 and 200 µg/mL) were added to the liquid phase of a calcium phosphate cement (CPC). The cements were immersed in simulated body fluid (SBF) for different intervals and physical, physicochemical and mechanical properties of them were evaluated. The release of added ascorbic acid from CPCs into the SBF solution was also studied. From the results, both setting time and injectability of CPC decreased by adding ascorbic acid, however the compressive strength was sharply increased before soaking in SBF solution. But, the compressive strength values of all cements (with or without ascorbic acid) soaked in SBF solution for more than 7 d duration were comparable. The X-ray diffractometry results showed that in vitro apatite formation ability of cement reactants did not change by adding ascorbic acid. The scanning electron microscopy images indicated that morphology of the formed apatite crystals was nano-needlelike and needle diameter was less than 100 nm. The loaded ascorbic acid was slowly released from CPC into the SBF solution so that about 10% and 20% of the loaded drug was released after 504 h for the cements containing 100 and 200 µg/mL ascorbic acid, respectively. The release rate was increased when the amount of added ascorbic acid decreased by 50 µg/mL.     

Evaluation of phase transformation behavior in biphasic calcium phosphate with controlled spherical micro-granule architecture

Spherical biphasic calcium phosphate (BCP) micro-granules consisting of hydroxyapatite (HAp) and tricalcium phosphate (TCP) were prepared using a rotary spray drying and sintering process. A significant phase transformation of TCP in BCP granules occurred according to the relative content of HAp and TCP in the BCP granules depending on the sintering temperature. The crystal growth of HAp in BCP was suppressed by a phase transformation of TCP with a change in the electron density distribution in a crystal unit cell. In addition, these progressive changes in the hydroxyl and phosphate group of BCP granules might be due to the phase transformation of TCP. Consequently, such structural changes in the BCP granules led to an unexpected change in granule size and micro-cracks on their surface at high temperatures.     

Investigation of biphasic calcium phosphate/gelatin nanocomposite scaffolds as a bone tissue engineering

The porous scaffold of nanobiphasic calcium phosphate (n-BCP) and gelatin from bovine skin type B was prepared by freeze-drying method. The porogen which used was Naphthalene. EDC (N-(3-dimethyl aminopropyl)-N′-ethyl carbodiimide hydrochloride) for stabilization of gelatin by cross-linking method was used. The scaffold was characterized by SEM, XRD and FTIR. As a result, a biocompatible scaffold with good cell attachment, facility in formation in desired shapes and simplicity in production were prepared for bone tissue engineering.     

Comparison of DOPA and DPPA liposome templates for the synthesis of calcium phosphate nanoshells

The aim of this paper is to synthesise calcium phosphate (CaP) nanoshells by controlling their particle size and shape using negatively charged liposomes (1,2 dioleoyl-sn-glycero-3 phosphate sodium salt (DOPA) and 1,2-dipalmitoyl-sn-glycero-3-phosphate sodium salt (DPPA)) as a template. The morphology, particle size, size distribution and zeta potential properties of DOPA and DPPA liposome templates were determined. The results showed that both DOPA and DPPA formed spherical nanoshell structures to be used as templates for the synthesis of CaP nanoshells. By using the DOPA template, spherical CaPs structures with a mean particle size of 197.5 ± 5.8 nm were successfully formed. In contrast, needle or irregularly shaped CaP particles were observed when using the DPPA template.     

Dual-scale porous biphasic calcium phosphate gyroid scaffolds using ceramic suspensions containing polymer microsphere porogen for digital light processing

This study demonstrates a novel type of biphasic calcium phosphate (BCP) gyroid scaffolds featuring of gyroid macroporous structure and micropous BCP walls using poly(methyl methacrylate) (PMMA) microspheres as the porogen for ceramic digital light processing (DLP) technique. To tailor the microporosity of the BCP walls and the overall porosity of the dual-scale porous BCP scaffolds, the PMMA content with regard to the BCP powder was controlled in the range of 40 vol% to 70 vol%. After debinding at 600 °C and sintering at 1200 °C for 3 h, micropores were uniformly created throughout each BCP framework, while preserving 3?dimensional gyroid macroporous structures. As the PMMA content increased from 40 vol% to 70 vol%, the microporosity remarkably increased from 31.9 (±2.5) vol% to 55.2 (±1.4) vol%. This approach allowed the achievement of very high overall porosities (82.2–89.7 vol%) for the dual-scale porous scaffolds. However, all the scaffolds showed reasonable compressive strengths (0.8 MPa ?2.1 MPa), which are comparable to those of cancellous bones.     

Enhanced mechanical properties and biological responses of SLA 3D printed biphasic calcium phosphate bioceramics by doping bioactive metal elements

Favorable mechanical properties and outstanding bioactivity are necessary for bioceramics used for bone defect repair. The doping of Mg²⁺ and Fe³⁺ can improve the mechanical properties and bone regeneration capacity of calcium phosphate ceramics. In this study, magnesia oxide (MgO), ferric oxide (Fe₂O₃), and iron (Fe) powders are chosen as dopants to enhance biphasic calcium phosphate (BCP) bioceramics, and the MgO-BCP, Fe₂O₃-BCP, Fe-BCP bioceramics are prepared by stereolithography (SLA) for the first time. The effects of these dopants on the curing behavior of bioceramic slurries, mechanical properties, biodegradation, and cytocompatibility of BCP bioceramics are studied. The addition of 1 wt% Fe well enhances the flexural strength of BCP from 91.61 MPa to 122.60 MPa sintered at 1250 °C. The addition of 1 wt% MgO effectively promotes the biodegradation of BCP in simulated body solution (SBF), and enhances the proliferation of mouse pre-osteoblast (MC3T3-E1) cells in vitro. In addition, Fe powder is more suitable as a dopant for SLA 3D printed BCP than Fe₂O₃ powder, and all the performances of Fe-BCP are better than those of Fe₂O₃-BCP. The less microstructure defects and slower Fe³⁺ release rate make Fe-BCP have higher flexural strength and less cytotoxic compared with Fe₂O₃-BCP. This novel way exhibits beneficial effects of bioactive metal elements on mechanical properties and bioactivity, and indicates SLA 3D printed BCP bioceramic doped with MgO, Fe can be promising candidates for bone defect repair.     

Development and characterisation of microporous biomimetic scaffolds loaded with magnetic nanoparticles as bone repairing material

Fine-tuning of the scaffolds structural features for bone tissue engineering can be an efficient approach to regulate the specific response of the osteoblasts. Here, we loaded magnetic nanoparticles aka superparamagnetic iron oxide nanoparticles (SPIONs) into 3D composite scaffolds based on biological macromolecules (chitosan, collagen, hyaluronic acid) and calcium phosphates for potential applications in bone regeneration, using a biomimetic approach. We assessed the effects of organic (chitosan/collagen/hyaluronic acid) and inorganic (calcium phosphates, SPIONs) phase over the final features of the magnetic scaffolds (MS). Mechanical properties, magnetic susceptibility and biological fluids retention are strongly dependent on the final composition of MS and within the recommended range for application in bone regeneration. The MS architecture/pore size can be made bespoken through changes of the final organic/inorganic ratio. The scaffolds undertake mild degradation as the presence of inorganic components hinders the enzyme catalytic activity. In vitro studies indicated that osteoblasts (SaOS-2) on MS9 had similar cell behaviour activity in comparison with the TCP control. In vivo data showed an evident development of integration and resorption of the MS composites with low inflammation activity. Current findings suggest that the combination of SPIONs into 3D composite scaffolds can be a promising toolkit for bone regeneration.     

The effects of BaTiO3 on the handleability and mechanical strength of the prepared piezoelectric calcium phosphate silicate for bone tissue engineering

Natural bone is a piezoelectric material that can generate electrical signals when subjected to an external force. Although many studies have attempted to develop piezoelectric biomaterials for bone regeneration, post-treatment steps, such as sintering, are always needed. In this study, we prepared an injectable and piezoelectric bone substitute based on nanosized BaTiO₃ (nBT)-added calcium phosphate silicate (CPS). The impacts of nBT on the CPS handleability and mechanical strength were characterized, and show that adding nBT could improve the CPS handleability but affect the CPS mechanical strength in a concentration-dependent manner (from 25.3 ± 1.0 MPa for 10BC to 13.5 ± 1.0 MPa for 40BC). In addition, our approach could fabricate a piezoelectric bone substitute with comparable piezoelectricity to the native bone without any post-treatment. The in vitro analyses demonstrated that nBT/CPS was biocompatible and could promote osteoblast differentiation. In conclusion, our results strongly indicate that the injectable formulation based on nBT/CPS can be a promising candidate in bone tissue engineering, and further research is needed to investigate the biomaterial's performance in bone defect animal models.     

Fabrication of asymmetric membranes from polyhydroxybutyrate and biphasic calcium phosphate/chitosan for guided bone regeneration

Chitosan (CS) is known for its biocompatibility, antibacterial function, and wound healing acceleration, while calcium phosphate (CP) can promote bone regeneration. However, to be useful as barrier membrane for guided bone regeneration (GBR) in periodontal treatments, the membranes must have suitable mechanical strength in addition to good barrier properties. Therefore, a dense polyhydroxybutyrate (PHB) layer was integrated with a porous biphasic calcium phosphate/chitosan (BCP/CS) layer to form an asymmetric PHB-BCP/CS membrane. Moreover, for enhancing the interfacial strength, the PHB layer was chemically bonded to the BCP/CS layer through plasma-induced grafting of poly(acrylic acid) on its surface and followed by amidation with CS via carbodiimide activation. The incorporation of the PHB layer greatly increased the initial modulus and ultimate tensile strength of the membrane up to 524 and 16.5 MPa, respectively. In addition, the human gingival fibroblast (HGF) cells could proliferate very well on the PHB layer of the membrane, yet they were prohibited from down-growing through the membrane. Also, the addition of BCP particles in the CS layer increased the proliferation of osteoblast cells. Thus, the asymmetric PHB-BCP/CS membrane has the potential to be used as a barrier membrane for GBR in periodontal tissue engineering.     

Study of the chemical activation of hydroxyapatite rich ashes as raw materials for geopolymers

The behaviour of ashes, deriving from mixed vegetal and animal biomass incineration, was studied in different alkaline and acidic environments, in order to assess their suitability as primary raw material for low-temperature chemical consolidation. Mixed biomass ashes are mainly based on calcium phosphate and secondly on aluminosilicate compounds; they still represent an unexplored source material to be used in alternative ceramics production. Their chemical behaviour was studied as a function of pH and chemical nature of the leaching solutions, to identify the suitable conditions for ashes chemical consolidation. Preliminary results indicated that acidic digestion of ashes, regardless acid counterion nature, is able to determine the complete decomposition of complex calcium-phosphate phases. Then it allows recombination of dissolved phases into new ones, thus promoting chemical consolidation. Alkaline media were found to be less effective, however, biomass ashes might be successfully regarded as partially reactive fillers for alkali-activated materials.