Calcium Phosphate Particles Research Articles

A fast degrading PLLA composite with a high content of functionalized octacalcium phosphate mineral phase induces stem cells differentiation

Octacalcium phosphate (OCP) was synthesized yielding a combination of OCP and hydroxyapatite (HA) with a ratio of 90:10 (OCP/HA). A method was developed to functionalize the surface of the apatite using lauroyl chloride to improve the dispersion of the mineral phase in a poly(L-lactide) (PLLA) polymeric matrix. Infrared spectra and thermal gravimetric analysis confirmed the presence of laurate on the surface of calcium phosphate (CaP) particles. Neat HA particles were also functionalized with lauryl chloride for comparative purposes. PLLA/OCP/HA-laurate (PLLA/OCP/HA-L) and PLLA/HA-laurate (PLLA/HA-L) composites were fabricated by electrospinning method. The presence of the functional groups resulted in significant improvement of the dispersion of OCP/HA and HA particles into the polymeric matrix, allowing inclusion of up to 40% of mineral phase. Electrospun fibrous biocomposites of PLLA/CaP containing up to 40% in mineral phase were obtained without compromising their mechanical properties. Measurements of mass loss and calcium release in vitro showed that OCP/HA is more soluble than HA. The bioactivity of the composites was investigated by simulated body fluid test (SBF). Although both PLLA/OCP/HA-L and PLLA/HA-L fibers can form CaP crystals on their surface after exposition in SBF, the results demonstrate a significant enhancement in mineralization when OCP/HA is the mineral phase in the composite instead neat HA. Furthermore, the obtained PLLA/OCP/HA-L electrospun fibers favored the proliferation and differentiation of stem cells from human exfoliated deciduous teeth and mouse calvaria-derived preosteoblastic cells into mineralized osteoblasts. This new material is proposed as fast degrading CaP biocomposite for bone and teeth applications.

Calcium/Phosphate Solubility Curves for Premasol and Trophamine Pediatric Parenteral Nutrition Formulations.

Calcium and phosphate incompatibility in parenteral nutrition formulations remains a critical concern for patient safety. This study examined calcium phosphate solubility for 2-in-1 admixtures prepared using 2 commercially available pediatric amino acid solutions (Premasol, Baxter Healthcare Corp; or Trophamine, B. Braun Medical Inc), applying identical test methods, storage conditions, and acceptance criteria. Parenteral 2-in-1 admixtures included amino acid; dextrose; static concentrations of sodium, potassium, and magnesium, and varying concentrations of calcium (0-60 mEq/L), phosphate (15-50 mmol/L), and cysteine. Three replicate samples were stored for 48 hours at 40°C ± 2°C and then visually inspected for particulate matter, evaluated for subvisible particulate matter, when particulate matter was noted, microscopic examination was performed to confirm the presence of calcium phosphate crystals. Pass criteria were: all replicates free of visible particulate matter related to calcium phosphate crystals and particle counts below US Pharmacopeia <788> limits. Premasol and Trophamine generated identical calcium phosphate curves for 2% amino acid formulations containing 20% dextrose with/without cysteine, and similar curves for the 1% or 3% amino acid formulations containing 10% or 20% dextrose with/without cysteine. Calcium phosphate particles were identified in failed samples by scanning electron microscopy/energy dispersive X-ray spectroscopy. Calcium phosphate solubility was higher in formulations containing cysteine 40 mg/g amino acid vs. cysteine 20 mg/g amino acid and in cysteine 20 mg/g amino acid vs. no cysteine. Admixtures made with 1%, 2%, or 3% Premasol or Trophamine have essentially equivalent calcium phosphate solubility curves when tested with identical methods, storage conditions, and acceptance criteria.

Enzyme immobilisation on poly-l-lysine-containing calcium phosphate particles for highly sensitive glucose detection.

High catalytic activities of enzymes are necessary for enzyme immobilising technology for the development of glucose sensors. The aim of this study is to synthesise two types of poly(l-lysine)-containing calcium phosphate particles (pLys-HAp) and to achieve the immobilisation of glucose oxidase (GOX) on them. The oxidation activity of GOX immobilised on these particles was more than 80% compared to that of native GOX (considered to be 100%). Additionally, the relative activity of GOX immobilised on poly-ε-lysine-containing HAp (ε-pLys-HAp) remained approximately 70% after ten cycles. Moreover, glucose detection was able to be performed in the linear range of 4–400 μM using GOX immobilised on pLys-HAp composites. In the direct electrochemistry measurement using the cyclic voltammetry (CV) method, a glassy carbon electrode (GCE) modified by ε-pLys-HAp was a good enzyme electrode and can be used for glucose detection with high sensitivity. From these results, poly(l-lysine)-containing HAp composites can be expected to be enzyme immobilisation agents with high stability and biosensors with high sensitivity.

Formation of calcium phosphate nanoparticles mediated by animal protein hydrolysates enhances calcium absorption by murine small intestine ex vivo.

Animal protein intake appears to exert beneficial effects on bone health. Here, animal protein hydrolysates (APHs) originated from casein, whey protein isolate, egg white, myofibrillar protein, sarcoplasmic protein and gelatin were shown to prevent calcium phosphate (CaP) precipitation by increasing the surface charge and slowing the crystal growth of CaP particles. Dynamic light scattering, transmission electron microscopy and energy-dispersive X-ray analysis revealed the APH-mediated formation of irregularly shaped secondary nanoparticles aggregated from rather small amorphous CaP nanoclusters. APHs effectively counteracted the detrimental effect of the low calcium-to-phosphorus ratio on calcium transportation across ligated murine ileum, and by treating with NaN3, amiloride and low temperature, macropinocytosis was found to involve the intestinal uptake of CaP nanoparticles. APHs of meat origin had weaker potential to increase CaP solubility and ileal calcium transportation than those of dairy and egg origins. Overall, our study reports a novel mechanism for animal protein intake to promote intestinal calcium absorption.

3-D printing of chitosan-calcium phosphate inks: rheology, interactions and characterization.

Bone substitute fabrication is of interest to meet the worldwide incidence of bone disorders. Physical chitosan hydrogels with intertwined apatite particles were chosen to meet the bio-physical and mechanical properties required by a potential bone substitute. A set up for 3-D printing by robocasting was found adequate to fabricate scaffolds. Inks consisted of suspensions of calcium phosphate particles in chitosan acidic aqueous solution. The inks are shear-thinning and consist of a suspension of dispersed platelet aggregates of dicalcium phosphate dihydrate in a continuous chitosan phase. The rheological properties of the inks were studied, including their shear-thinning characteristics and yield stress. Scaffolds were printed in basic water/ethanol baths to induce transformation of chitosan-calcium phosphates suspension into physical hydrogel of chitosan mineralized with apatite. Scaffolds consisted of a chitosan polymeric matrix intertwined with poorly crystalline apatite particles. Results indicate that ink rheological properties could be tuned by controlling ink composition: in particular, more printable inks are obtained with higher chitosan concentration (0.19 mol·L-1).

Chitosan and polyethylene glycol based membranes with antibacterial properties for tissue regeneration

The prevention of microbial infections associated with implantable medical devices and superficial wounds represents one of the main research strategies in the field of biomaterials. The present study reports on the development of composite membranes of Chitosan (CS)-Polyethylene glycol (PEG) matrix, incorporating particles of biphasic calcium phosphate (BCP), zinc oxide (ZnO) and copper oxide (CuO). The properties that are relevant for intended applications in tissue regeneration and antibacterial coatings of implants were assessed. It was found that the addition of 1% (w/w - relative to the mass of CS) of each metal oxide promoted satisfactory bacteriostatic activity and exhibited no cytotoxic effects towards the Vero cell line. The formation of bonds between the CS/PEG matrix and ionic species from the powders enhanced the cross-linking degree and mechanical properties of composite membranes in comparison to the non-doped membrane with the same polymer matrix (CS/PEG = 70/30%). A gradual degradation of the composite membranes over the immersion time in simulated body fluid (SBF) was accompanied by a continuous surface deposition of uniform apatite layer.

In vitro remineralization of artificial enamel caries with resin composites containing calcium phosphate particles.

The aim of the study was to evaluate the effect of experimental composites containing dicalcium phosphate dihydrate (DCPD) on remineralization of enamel lesions. Five resin-based composites containing equal parts (in mols) of bisphenol-A glycidyl dimethacrylate (BisGMA), triethylene glycol dimethacrylate (TEGDMA), and 60 vol % of fillers were manipulated. Filler phase was constituted by silanized barium glass and 0, 10, or 20 vol % of DPCD particles, either functionalized (F) or nonfunctionalized (NF) with TEGDMA. Artificial subsurface lesions were produced in human enamel fragments and divided according to the resin composite applied on the lesion (no DCPD, 20% NF, 20% F, 10% NF, 10% F) plus a group without composite build-up (nontreated, NT). Fragments were exposed to 16 days of pH cycling. Specimens were evaluated using transverse microradiography (TMR). Calcium and phosphate concentrations in pH-cycling solutions were determined by spectrophotometry. TMR and ionic concentrations were analyzed using one-way ANOVA/Tukey and Kruskal-Wallis/Dunn test, respectively (alpha: 0.05). All composite groups showed enamel remineralization (3%-23%). Higher mineral recovery in the middle (7%-11%) and bottom (2%-7%) thirds of the lesion was observed in groups with DCPD-containing composites compared to the "no DCPD" group (middle: 1%, bottom: -3%). Lesion depth was significantly reduced in groups using DCPD-containing composites compared to NT group. No noticeable increase in calcium and phosphate ions was observed in the pH-cycling solutions due to the presence of DCPD in the composites. In conclusion, composites with DCPD fractions as low as 10%, regardless of functionalization, were able to promote mineral recovery and reduce lesion depth of enamel lesions. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1542-1550, 2019.

Bioactivity Assessment of Apatite Nuclei-PVDF Composite Thin Films

Particles of calcium phosphate were precipitated by raising the temperature and the pH of simulated body fluid (SBF) named Apatite Nuclei (AN). AN and polyvinylidene fluoride (PVDF) composites thin films with different weight percentages of AN in PVDF were fabricated by solution casting technique, using doctor blade method. In order to assess the bioactivity, the thin films were soaked in simulated body fluid (SBF). It was found that the film containing 30 wt.% of AN in PVDF actively induced hydroxyapatite formation in 3 days soaking period in SBF.

Fabrication of Bioactive Polycaprolactone by Incorporation of Apatite Nuclei

When the pH or the temperature of a simulated body fluid is raised, fine particles of calcium phosphate are precipitated, which are named apatite nuclei (AN). In this study, we aimed to impart bioactivity to polycaprolactone by incorporating AN. AN-PCL composite films having different weight percentages of AN in PCL were fabricated by solution casting technique, using doctor blade method. Results showed that more than 20 wt.% AN-PCL films were fully covered with hydroxyapatite layer in three days soaking period in SBF.

Dependence of calcium phosphate formation on nanostructure of rutile TiO2(110) surfaces

To demonstrate the effect of the surface nanostructures on the osteoconductivity of titanium dioxide (TiO2), formation of calcium phosphate on the rutile TiO2(110) surfaces in Hanks’ balanced salt solution (HBSS) was examined using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) techniques. When the TiO2(110) surfaces annealed in air were immersed in HBSS, calcium phosphate particles were formed on the surface. The calcium phosphate formation was suppressed by further annealing of the surfaces in O2 before the immersion in HBSS. The suppression of calcium phosphate formation is attributed to the transformation of the topmost layer of the air-annealed TiO2(110) surface from amorphous to rutile. The arrangement of the phosphate ions adsorbed on the rutile (1 × 1) surface does not match the hydrogen phosphate ion lattice of the CaHPO4 sheet of brushite.

Porous Inorganic Carriers Based on Silica, Calcium Carbonate and Calcium Phosphate for Controlled/Modulated Drug Delivery: Fresh Outlook and Future Perspectives.

Porous inorganic nanostructured materials are widely used nowadays as drug delivery carriers due to their adventurous features: suitable architecture, large surface area and stability in the biological fluids. Among the different types of inorganic porous materials, silica, calcium carbonate, and calcium phosphate have received significant attention in the last decade. The use of porous inorganic materials as drug carriers for cancer therapy, gene delivery etc. has the potential to improve the life expectancy of the patients affected by the disease. The main goal of this review is to provide general information on the current state of the art of synthesis of the inorganic porous particles based on silica, calcium carbonate and calcium phosphate. Special focus is dedicated to the loading capacity, controllable release of drugs under internal biological stimuli (e.g., pH, redox, enzymes) and external noninvasive stimuli (e.g., light, magnetic field, and ultrasound). Moreover, the diverse compounds to deliver with silica, calcium carbonate and calcium phosphate particles, ranging from the commercial drugs to genetic materials are also discussed.

A new and prospective resource for scandium: Evidence from the geochemistry of deep-sea sediment in the western North Pacific Ocean

Scandium is a critical element in state-of-the-art, green technologies. However, it is also extremely expensive due to its scarcity. Recently, some deep-sea sediments that contain significant amounts of rare-earth elements that are indispensable to modern industries have been discovered in the western North Pacific Ocean, and the concentration of Sc in these sediments is comparable with that of land-based mines. A simple leaching experiment has demonstrated that up to 70% of Sc in these sediments, contained within biogenic calcium phosphate particles, can be recovered in 5 min, using dilute acids. On the basis of available core samples, we propose that a promising target area to mine Sc is along a west–east transect between longitudes 153°30′ E and 154°00′ E, at an approximate latitude of 21°58′ N. Depending on the depth of the most enriched sediment layer, just 1 km2 of the sediment could potentially yield 3.3–6.6 times the current global annual consumption of Sc. Because these sediments contain an unusually high concentration, at commercial quantities, of industrially important, heavy rare-earth elements, apart from Sc, they could be a productive source for the long-term extraction of these critical metals.

Osteogenesis: The Effect of Addition of Calcium Phosphate Particles to Hydrogel-Based Composite Materials on Stiffness and Differentiation of Mesenchymal Stromal Cells toward Osteogenesis (Adv. Healthcare Mater. 18/2018)

Osteogenesis: The Effect of Addition of Calcium Phosphate Particles to Hydrogel-Based Composite Materials on Stiffness and Differentiation of Mesenchymal Stromal Cells toward Osteogenesis (Adv. Healthcare Mater. 18/2018)

Scanning electron microscopy for blood micro-crystals in aortic stenosis patients.

BackgroundMicro-crystals of calcium phosphate have been detected on the aortic valve of patients with aortic stenosis using scanning electron microscopy. It is not known whether crystalisation is specific to heart valve tissue or a general blood-derived process.MethodsTo this end we modified the method to determine whether calcium phosphate micro-crystals were present in the blood of patients with aortic stenosis. The method was first validated by adding synthetic calcium phosphate hydroxyapatite micro-crystals to healthy volunteer blood samples and determining the lower limit of detection. Then the method was used to examine the blood of 63 patients with echocardiographically confirmed aortic stenosis and 69 unaffected controls undergoing echocardiography for other reasons. Serum calcium and phosphate were measured and the calcium phosphate product compared in cases and controls.ResultsIn the validation study, synthetic hydroxyapatite micro-crystals were identified down to a lower concentration limit of 0.008mg/mL. In the experimental study no particles were identified in any patient, with or without aortic stenosis, even though serum calcium phosphate was higher in cases compared with controls 2.6mmol/L (2.58–2.77) versus 2.47mmol/L (2.36–2.57), p = 0.005 for the difference.ConclusionThe results of our study confirm a positive association between serum calcium phosphate and aortic stenosis, but indicate that the calcium phosphate particles found in valve tissue do not precipitate freely in the blood.

Calcium phosphate particles stimulate exosome secretion from phagocytes for the enhancement of drug delivery.

Exosomes are attractive potential carriers for drug delivery because of their natural function of transferring biomolecules among cells without eliciting immune responses. However, an obstacle to the application of exosomes for drug delivery is the difficulty in collecting sufficient numbers of these vesicles. In this study, we demonstrate treatment with calcium phosphate (CaP) particles could increase over two-fold the number of exosomes secreted from macrophage-like RAW264.7 cells and monocyte-like THP-1 cells. CaP particles were easily internalized into cells and dissolved in acidic late-endosomes or lysosomes, resulting in the rupture of their membranes followed by the release of Ca2+ into cytosol. Moreover, we found that exosomes secreted from cells treated with CaP particles are not contaminated by the Ca2+ released from CaP; the Ca2+ contents in exosomes secreted from CaP particle-treated cells were similar to that in exosomes from untreated control cells. This study highlights the potential for the efficient production of exosomes using CaP particles for drug delivery.

Lanthanum-containing bioparticles are associated with the influence of lanthanum on high phosphate mediated bone marrow stromal cells viability.

Lanthanum (La)-based binder appears effective in reducing serum inorganic phosphate (Pi) levels among chronic dialysis patients, yet concern remains about La accumulation in bone during long-term oral administration. In this study, the effect of lanthanum chloride (LaCl3) on bone marrow stromal cells (BMSCs) viability was investigated under high Pi situation. We found low concentration (10-9M) of LaCl3 increased BMSCs viability, while high concentration (10-5M) of LaCl3 not only inhibited BMSCs viability but also stimulated high Pi induced cell apoptosis. In addition, La-containing calcium phosphate (CaP) particles can be detected on cell surfaces and inside cells. We also found that inhibition of CaP formation by adding sodium pyrophosphate, a recognized inhibitor of hydroxyapatite formation, abrogated LaCl3 induced the BMSCs viability. For isolated La-containing CaP particles, the particle size increased and crystal phase switched with elevated concentration of LaCl3. These results demonstrated that La-containing CaP particles were associated with the process of LaCl3 mediated BMSCs viability and the physicochemical properties of these particles played an important role in modulating BMSCs function.

Wettability Study on Natural Rubber Surfaces for Applications as Biomembranes

This manuscript reports an experimental study on surfaces of natural rubber membranes modified by incorporation of calcium phosphate particles. In particular, we focused on the wettability, a subject for biological aspects. Five surfaces of natural rubber (NR) membranes (pure, polymer-bioceramic composite (NR-CaP), and three modified surfaces subjected to a simulated body fluid (NR-SBF)) were produced and characterized by confocal Raman-spectroscopy, AFM, SEM, and XPS, and the results were correlated with the wetting properties. Seven liquids (water, formamide, di-iodomethane, ethylene glycol, hexadecane, simulated body fluid, and human blood droplets) were used in different experimental sections. Static and dynamic contact angle measurements were conducted to obtain the solid-liquid tensions, work of adhesion, and depinning forces. The incorporation of CaP particles in the polymer decreases the roughness and increases the interfacial adhesion, and there was no dependence between the morphology and equilibrium contact line. The hydrophobic state of the NR surfaces is preserved. After exposure to a biological environment, the NR surfaces were chemically modified increasing blood wettability and decreasing the negative surface charges and the contact angle to values close to those associated with protein adsorption and cell adhesion, therefore opening possibilities for applications of these materials as biomembranes. On the other hand, the concepts applied, regarding different wettability aspects, should enable the evaluation of biomaterial surfaces and provide new insights allowing a better understanding of body fluid-material interfaces.

The Effect of Addition of Calcium Phosphate Particles to Hydrogel-Based Composite Materials on Stiffness and Differentiation of Mesenchymal Stromal Cells toward Osteogenesis.

The stiffness of a hydrogel has a significant role on the mechanical stability of a scaffold. However, the stiffness of pure hydrogels can be tuned only within a limited range. Herein, it is hypothesized that the range of hydrogel stiffness can be greatly increased by the addition of calcium phosphate particles and that such composites promote the osteogenic differentiation of human mesenchymal stem cells (hMSCs). Beta-tricalcium phosphate (β-TCP) particles are incorporated at concentrations of 0.5 and 5 mg mL-1 into various agarose and agarose-collagen blends. These composites are characterized with respect to stiffness, viscosity, degradation, cell morphology, viability, and osteogenesis. The osteogenic hMSCs in less stiff composites with 0.5 mg mL-1 β-TCP show the highest alkaline phosphatase expression compared to blends without β-TCP and stiffer composites with 5 mg mL-1 β-TCP. Quantitative polymerase chain reaction also shows higher expression of ALP, RUNX2, and collagen I by hMSCs in less stiff composites with 0.5 mg mL-1 β-TCP compared to blends without β-TCP and stiffer composite blends. It is concluded that by addition of calcium phosphate to specific hydrogels the stiffness can be tuned in a desired range and thus the osteogenic differentiation of embedded hMSCs can be better controlled and adjusted compared to pure hydrogels.

Mechanical characterization and ion release of bioactive dental composites containing calcium phosphate particles.

to verify the effect of the addition of dicalcium phosphate dihydrate (DCPD) particles functionalized with di- or triethylene glycol dimethacrylate (DEGDMA or TEGDMA) on the degree of conversion (DC), post-gel shrinkage (PS), mechanical properties, and ion release of experimental composites. Four composites were prepared containing a BisGMA/TEGDMA matrix and 60 vol% of fillers. The positive control contained only barium glass fillers, while in the other composites 15 vol% of the barium was replaced by DCPD. Besides the functionalized particles, non-functionalized DCPD was also tested. DC after 24 h (n = 3) was determined by FTIR spectroscopy. The strain gage method was used to obtain PS 5 min after photoactivation (n = 5). Flexural strength and modulus (n = 10) were calculated based on the biaxial flexural test results, after specimen storage for 24 h or 60 days in water. The same storage times were used for fracture toughness testing (FT, n = 10). Calcium and phosphate release up to 60 days was quantified by ICP-OES (n = 3). Data were analyzed by ANOVA/Tukey test (alpha: 5%). Composites containing functionalized DCPD presented higher DC than the control (p < 0.001). The material containing DEGDMA-functionalized particles showed higher PS than the other composites (p < 0.001). After 60 days, only the composite with DEGDMA-functionalized DCPD presented fracture strength similar to the control, while for flexural modulus only the composite with TEGDMA-functionalized particles was lower than the control (p < 0.001). FT of all composites containing DCPD was higher than the control after 60 days (p < 0.005). Calcium release was higher for the composite with non-functionalized DCPD at 15 days and no significant reductions were observed for composites with functionalized DCPD during the observation period (p < 0.001). For all the tested composites, phosphate release was higher at 15 days than in the subsequent periods, and no difference among them was recorded at 45 and 60 days (p < 0.001). DCPD functionalization affected all the studied variables. The composite with DEGDMA-functionalized particles was the only material with strength similar to the control after 60 days in water; however, it also presented the highest shrinkage. The presence of DCPD improved FT, regardless of functionalization. DCPD functionalization reduced ion release only during the first 15 days.

Fibulin-7, a heparin binding matricellular protein, promotes renal tubular calcification in mice

Ectopic calcification occurs during development of chronic kidney disease and has a negative impact on long-term prognosis. The precise molecular mechanism and prevention strategies, however, are not established. Fibulin-7 (Fbln7) is a matricellular protein structurally similar to elastogenic short fibulins, shown to bind dental mesenchymal cells and heparin. Here, we report that Fbln7 is highly expressed in renal tubular epithelium in the adult kidney and mediates renal calcification in mice. In vitro analysis revealed that Fbln7 bound heparin at the N-terminal coiled-coil domain. In Fbln7-expressing CHO-K1 cells, exogenous heparin increased the release of Fbln7 into conditioned media in a dose-dependent manner. This heparin-induced Fbln7 release was abrogated in CHO-745 cells lacking heparan sulfate proteoglycan or in CHO-K1 cells expressing the Fbln7 mutant lacking the N-terminal coiled-coil domain, suggesting that Fbln7 was tethered to pericellular matrix via this domain. Interestingly, Fbln7 knockout (Fbln7−/−) mice were protected from renal tubular calcification induced by high phosphate diet. Mechanistically, Fbln7 bound artificial calcium phosphate particles (aCPP) implicated in calcification and renal inflammation. Binding was decreased significantly in Fbln7−/− primary kidney cells relative to wild-type cells. Further, overexpression of Fbln7 increased binding to aCPP. Addition of heparin reduced binding between aCPP and wild-type cells to levels of Fbln7−/− cells. Taken together, our study suggests that Fbln7 is a local mediator of calcium deposition and that releasing Fbln7 from the cell surface by heparin/heparin derivatives or Fbln7 inhibitory antibodies may provide a novel strategy to prevent ectopic calcification in vivo.