Calcium Phosphate Bioceramics Research Articles

Effects of Stirring and Fluid Perfusion on the In Vitro Degradation of Calcium Phosphate Cement/PLGA Composites.

In vitro degradation rates of calcium phosphate bioceramics are investigated using a large variation of soaking protocols that do not all match the dynamic conditions of the perfused physiological environment. Therefore, we studied the effect of stirring and fluid perfusion on the in vitro degradation rate of apatitic calcium phosphate cements (CPC) containing poly(lactic-co-glycolic acid) (PLGA) microspheres. The composites were soaked in phosphate-buffered saline up to 6 weeks under unstirred, stirred, or perfused conditions followed by analysis of mass loss, compression strength, porosity, crystal phase composition, and morphology of the cement composites. The results showed that fluid perfusion reduced the decrease in pH and corresponding degradation rates, while nonperfused soaking conditions (i.e., stirred and unstirred conditions) resulted into more extensive acidification, the rate of which increased with stirring. After 2 weeks, the formation of a secondary brushite phase was observed for cement composites soaked under nonperfused (i.e., stirred and unstirred) conditions, whereas this phase was not detected in cements soaked under perfused conditions. The degradation rate of cement composites decreased in the order unstirred>stirred>perfused, as evidenced by quantification of mass loss, compression strength, and pore morphology. To summarize, we have demonstrated that soaking conditions strongly affected the in vitro degradation process of CPCs. As a consequence, it can be concluded that the experimental design of current in vitro degradation studies does not allow for correlation to (pre-)clinical studies.

Sol-Gel Derived Zinc Doped Calcium Phosphate Bioceramics Prepared via Different Sintering Strategies

Sinterability of zinc doped calcium phosphate (CaP) sintered via conventional pressureless and two step sintering methods was investigated. The starting powders have been synthesized through sol-gel method with zinc concentration varied in the range of 0, 1, 4, and 10mol%. After uniaxial pressing, the compacts were sintered via conventional pressureless sintering. The dense samples were studied in terms of the phase stability, relative density, Vickers hardness, and fracture toughness. The phase of samples was hydroxyapatite and β-tricalcium phosphate. The presence of Zn caused a change in the sintering behaviour of dense calcium phosphate. Zn doping has improved relative density of 4mol% Zn-doped by 8.1% compared to undoped fired at 1300°C. The maximum fracture toughness of 1.43 MPa.m1/2 and Vickers hardness of 3.44 GPa were achieved at 1200°C by 4 mol% Zn-doped. The low Vickers hardness and fracture toughness obtained for dense samples sintered of two step sintering compared to conventional pressureless sintering is due to the low measured bulk density. This study showed that conventional pressureless sintering was beneficial in producing high toughness of Zn doped CaP.

Accelerated Self-Hardening Tetracalcium Phosphate Based Bone Cement with Enhanced Strength and Biological Behaviour

Tetracalcium phosphate (TTCP) is the most basic phase among the calcium phosphate bioceramics. TTCP has been used as self setting bone cement and its relatively high surface pH seems to be very beneficial for cell adhesion and bone formation. Various attempts have been made to improve the cement properties for the ease of use during the surgical procedures. The effect of citric acid (CA) on the setting properties, apatite forming ability, strength and biological behavior of TTCP based bone cement has been studied in detail in the present study. The cement formulation containing 15 % CA has been found to have setting time between 9 and 16 min suitable for bone cement. It also showed complete conversion to apatite phase and highest compressive strength after 28 days of immersion in phosphate buffer at physiological conditions. Cell culture studies using rat skeletal muscle cells confirm higher cell viability in the CA containing cements compared to the pure cement without CA content. The results suggest that self-hardening tetracalcium phosphate based bone cement modified with CA holds a promise for its use in orthopaedic and trauma surgery.

Validation of an in vitro 3D bone culture model with perfused and mechanically stressed ceramic scaffold.

An engineered three dimensional (3D) in vitro cell culture system was designed with the goal of inducing and controlling in vitro osteogenesis in a reproducible manner under conditions more similar to the in vivo bone microenvironment than traditional two-dimensional (2D) models. This bioreactor allows efficient mechanical loading and perfusion of an original cubic calcium phosphate bioceramic of highly controlled composition and structure. This bioceramic comprises an internal portion containing homogeneously interconnected macropores surrounded by a dense layer, which minimises fluid flow bypass around the scaffold. This dense and flat layer permits the application of a homogeneous loading on the bioceramic while also enhancing its mechanical strength. Numerical modelling of constraints shows that the system provides direct mechanical stimulation of cells within the scaffold. Experimental results establish that under perfusion at a steady flow of 2 µL/min, corresponding to 3 ≤ Medium velocity ≤ 23 µm/s, mouse calvarial cells grow and differentiate as osteoblasts in a reproducible manner, and lay down a mineralised matrix. Moreover, cells respond to mechanical loading by increasing C-fos expression, which demonstrates the effective mechanical stimulation of the culture within the scaffold. In summary, we provide a "proof-of-concept" for osteoblastic cell culture in a controlled 3D culture system under perfusion and mechanical loading. This model will be a tool to analyse bone cell functions in vivo, and will provide a bench testing system for the clinical assessment of bioactive bone-targeting molecules under load.

Microwave assisted synthesis and characterization of magnesium substituted calcium phosphate bioceramics.

Hydroxyapatite is used extensively in hard tissue repair due to its biocompatibility and similarity to biological apatite, the mineral component of bone. It differs subtly in composition from biological apatite which contains other ions such as magnesium, zinc, carbonate and silicon (believed to play biological roles). Traditional methods of hydroxyapatite synthesis are time consuming and require strict reaction parameter control. This paper outlines synthesis of magnesium substituted hydroxyapatite using simple microwave irradiation of precipitated suspensions. Microwave irradiation resulted in a drastic decrease in ageing times of amorphous apatitic phases. Time taken to synthesize hydroxyapatite (which remained stable upon heat treatment at 900°C for 1h) reduced twelve folds (to 2h) as compared to traditionally required times. The effects of increasing magnesium concentration in the precursors on particle size, surface area, phase-purity, agglomeration and thermal stability, were observed using scanning electron microscopy, BET surface area analysis, X-ray diffraction and photo acoustic Fourier transform infra-red spectroscopy. Porous agglomerates were obtained after a brief heat-treatment (1h) at 900°C.

Dual mode antibacterial activity of ion substituted calcium phosphate nanocarriers for bone infections.

Nanotechnology has tremendous potential for the management of infectious diseases caused by multi-drug resistant bacteria, through the development of newer antibacterial materials and efficient modes of antibiotic delivery. Calcium phosphate (CaP) bioceramics are commonly used as bone substitutes due to their similarity to bone mineral and are widely researched upon for the treatment of bone infections associated with bone loss. CaPs can be used as local antibiotic delivery agents for bone infections and can be substituted with antibacterial ions in their crystal structure to have a wide spectrum, sustained antibacterial activity even against drug resistant bacteria. In the present work, a dual mode antibiotic delivery system with antibacterial ion substituted calcium deficient hydroxyapatite (CDHA) nanoparticles has been developed. Antibacterial ions such as zinc, silver, and strontium have been incorporated into CDHA at concentrations of 6, 0.25–0.75, and 2.5–7.5 at. %, respectively. The samples were found to be phase pure, acicular nanoparticles of length 40–50 nm and width 5–6 nm approximately. The loading and release profile of doxycycline, a commonly used antibiotic, was studied from the nanocarriers. The drug release was studied for 5 days and the release profile was influenced by the ion concentrations. The release of antibacterial ions was studied over a period of 21 days. The ion substituted CDHA samples were tested for antibacterial efficacy on Staphylococcus aureus and Escherichia coli by MIC/MBC studies and time-kill assay. AgCDHA and ZnCDHA showed high antibacterial activity against both bacteria, while SrCDHA was weakly active against S. aureus. Present study shows that the antibiotic release can provide the initial high antibacterial activity, and the sustained ion release can provide a long-term antibacterial activity. Such dual mode antibiotic and antibacterial ion release offers an efficient and potent way to treat an incumbent drug resistant infection.

Phase stability of silver particles embedded calcium phosphate bioceramics

In this paper, we report the compositional variation-dependent phase stability of hydroxyapatite (Ca10(PO4)6(OH)2) on doping with silver. The transformation of hydroxyapatite to (β/α) tricalcium phosphate phases during sintering has been explored using Raman spectroscopy and X-ray diffraction techniques. The optical absorption spectroscopy analysis reveals the presence of Ag+ ions at low doping levels. As the doping increases, abundance of Ag particles is enhanced.

Effects of ions traces on the dissolution of bioceramics composed of hydroxyapatite and β-tricalcium phosphate

The aim of this work is to study the effect of trace ions as Mg2+, Sr2+, (SiO4)4- and Al3+, brought by the raw material, on the dissolution of the calcium-phosphate bioceramics.  The precursor powders prepared by aqueous precipitation with molar ratio Ca / P = 1.630 ± 0.002, were calcined at 1100°C resulting in the formation of a mixture of hydroxyapatite (Ca10(PO4)6(OH)2) and β-tricalcium phosphate (β-Ca3(PO4)2).  Two mixtures which differ by the content of trace ions were characterized for phase purity, chemical composition and morphology.  The dissolution tests were performed at 37 °C in acidic buffers solution at pH 4.8.  The results showed that the dissolution of calcium ions is more important in the presence of ions trace while the phosphor ions were not affected.  The dissolution and dissolution-reprecipitation observed of various ions trace can modify the surface properties of calcium phosphate bioceramics and therefore the properties of biological products, such as resorbtion and reactivity can be affected.

Response of stem cells from different origins to biphasic calcium phosphate bioceramics.

Biphasic calcium phosphate (BCP) bioceramics have been successfully applied in a broad variety of presentation forms and with different ratios of hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP). BCPs have been loaded with stem cells from different origins for bone tissue engineering purposes, but evidence of stem cell behavior on different compositions (various HA/β-TCP ratios) and physical features of BCPs is limited. We compared the adhesion, proliferation, viability and osteogenic potential of human mesenchymal stem cells (MSCs) on granular BCPs with equal HA/β-TCP ratio of diverse particle sizes and on porous blocks which had different chemical compositions. In addition, the osteogenic differentiation of MSCs was compared to adipose-derived (ADSC) and dental pulp (DPSC) stem cells, as well as to pre-osteoblasts on a particulate BCP. MSCs growing on granular BCPs demonstrated increased number as compared to MSCs growing on blocks. Cells proliferated to a greater extent on small granular BCPs, while large granular BCPs and blocks promoted cell differentiation. Surprisingly, the expression of genes involved in osteogenesis was upregulated in MSCs on bioceramics in basal medium which indicates that BCPs may have osteoinductive potential. This was confirmed with the upregulation of osteochondrogenic markers, at different time points, when stem cells from various tissues were grown on the BCP. This study demonstrates that BCPs, depending on their physical features and chemical composition, modulate stem cell behavior, and that stem cells from different origins are inherently distinct in their gene expression profile and can be triggered toward osteochondrogenic fate by BCPs.

Elucidating the individual effects of calcium and phosphate ions on hMSCs by using composite materials

The biological performance of bone graft substitutes based on calcium phosphate bioceramics is dependent on a number of properties including chemical composition, porosity and surface micro- and nanoscale structure. However, in contemporary bioceramics these properties are interlinked, therefore making it difficult to investigate the individual effects of each property on cell behavior. In this study we have attempted to investigate the effects of calcium and inorganic phosphate ions independent from one another by preparing composite materials with polylactic acid (PLA) as a polymeric matrix and calcium carbonate or sodium phosphate salts as fillers. Clinically relevant bone marrow derived human mesenchymal stromal cells (hMSCs) were cultured on these composites and proliferation, osteogenic differentiation and ECM mineralization were investigated with time and were compared to plain PLA control particles. In parallel, cells were also cultured on conventional cell culture plates in media supplemented with calcium or inorganic phosphate to study the effect of these ions independent of the 3D environment created by the particles. Calcium was shown to increase proliferation of cells, whereas both calcium and phosphate positively affected alkaline phosphatase enzyme production. QPCR analysis revealed positive effects of calcium and of inorganic phosphate on the expression of osteogenic markers, in particular bone morphogenetic protein-2 and osteopontin. Higher levels of mineralization were also observed upon exposure to either ion. Effects were similar for cells cultured on composite materials and those cultured in supplemented media, although ion concentrations in the composite cultures were lower. The approach presented here may be a valuable tool for studying the individual effects of a variety of soluble compounds, including bioinorganics, without interference from other material properties.

PH Effect on the Dissolution Behavior of the Microspheres Containing Strontium Ranelate

Strontium is known for efficient actions on bone formation and resorption. Strontium ranelate (SrR) is a commercial drug which maintains this balance during bone turnover, reducing the risks of vertebral fractures in the patients. Calcium phosphate bioceramics associated with alginate matrices containing strontium (Sr) could improve bone regeneration due to gradual Sr release. In this report, the strontium ranelate was incorporated on microspheres of alginate (ALG)/β-tricalcium phosphate in a single route of the production. Energy dispersive spectroscopy showed that strontium was incorporated on the surface of the microspheres produced. Thedissolution behaviour into a buffer solution at pH 4.0 and at 7.4 was evaluated, measuring Sr content on the microspheres after in vitro assays by atomic absorption spectrometry. Dissolution tests showed a rapid strontium release in both assays, however, it was more pronounced at pH 4.0. Fourier transform infrared spectra indicated the presence of a new precipitated phase at pH 7.4 up after 14 days. Scanning electron microscopy of the microspheres submitted to in vitro revealed that the microspheres at pH 4.0 buffer underwent erosion up to 7 days, while the ones in pH 7.4 buffer, eroded in 48h. This behaviour is due to the high swelling rate of the microspheres in neutral pH. The solubility of the microspheres favors its use as a great material for a local strontium release and remodeling bone.

Strontium Incorporation on Microspheres of Alginate/β-tricalcium Phosphate as Delivery Matrices

Strontium (Sr) is known to positively affect the mechanism of bone remodelling. Consequently, calcium phosphate bioceramics associated with alginate matrices containing strontium could improve bone regeneration due to gradual strontium release. This work aims to incorporate Sr on microspheres of alginate (ALG)/β-tricalcium phosphate (β-TCP) and evaluates the in vitro release of strontium ions into a buffer solution at pH 4.0 and 7.4. In this study, strontium chloride was employed as a cross-linking agent and Sr source. Energy dispersive spectroscopy (EDS) showed that strontium was incorporated mainly at the surface of the microspheres produced. The in vitro experiments revealed that there is a rapid strontium release up to 24 h at pH 7.4 due to Sr location on microspheres' surface. At pH 4.0 both calcium and strontium were released due to the β-TCP dissolution.

Sintering and Characterization of Calcium Phosphate Biomaterials Elaborated from Fossilized Calcareous Shell

Bioceramics of calcium phosphate, obtained from natural raw materials, are promising as bone substitutes because they exhibit crystallographic similarity with the bone tissue. This work deals with the sintering and characterization of calcium phosphate biomaterials from fossilized calcareous shells. Four compositions of biomaterials were prepared with Ca/P molar ratio ranging from 1.4 to 1.67. They were synthesized using a wet method and calcined at 900°C/2h providing calcium phosphate powder, then compressed into a metallic mould. The samples obtained from this compression were sintered at 1200oC for 2h. The biomaterials recovered from sintering were subjected to a microstructural characterization by scanning electron microscopy [SE and by X-ray diffraction [XR. Mechanical properties were determined by compression tests. Finally, the Arthur method was used for determining the hydrostatic density and open porosity from these biomaterials. The value of fracture strength was between 54 and 84 MPa for compositions 1.5, 1.67 and 1.6 molar and for composition 1.4 molar about 328 Mpa. The results also showed was the amount of open porosity which ranged between 35 and 54% with increasing Ca/P molar ratio. These studies demonstrate that the production of biomaterials from fossilized calcareous shells may be a new alternative to the production of biomaterials for bone reconstruction.

Electrophoretic deposition of magnesium silicates on titanium implants: Ion migration and silicide interfaces

Magnesium silicates (MgxSiOy) and in particular forsterite (Mg2SiO4) owing to their low thermal expansion mismatch with metals are promising materials for bioactive coating of implants. Here, we report the electrophoretic deposition (EPD) of forsterite onto titanium substrates using different precursors. Unlike bulk samples which achieve full stoichiometry only beyond 1400 °C, non-stoichiometric magnesium silicate rapidly decomposes into magnesium oxide nanowires during sintering. Elemental mapping and X-ray diffraction suggest that oxygen diffusion followed by ion exchange near the substrate leads to formation of an interfacial Ti5Si3 layer. Pre-annealed forsterite powder on the other hand shows a comparatively lower diffusion rate. Overall, magnesium silicate coatings do not exhibit thermally induced microcracks upon sintering as opposed to calcium phosphate bioceramics which are currently in use.

Development and Characterization of Biphasic Hydroxyapatite/β-TCP Cements.

Biphasic calcium phosphate bioceramics composed of hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) have relevant properties as synthetic bone grafts, such as tunable resorption, bioactivity, and intrinsic osteoinduction. However, they have some limitations associated to their condition of high-temperature ceramics. In this work self-setting Biphasic Calcium Phosphate Cements (BCPCs) with different HA/β-TCP ratios were obtained from self-setting α-TCP/β-TCP pastes. The strategy used allowed synthesizing BCPCs with modulated composition, compressive strength, and specific surface area. Due to its higher solubility, α-TCP was fully hydrolyzed to a calcium-deficient HA (CDHA), whereas β-TCP remained unreacted and completely embedded in the CDHA matrix. Increasing amounts of the non-reacting β-TCP phase resulted in a linear decrease of the compressive strength, in association to the decreasing amount of precipitated HA crystals, which are responsible for the mechanical consolidation of apatitic cements. Ca2+ release and degradation in acidic medium was similar in all the BCPCs within the timeframe studied, although differences might be expected in longer term studies once β-TCP, the more soluble phase was exposed to the surrounding media.

Experimental evaluation of hardening strains in a bioceramic material using an embedded optical sensor

The paper introduces the use of optical fiber Bragg grating sensors for investigating the magnitude of the generated strains induced during the hardening process of calcium phosphate bioceramics such as hydroxyapatite cements. It is demonstrated that such optical sensors can provide important information on the magnitude of the generated internal strains during the entire hardening period. Their potential in capturing any phase transformations that the cement structure undergoes during the process of hardening is also examined and discussed. Mechanical properties of moist cements before and after the appearance of an observed transition point during hardening are also reported.

In vitro osteoinductive potential of porous monetite for bone tissue engineering

Tissue engineering–based bone grafts are emerging as a viable alternative treatment modality to repair and regenerate tissues damaged as a result of disease or injury. The choice of the biomaterial component is a critical determinant of the success of the graft or scaffold; essentially, it must induce and allow native tissue integration, and most importantly mimic the hierarchical structure of the native bone. Calcium phosphate bioceramics are widely used in orthopaedics and dentistry applications due to their similarity to bone mineral and their ability to induce a favourable biological response. One such material is monetite, which is biocompatible, osteoconductive and has the ability to be resorbed under physiological conditions. The osteoinductive properties of monetite in vivo are known; however, little is known of the direct effect on osteoinduction of human mesenchymal stem cells in vitro. In this study, we evaluated the potential of monetite to induce and sustain human mesenchymal stem cells towards osteogenic differentiation. Human mesenchymal stem cells were seeded on the monetite scaffold in the absence of differentiating factors for up to 28 days. The gene expression profile of bone-specific markers in cells on monetite scaffold was compared to the control material hydroxyapatite. At day 14, we observed a marked increase in alkaline phosphatase, osteocalcin and osteonectin expressions. This study provides evidence of a suitable material that has potential properties to be used as a tissue engineering scaffold.

In Vivo Preliminary Study on Bone Neoformation Behavior of Three Types of Calcium Phosphate Bioceramics

Calcium phosphate microporous bioceramics and biphasic compositions of hydroxyapatite and β-calcium phosphate, in the form of microporous granular biomaterials, are research topics and present themselves as potential orthopedic and biomedical applications in rebuilding and repairing maxillofacial bones and tooth structure. This is associated with the characteristics of microstructure, biocompatibility, bioactivity and bone conductivity properties which these materials offer when applied in vivo or in a simulation environment. This study aimed to assess the behavior of bone neoformation of three types of calcium phosphate biomaterials in in vivo tests with sheep within 60 and 90 days, with the help of a scanning electron microscope. The biomaterials used were provided by the Group of Biomaterials at the Santa Catarina State University. The in vivo tests were carried out by generating, on sheep, tibial bone defects, three of which were filled with biomaterial (one different biomaterial for each bone defect generated), whilst the fourth received a bone fragment obtained during the generation of the defect in question, to serve as a control group. The scanning electron microscopy (SEM) technique was used for carrying out the preliminary characterization studies so as to observe new bone formation and osseointegration. The X-ray diffractometry (XRD) served as a support for the characterization of crystalline phases. The results obtained are encouraging and show that the biomaterials presented good performance in the process of bone formation, biomaterial osseointegration by a new tissue and bone mineralization.

Preparation of hydroxyapatite/poly(lactic acid) hybrid microparticles for local drug delivery

Calcium phosphate (CaP) bioceramic is well known as bioactive and biocompatible material in bone tissue regeneration applications. Apatitic CaP, especially nano sized hydroxyapatite (NHAp), is more similar to the natural apatite presented in the bone tissue than CaP bioceramics. In the current research NHAp was modified using biodegradable polymer – poly(lactic acid) (PLA) to develop composites providing bone regeneration and local drug delivery. NHAp/PLA microcapsules were prepared using solid-in-water-in-oil-in-water (s/w1/o/w2) encapsulation technology. The impact of primary and secondary emulsion stability on the emulsion droplet and microparticle properties was evaluated. The stability of final emulsion can be increased by varying the process parameters. Stable s/w1/o/w2 emulsion using 3ml of NHAp suspension, not less than 100ml of 4% PVA water solution and 10ml of 10% PLA solution in dichloromethane can be obtained. S/w1/o/w2 microencapuslation method can be effectively used for the preparation of multi-domain microcapsules achieving high NHAp encapsulation efficacy (93%).

Human mesenchymal stem cells response to multi-doped silicon-strontium calcium phosphate coatings

The search for apatitic calcium phosphate coatings to improve implants osteointegration is, nowadays, preferentially focused in the obtaining of compositions closer to that of the inorganic phase of bone. Silicon and strontium are both present in trace concentrations in natural bone and have been demonstrated, by separate, to significantly improve osteoblastic response on calcium phosphate bioceramics. This work aims the controlled and simultaneous multi-doping of carbonated calcium phosphate coatings with both elements, Si and Sr, by pulsed laser deposition technique and the biological response of human mesenchymal stem cells to them. A complete physicochemical characterization has been also performed to analyze the coatings and significant positive effect was obtained at the osteogenic differentiation of cells, confirming the enormous potential of this multi-doping coating approach.