Apatitic Calcium Phosphate Research Articles

Energetics of lanthanide-doped calcium phosphate apatite

Lanthanides “Ln” (rare earths) are critical elements found in natural minerals such as calcium phosphate apatites, in sedimentary and igneous settings as well as in skeletal diagenesis. From a medical point of view, nanoparticles of lanthanide-doped apatites can be produced for conferring luminescence properties of interest in cancer cells detection. However, the impact of the substitution of Ln for Ca on the stability and solubility of related apatite phases is still essentially unknown. To investigate the thermochemical effects of such lanthanide substitution for calcium in apatite, we prepared and analyzed four series of apatites with up to 10% lanthanide substitution for calcium. After thorough physicochemical characterization via complementary techniques (XRD, FTIR, TG/DSC, and IPC-AES), high-temperature oxide melt solution calorimetry in molten sodium molybdate at 973 K was performed to determine their enthalpies of formation from constituent oxides and from the elements, at 298 K. Our results indicate that although enthalpies of formation are strongly exothermic in all cases, Ln-doping has a destabilizing effect, which increases with dopant concentration and with the size of the incorporated Ln3+ ion. After estimating standard entropies, Gibbs free energies of formation and equilibrium constants for Ca2+/Ln3+ exchange reactions in apatite were then evaluated, for the first time allowing access to quantitative thermodynamic data that may be used in various fields for stability calculations or partitioning estimates between fluids and solids.

Characterization of calcium phosphate apatite with variable Ca/P ratios sintered at low temperature

The characterization of calcium phosphate apatite with variable Ca/P ratios synthesized by the hydrolysis method and sintered at 1073K has been investigated. When the precipitate powders are sintered at 1073K for 4h, the major phase observed is hydroxyapatite (Ca10(PO4)6(OH)2, HA), and the minor phases are rhenanite (NaCaPO4) and β-tricalcium phosphate (β-TCP, β-Ca3(PO4)). The shrinkage increased from 5.15 to 21.00%, and the grain size of the pellets also increased from 31 to 348nm along with the ratios of Ca/P. The TEM microstructure identified with SAED and EDS analyses show that an amorphous phase of NaCaPO4 is present at the grain boundaries of HA, which promotes the sintering of calcium phosphate, but reduces the mechanical properties.

Artefactual nanoparticle activation of the inflammasome platform: in vitro evidence with a nano-formed calcium phosphate.

To determine whether in vitro experimental conditions dictate cellular activation of the inflammasome by apatitic calcium phosphate nanoparticles. The responses of blood-derived primary human cells to in situ-formed apatite were investigated under different experimental conditions to assess the effect of aseptic culture, cell rest and duration of particle exposure. Cell death and particle uptake were assessed, while IL-1β and caspase 1 responses, with and without lipopolysaccharide prestimulation, were evaluated as markers of inflammasome activation. Under carefully addressed experimental conditions, apatitic nanoparticles did not induce cell death or engage the inflammasome platform, although both could be triggered through artefacts of experimentation. In vitro studies often predict that engineered nanoparticles, such as synthetic apatite, are candidates for inflammasome activation and, hence, are toxic. However, the experimental setting must be very carefully considered as it may promote false-positive outcomes.

Modification of Ti6Al4V substrates with well-defined zwitterionic polysulfobetaine brushes for improved surface mineralization.

Osteoconductive mineral coatings are beneficial for improving the osteointegration of metallic orthopedic/dental implants, but achieving adequate structural integration between the surface minerals and underlying metallic substrates has been a significant challenge. Here, we report covalent grafting of zwitterionic poly(sulfobetaine methacrylate) (pSBMA) brushes on the Ti6Al4V substrates to promote the surface-mineralization of hydroxyapatite with enhanced surface mineral coverage and mineral-substrate interfacial adhesion. We first optimized the atom transfer radical polymerization (ATRP) conditions for synthesizing pSBMA polymers in solution. Well-controlled pSBMA polymers (relative molecular weight up to 26kD, PDI = 1.17) with high conversions were obtained when the ATRP was carried out in trifluoroethanol/ionic liquid system at 60 °C. Applying identical polymerization conditions, surface-initiated atom transfer radical polymerization (SI-ATRP) was carried out to graft zwitterionic pSBMA brushes (PDI < 1.20) from the Ti6Al4V substrates, generating a stable superhydrophilic and low-fouling surface coating without compromising the bulk mechanic property of the Ti6Al4V substrates. The zwitterionic pSBMA surface brushes, capable of attracting both cationic and anionic precursor ions during calcium phosphate apatite mineralization, increased the surface mineral coverage from 32% to 71%, and significantly reinforced the attachment of the apatite crystals on the Ti6Al4V substrate. This facile approach to surface modification of metallic substrates can be exploited to generate multifunctional polymer coatings and improve the performance of metallic implants in skeletal tissue engineering and orthopedic and dental care.

Adsorption on apatitic calcium phosphates for drug delivery: interaction with bisphosphonate molecules.

Bisphosphonates (BPs) are well established as an important class of drugs for the treatment and prevention of several bone disorders including osteoporosis. This work investigated the interaction of two bisphosphonates, risedronate and tiludronate, with several apatitic supports, a well-crystallised hydroxyapatite (HA) and nanocrystalline apatites with varying maturation times, chemical composition and surface characteristics. The purpose was to fully understand the adsorption mechanism and desorption process, by the evaluation of the effect of several physicochemical parameters (temperature, pH and concentration of calcium and phosphate ions). Whatever the nature of the BP and the structure and composition of the apatite, the adsorption of such anti-resorptive agents can be well described as an ion exchange-reaction between phosphates species on the apatitic surface and BP molecules in solution. However, the parameters of adsorption can vary depending on the physicochemical conditions of the adsorption reaction. In addition, the structure and composition of the apatitic surface also influence the adsorption properties. Finally, BPs molecules are slowly released from apatitic supports, because most of the adsorbed molecules are irreversibly bound and not spontaneously released by dilution or simple washing. Moreover, similar to their adsorption, the release of bisphosphonates is strongly affected not only by the chemical properties of the molecule, but also by the chemical and structural characteristics of the apatitic substrates. The understanding of the adsorption and release processes provides fundamental tools for the development of drug delivery systems using apatite materials.

Mechanisms of Medial Arterial Calcification in Diabetes

Medial artery calcification (MAC) is a characteristic feature of diabetes. MAC represents a concentric calcification that proceeds via matrix vesicle-nucleated mineralization accompanied with apatitic calcium phosphate deposits in the arterial tunica media in the absence of atheroma and neointima. Multiple factors contribute to the induction and progression of diabetic MAC including inflammation, oxidative stress, adiposity, insulin resistance, advanced glycation end-products, and hyperphosphatemia. Osteoblast-like cells form in the vessel wall from vascular smooth muscle cells and multipotent vascular mesenchymal progenitors. These mineralizing cells as well as the recruitment of undifferentiated progenitors to the osteochondrocyte lineage play a critical role in the calcification process. Important transcription factors such as Msx 2, Osterix, and RUNX2 are crucial in the programming of osteogenesis. Currently, no therapy is available to reverse vascular calcification. Available therapies can only reduce and slow the progression of vascular calcification. Targeting regulatory proteins and enzymes directly involved in osteochondrogenesis and hydroxyapatite accumulation in the vascular wall may be beneficial for generating new efficient anti-calcific drugs.

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%).

Grade-1 titanium soaked in a DMEM solution at 37 °C

DMEM (Dulbecco's modified Eagle medium) solutions are used in performing in vitro cell culture experiments to assess the cell biocompatibility of synthetic biomaterials. In this study, Hepes-buffered, phenol red- and sodium pyruvate-free DMEM solutions were used, for the first time as immersion media at 37°C, to test alkali-treated (5M NaOH, 60°C, 24h) grade-1 titanium substrates. Such DMEM solutions were found to deposit X-ray-amorphous calcium phosphate (ACP), in one or two weeks, on the soaked grade-1 Ti substrates. A limited number of previous studies focusing on the biomimetic coating of alkali-treated Ti6Al4V coupons in DMEM have actually used different DMEM solutions, which were not Hepes-buffered and containing phenol red and sodium pyruvate. The previous studies with such DMEM solutions reported the deposition of cryptocrystalline apatitic calcium phosphate (Ap-CaP) on Ti6Al4V substrates, but not ACP. An inorganic solution (free of amino acids, vitamins, glucose, sodium pyruvate and phenol red), simulating the ion concentrations of the DMEM solutions, was also used for the first time in depositing ACP on grade-1 Ti substrates upon soaking at 37°C for only 24h. The solutions and deposits of this study were analyzed by AAS, ICP-AES, FTIR, XRD, XPS, and surface profilometry.

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.

Peroxide-doped apatites: Preparation and effect of synthesis parameters

This contribution focused on the preparation of peroxide-doped calcium phosphate apatites — in view of potential uses as bioactive bioceramics with antimicrobial functions, and on their main physico-chemical characteristics. Two synthesis routes were investigated. First, the hydrolysis of β-TCP in the presence of H2O2 was followed. However, only elevated concentrations in H2O2 in the medium or temperatures around 150°C allowed us to reach the complete β-TCP-to-apatite hydrolysis process, and the obtained samples exhibited a high crystallinity state with no non-apatitic chemical environments. The second protocol tested consisted in the direct apatite precipitation in the presence of H2O2 in the medium (at room temperature). This protocol led to single-phased nanocrystalline apatites, and our data indicate that part of the apatitic OH− ions were substituted by oxygenated species, and typically by peroxide ions (quantified). Physico-chemical modifications in the form of an improvement in crystallinity state, an increase in unit cell volume, and the presence of additional Raman bands were noticed and discussed.

Injectable biphasic calcium phosphate cements as a potential bone substitute

Apatitic calcium phosphate cements (CPCs) have been widely used as bone grafts due to their excellent osteoconductive properties, but the degradation properties are insufficient to stimulate bone healing in large bone defects. A novel approach to overcome the lack of degradability of apatitic CPC involves the development of biphasic CPCs (BCPC) based on tricalcium phosphate (TCP) in both α- and β-polymorphs. The aim of the current study was to prepare and analyze the physicochemical properties of BCPCs based on dual phase α/β-TCP as obtained by heat treatment of pure α-TCP. The handling and mechanical characteristics of the samples as well as the degradation behavior under in vitro condition were investigated and compared with a standard monophasic α-TCP-based CPC. The results showed that different heat treatments of commercially available α-TCP allowed the formation of biphasic calcium phosphate powder with a variety of α/β-TCP ratios. The use of biphasic powder particles as a reactant for CPCs resulted into increased setting and injectability times of the final BCPCs. During hardening of the cements, the amount of apatite formation decreased with increasing β-TCP content in the biphasic precursor powders. The morphology of the monophasic CPC consisted of plate-like crystals, whereas needle-like crystals were observed for BCPCs. In vitro degradation tests demonstrated that dissolution rate and corresponding calcium release from the set cements increased considerably with increasing β-TCP content, suggesting that apatitic CPCs can be rendered degradable by using biphasic α/β-TCP as powder precursor phase.

Synthesis, characterization, and thermo-mechanical properties of copper-loaded apatitic calcium phosphates

Copper is well known as a classical transition metal used in heterogeneous catalysis. In this study, copper-loaded apatitic calcium phosphates were prepared using incipient wetness impregnation (IWI) and ionic exchange (IE) methods. The interaction between copper precursor (copper nitrate trihydrate, Cu(NO3)2∙3H2O) and apatitic calcium phosphate (CaP) depended strongly on the preparation method and the content of copper-loaded. Using IE, copper(II) cations (Cu2+) were incorporated in the apatitic structure of CaP. The content of copper(II) cations seemed to be limited at about 2.2 wt.%. Calcination at 400 °C had no influence on the solids obtained by the IE method. Using IWI, the deposition of a theoretical copper content of 2 wt.% led to the incorporation of copper(II) cations in the apatitic structure of CaP by IE with Ca2+, despite the low quantity of aqueous solvent used. Therefore the resulting product was similar to that obtained by IE. When the theoretical copper content rose to 20 wt.%, the entire amount of copper precursor molecules were largely deposited, which resulted in the formation of copper oxide particles (CuO) after air calcination at 400 °C. Thermo-mechanical analysis study showed that the presence of copper oxide did not modify the thermal shrinkage of the initial calcium phosphate. On the other hand, thermal shrinkage was much more important in the case of CaP substituted with copper(II) cations.

Influence of polymer molecular weight in osteoinductive composites for bone tissue regeneration

In bone tissue regeneration, certain polymer and calcium-phosphate-based composites have been reported to enhance some biological surface phenomena, facilitating osteoinduction. Although the crucial role of inorganic fillers in heterotopic bone formation by such materials has been shown, no reports have been published on the potential effects the polymer phase may have. The present work starts from the assumption that the polymer molecular weight regulates the fluid uptake, which determines the hydrolysis rate and the occurrence of biological surface processes. Here, two composites were prepared by extruding two different molecular weight l/d,l-lactide copolymers with calcium phosphate apatite. The lower molecular weight copolymer allowed larger fluid uptake in the composite thereof, which was correlated with a higher capacity to adsorb proteins in vitro. Further, the large fluid absorption led to a quicker composite degradation that generated rougher surfaces and enhanced ion release. Following intramuscular implantation in sheep, only the composite with the lower molecular weight polymer could induce heterotopic bone formation. Besides influencing the biological potential of composites, the molecular weight also regulated their viscoelastic behaviour under cyclic stresses. The results lead to the conclusion that designing biomaterials with appropriate physico-chemical characteristics is crucial for bone tissue regeneration in mechanical load-bearing sites.

Blood vessels are concentrated within the implant surface concavities: a histologic study in rabbit tibia

Angiogenesis plays a key role in bone formation and maintenance. Bone formation has been reported to initiate in the concavities rather than the convexities in a hydroxyapatite substratum and the implant threads of dental implants. The aim of the present study was to evaluate the number of the blood vessels inside the concavities and around the convexities of the threads of implants in a rabbit tibia model. A total of 32 thread-shaped implants blasted with apatitic calcium phosphate (TCP/HA blend) (Resorbable Blast Texturing, RBT) (Maestro, BioHorizons(®), Birmingham, AL, USA) were inserted in 8 rabbits. Each rabbit received 4 implants, 2 in the right and 2 in left tibia. Implants were retrieved after 1, 2, 4, and 8 weeks and treated to obtain thin ground sections. Statistically significant differences were found in the number of vessels that had formed in the concavities rather than the convexities of the implants after 1 (p = 0.000), and 2 weeks (p = 0.000), whilst no significant differences after 4 (p = 0.546) and 8 weeks (p = 0.275) were detected. The present results supported the hypothesis that blood vessel formation was stimulated by the presence of the concavities, which may provide a suitable environment in which mechanical forces, concentrations and gradients of chemotactic molecules and blood clot retention may all drive vascular and bone cell migration.

Effects of phosphates on microstructure and bioactivity of micro-arc oxidized calcium phosphate coatings on Mg–Zn–Zr magnesium alloy

Calcium phosphate (CaP) coatings were prepared on Mg–Zn–Zr magnesium alloy by micro-arc oxidation (MAO) in electrolyte containing calcium acetate monohydrate (CH3COO)2Ca·H2O) and different phosphates (i.e. disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), sodium phosphate (Na3PO4·H2O) and sodium hexametaphosphate((NaPO3)6)). Scanning electron microscope (SEM), energy-dispersive X-ray spectrometry (EDS) and X-ray diffractometer (XRD) were employed to characterize the microstructure, elemental distribution and phase composition of the CaP coatings. Simulated body fluid (SBF) immersion test was used to evaluate the coating bioactivity and degradability. Systemic toxicity test was used to evaluate the coating biocompatibility. Fluoride ion selective electrode (ISE) was used to measure F− ions concentration during 30 days SBF immersion. The CaP coatings effectively reduced the corrosion rate and the surfaces of CaP coatings were covered by a new layer formed of numerous needle-like and scale-like apatites. The formation of these calcium phosphate apatites indicates that the coatings have excellent bioactivity. The coatings formed in (NaPO3)6-containging electrolyte exhibit thicker thickness, higher adhesive strength, slower degradation rate, better apatite-inducing ability and biocompatibility.

Tumoral Calcinosis Form of Hydroxyapatite Deposition Disease in Related Red-Bellied Short-Necked Turtles, Emydura subglobosa

Ten of 12 red-bellied short-necked turtles from a single clutch presented at 9 months of age with multiple white to tan nodules on their feet. Histologically, the nodules were composed of large periarticular deposits of mineralized crystalline material that extended into the joint spaces of interphalangeal joints and was surrounded by granulomatous inflammation and fibrosis. Crystallographic analysis determined the material to be apatite (calcium phosphate hydroxide) consistent with the tumoral calcinosis form of hydroxyapatite deposition disease (HADD). HADD has previously been described in aquatic turtles and rarely lizards and must be differentiated from gout in reptiles. A cause for the tumoral calcinosis lesions in these turtles could not be determined; however, based on previous reports in this species, a species-specific predilection, in conjunction with unknown environmental factors, is suspected. The use of the terms HADD, pseudogout (calcium pyrophosphate crystal deposition disease), and calcinosis circumscripta has been inconsistent, creating confusion in the literature.

Novel contributions on luminescent apatite-based colloids intended for medical imaging

The setup of colloidal hybrid nanosystems based on biomimetic calcium phosphate apatites doped with europium ions has recently raised great interest in the pharmacological community, especially due to their bio-inspired character. This is especially relevant in relation with medical imaging for cancer diagnosis. Questions however remain in relation to a number of applicability aspects, some of which have been examined in this contribution. In a first part of this work, we explored further the luminescence properties of such colloidal nanoparticles. We pointed out, upon excitation of europium, the existence of some non-radiative de-excitation via the vibration of O-H oscillators located at the vicinity of the Eu(3+) luminescent centers. The replacement of Eu(3+) by Tb(3+) ions, less prone to non-radiative de-excitation, was then tested in a preliminary way and can be seen as a promising alternative. In a second part of this work, we inspected the possibility to store these colloids in a dry state while retaining a re-suspension ability preserving the nanometer size of the initial nanoparticles, and we propose a functional protocol involving the addition of glucose prior to freeze-drying. We finally showed for the first time, based on titrations of intracellular Ca(2+) and Eu(3+) ions, that folic acid-functionalized biomimetic apatite nanoparticles were able to target cancer cells that overexpress folate receptors on their membrane, which we point out here in the case of T-47-D breast carcinoma cells, as opposed to ZR-75-1 cells that do not express folate receptors. This contribution thus opens new exciting perspectives in the field of targeted cancer diagnosis, thus confirming the promise of biomimetic apatites-based colloidal formulations.

Experimental Evaluation in Rabbits of the Effects of Thread Concavities in Bone Formation with Different Titanium Implant Surfaces

Differences in implant microtexture are important in conditioning the bone response around dental implants. The aim of the present study was an evaluation of the bone response in machined (M), blasted with apatitic calcium phosphate (tricalcium phosphate/hydrossyapatite [HA] blend) particles (resorbable blast texturing [RBT]), and coated with HA implants. A total of 48 (16 M, 16 RBT, and 16 HA) threaded screw-shaped implants were inserted into the tibia of 12 rabbits. The specimens were retrieved after 1, 2, 4, and 8 weeks and processed for histology. All experimental groups showed an increase of the bone-implant contact percentages through the study period. Higher and highly statistically significant differences were found in the percentages of bone observed in the concavities rather than in the convexities of the implants retrieved after 1, 2, and 4 weeks, while no significant differences were found after 8 weeks. In the different time periods, higher percentages of bone-implant contact were found in the RBT and HA-coated implants both in the concavities and in the convexities, but these differences were not statistically significant. The newly formed bone present in the concavity of the threads was not influenced by the implant surface in the first healing period, while after 4 to 8 weeks, the percentage of bone observed in the concavities and convexities was similar. Additional histological studies are necessary to further evaluate the critical role of the concave geometry in bone differentiation and formation around dental implants.

Apatite Formation: Why It May Not Work as Planned, and How to Conclusively Identify Apatite Compounds

Calcium phosphate apatites are inorganic compounds encountered in many different mineralized tissues. Bone mineral, for example, is constituted of nanocrystalline nonstoichiometric apatite, and the production of “analogs” through a variety of methods is frequently reported. In another context, the ability of solid surfaces to favor the nucleation and growth of “bone-like” apatite upon immersion in supersaturated fluids such as SFB is commonly used as one evaluation index of the “bioactivity” of such surfaces. Yet, the compounds or deposits obtained are not always thoroughly characterized, and their apatitic nature is sometimes not firmly assessed by appropriate physicochemical analyses. Of particular importance are the “actual” conditions in which the precipitation takes place. The precipitation of a white solid does not automatically indicate the formation of a “bone-like carbonate apatite layer” as is sometimes too hastily concluded: “all that glitters is not gold.” The identification of an apatite phase should be carefully demonstrated by appropriate characterization, preferably using complementary techniques. This review considers the fundamentals of calcium phosphate apatite characterization discussing several techniques: electron microscopy/EDX, XRD, FTIR/Raman spectroscopies, chemical analyses, and solid state NMR. It also underlines frequent problems that should be kept in mind when making “bone-like apatites.”

Effects of storage in formalin on composition of canine and feline uroliths

To determine whether storage in neutral-buffered 10% formalin in vitro has any effect on the composition of biogenic minerals of canine and feline uroliths. Prospective in vitro study. Canine and feline uroliths submitted to the Minnesota Urolith Center from 34 dogs and 27 cats. Submissions from each dog or cat consisted of multiple uroliths of a single mineral type. After retrieval from the urinary tract, none of the uroliths had been placed in a preservative before submission. Evaluated uroliths were exclusively composed of the following: only struvite (uroliths from 5 dogs and 5 cats), calcium oxalate (5 dogs and 5 cats), calcium phosphate apatite (5 dogs and 5 cats), cystine (5 dogs and 5 cats), ammonium urate (5 dogs and 5 cats), or silica (5 dogs). One urolith from each dog or cat was quantitatively analyzed by polarized light microscopy, infrared spectroscopy, or both. Another urolith from the same animal was immersed in 1 mL of neutral-buffered 10% formalin for 48 hours at room temperature (22.5°C). Uroliths exposed to formalin were then air-dried for 30 minutes, and the analysis was repeated. After exposure to formalin, a portion of every struvite urolith was transformed into newberyite. This was not observed with any other urolith mineral type. Quantitative mineral analysis of nonstruvite uroliths revealed no detectable change in mineral composition. However, 3 of 10 ammonium urate uroliths dissolved when placed in formalin. To avoid misdiagnosis of mineral composition, uroliths should not be immersed in formalin prior to analysis.