Immunomodulatory activity of polycaprolactone nanoparticles with calcium phosphate salts against Leishmania infantum infection

Objective:To prepare and characterize polycaprolactone(PCL)nanoparticles loaded with sonicator fragmented(SLA)and freeze-thaw Leishmania antigens(FTLA)and to investigate the in vitro immunogenicity of antigen-encapsulated nanoparticles with calcium phosphate adjuvant.Methods:The water/oil/water binary emulsion solvent evaporation method was used to synthesize antigen-loaded PCL nanoparticles.Particles were characterized by scanning electron microscopy and zeta potential measurements.Their cytotoxicity in J774 macrophages in vitro was determined by MTT analysis.In addition,the amount of nitric oxide and the level of cytokines produced by macrophages were determined by Griess reaction and ELISA method,respectively.The protective effect of the developed formulations was evaluated by determining the infection index percentage in macrophages infected with Leishmania infantum.Results:Compared to the control group,SLA PCL and FTLA PCL nanoparticles with calcium phosphate adjuvant induced a 6-and 7-fold increase in nitric oxide,respectively.Additionally,the vaccine formulations promoted the production of IFN-γand IL-12.SLA PCL and FTLA PCL nanoparticles combined with calcium phosphate adjuvant caused an approximately 13-and 11-fold reduction in infection index,respectively,compared to the control group.Conclusions:The encapsulation of antigens obtained by both sonication and freeze-thawing into PCL nanoparticles and the formulations with calcium phosphate adjuvant show strong in vitro immune stimulating properties.Therefore,PCL-based antigen delivery systems and calcium phosphate adjuvant are recommended as a potential vaccine candidate against leishmaniasis.

Effect of Fluoride on the Ion-association of Calcium Phosphate and Crystallization of Hydroxyapatite

Using a titration setup to accurately control the reaction conditions and in situ monitor the reaction,we showed that fluoride exhibited negligible effects on the ion association process of calcium and phosphate and the formation of ACP nanospheres in a buffer solution with constant ionic strength.However,the stability of ACP increased with increasing fluoride concentration,which was ascribed to the inhibitory effect of fluoride on the aggregation of ACP nanospheres and the nucleation of nanocrystals on the surface of ACP nanospheres.Furthermore,fluoride could inhibit the lateral growth of HAP nanosheets and promote the formation of rod-like crystals.These results further improve our understanding of the crystallization pathway of HAP crystals and the regulatory effects of fluoride.

Corrosion resistance of cerium-doped zinc calcium phosphate chemical conversion coatings on AZ31 magnesium alloy

Zinc calcium phosphate （Zn-Ca-P） coating and cerium-doped zinc calcium phosphate （Zn-Ca-Ce-P） coating were prepared on AZ31 magnesium alloy. The chemical compositions, morphologies and corrosion resistance of coatings were investigated through energy-dispersive X-ray spectroscopy （EDS）, X-ray photoelectron spectroscopy （XPS）, X-ray diffraction （XRD）, electron probe micro-analysis （EPMA） and scanning electron microscopy （SEM） together with hydrogen volumetric and electrochemical tests. The results indicate that both coatings predominately contain crystalline hopeite （Zn3（PO4）2·4H2O）, Mg3（PO4）2 and Ca3（PO4）2, and traces of non-crystalline MgF2 and CaF2. The Zn-Ca-Ce-P coating is more compact than the Zn-Ca-P coating due to the formation of CePO4, and displays better corrosion resistance than the Zn-Ca-P coating. Both coatings protect the AZ31 Mg substrate only during an initial immersion period. The micro-galvanic corrosion between the coatings and their substrates leads to an increase of hydrogen evolution rate （HER） with extending the immersion time. The addition of Ce promotes the homogenous distribution of Ca and formation of hopeite. The Zn-Ca-Ce-P coating has the potential for the primer coating on magnesium alloys.

Novel rechargeable calcium phosphate nanoparticle-containing orthodontic cement

White spot lesions （WSLs）, due to enamel demineralization, occur frequently in orthodontic treatment. We recently developed a novel rechargeable dental composite containing nanoparticles of amorphous calcium phosphate （NACP） with long-term calcium （Ca） and phosphate （P） ion release and caries-inhibiting capability. The objectives of this study were to develop the first NACP- rechargeable orthodontic cement and investigate the effects of recharge duration and frequency on the efficacy of ion re-release. The rechargeable cement consisted of pyromellitic glycerol dimethacrylate （PMGDM） and ethoxylated bisphenol A dimethacrylate （EBPADMA）. NACP was mixed into the resin at 40% by mass. Specimens were tested for orthodontic bracket shear bond strength （SBS） to enamel, Ca and P ion initial release, recharge and re-release. The new orthodontic cement exhibited an SBS similar to commercial orthodontic cement without CaP release （P〉 0.1）. Specimens after one recharge treatment （e.g., 1 min immersion in recharge solution repeating three times in one day, referred to as ＂1 min 3 times＂） exhibited a substantial and continuous re-release of Ca and P ions for 14 days without further recharge. The ion re-release did not decrease with increasing the number of recharge/re-release cycles （P〉 0.1）. The ion re-release concentrations at 14 days versus various recharge treatments were as follows： 1 min 3 times〉3 min 2 times〉 1 min 2 times〉6 min 1 time〉3 min 1 time〉 1 min 1 time. In conclusion, although previous studies have shown that NACP nanocomposite remineralized tooth lesions and inhibited caries, the present study developed the first orthodontic cement with Ca and P ion recharge and long-term release capability. This NACP-rechargeable orthodontic cement is a promising therapy to inhibit enamel demineralization and WSLs around orthodontic brackets.

Microwave assisted synthesis & properties of nano HA-TCP biphasic calcium phosphate

Biphasic calcium phosphate （BCP） nanopowders were synthesized by using microwave and non-microwave irradiation assisted processes. The synthesized powders were pressed under a pressure of 90 MPa, and then were sintered at 1000-1200℃ for 1 h. The mechanical properties of the samples were investigated. The formed phases and microstructures were characterized by X-ray diffraction （XRD） and scanning electron microscopy （SEM）. The results showed that the synthesis time was shorter, along with a more homogeneous microstructure, when the microwave irradiation assisted method was applied. The compression strength and the Young＇s modulus of the samples synthesized with microwave irradiation were about 60 MPa and 3 GPa, but those of the samples synthesized without microwave irradiation were about 30 MPa and 2 GPa, respectively. XRD patterns of the microwave irradiation assisted and non-microwave irradiation assisted nanopowders showed the coexistence of hydroxyapatite （HA） and lricalcium phosphate （TCP） phases in the system.

Efficacy of two calcium phosphate pastes on the remineralization of artificial caries: a randomized controlled double-blind in situ study

To test the efficacy of two calcium phosphate pastes compared to that of fluoride toothpaste on remineralizing artificial caries in situ, this study had a double-blind crossover in situ design, involving three experimental phases of 14 days each, with an 8-day washout period between phases. Nine healthy subjects participated in the study. The subjects wore removable palatal appliances mounted with six human enamel slabs with artificial caries lesions, and in each of the experimental phases, used one of the following methods two times/day： group A, brushing with 1.0 g of Colgate Regular Flavor, followed by applying 0.25 g of Tooth Mousse Plus; group B, brushing with 0.25 g of Clinpro Tooth Crbme; and group C, brushing with 1.0 g of Colgate Regular Flavor. After 14 days, the enamel slabs （54 slabs/ group） were embedded in resin, sectioned and examined with a polarized-light microscope, and the lesion areas were quantified using Image-Pro Plus. All experimental groups showed a significant reduction in lesion area compared to the initial lesion area （paired t-test, P〈O.O01）. The mean reduction in lesion area of Groups A, B and C were （0.029__.0.010）, （0.030_＋0.009） and （0.027_＋0.009） mm2, respectively. There were no statistical differences between groups （KruskaI-Wallis test, P〉0.05）. All three groups remineralized the enamel slab lesions, indicating model sensitivity to fluoride. Given the differences in usage amounts and treated regimens, Clinpro Tooth Crbme provided similar benefits to the fluoride toothpaste; however, no additional benefit of Tooth Mousse Plus was observed when used in conjunction with the fluoride toothpaste.

Calcium Phosphate Biomaterials: An Update

Current calcium phosphate （ CaP ） biomaterials for bone repair, substitution, augmentation and regeneration include hydroxyapatite （ HA ） from synthetic or biologic origin, beta-tricaicium phosphate （ β- TCP ）, biphasic calcium phosphate （BCP）, and are available as granules, porous blocks, components of compashes （CaP/pollymer） cements, and as coatings on orthopedic and dental implants. Experimental calcium phosphate biomaterials include CO3^- and F-substituted apatites, Mg-and Zn-substituted β-TCP, calcium phosphate glasses, This paper is a brief review of the different types of CaP biomaterials and their properties such as bioactivity , osteoconductivity , osteoinductivity.

Three-dimensionally Perforated Calcium Phosphate Ceramics

Porous calcium phosphate ceramics were produced by compression molding using a special mold followed by sintering. The porous calcium phosphate ceramics have three-dimensional and penetrated open pores 380-400μm in diacneter spaced at intervals of 200μm. The layers of the linear penetration pores alternately lay perpendicular to pore direction. The porosity was 59%-65% . The Ca/ P molar ratios of the porous calcium phos phate ceramics range from 1.5 to 1.85. A binder cantaining methyl cellulose was most effective for preparing the powder compact among vinyl acetate, polyvinyl alcohol, starch, stearic acid, methyl cellulose and their mixtures . Stainless steel, polystyrene, nylon and bamboo were used as the long columnar dies for the penetrated open pores. When polystyrene, nylon and bamboo were used as the long columnar male dies, the dies were burned oat during the sintering process. Using stainless steel as the male dies with the removal of the dies before heat treatment resulted in a higher level of densification of the calcium phosphate ceramic.

The Effect of Premixed Schedule on the Crystal Formation of Calcium Phosphate Cement-chitosan Composite with Added Tetracycline

In this study, calcium phosphate cements （CPC） were prepared by mixing cement powders of tetracalcium phosphate （TTCP） with a cement liquid of phosphate acid saline solution. Tetracycline （TTC）-CPC, chitosan-CPC and chitosan-TTC-CPC were investigated with different premixed schedule. It was demonstrate that both TTC and chitosan worked on the phase transition and crystal characteristics. TTCP mixed with phosphate acid saline solution had similar features of Fourier transform-infrared spectrometry （FT-IR） no matter it was mixed with chitosan or TTC or both. TTC premixed with cement liquid or powder had significant different features of FT-IR and 876 cm-1 seemed to be a special peak for TTC when TTC was premixed with cement liquid. This was also supported by XRD analysis, which showed that TTC premixed with cement liquid improved phase transition of TTCP to OCP. Chitosan, as organic additive, regulates the regular crystal formation and inhibits the phase transition of TTCP to OCP, except when it is mingled with cement liquid premixed with TTC in field scanning electron microscope. It was concluded that the premixed schedule influences the crystal formation and phase transition, which may be associated with its biocompatibility and bioactivities in vivo.

Highly Efficient and Selective Removal of Pb（II） ions by Sulfur-Containing Calcium Phosphate Nanoparticles

A facile one-step co-precipitation method was demonstrated to fabricate amorphous sulfurcontaining calcium phosphate （SCP） nanoparticles, in which the sulfur group was in-situ introduced into calcium phosphate. The resulting SCP exhibited a noticeable enhanced performance for Pb（II） removal in comparison with hydroxyapatite （HAP）, being capable of easily reducing 20 ppm of Pb（II） to below the acceptable standard for drinking water within less than 10 min. Remarkably, the saturated removal capacities of Pb（II） on SCP were as high as 1720.57 mg/g calculated by the Langmuir isotherm model, exceeding largely that of the previously reported absorbents. Significantly, SCP displayed highly selective removal ability toward Pb（II） ions in the presence of the competing metal ions （Ni（II）, Co（II）, Zn（II）, and Cd（II））. Further investigations indicated that such ultra-high removal efficiency and preferable affinity of Pb（II） ions on SCP may be reasonably ascribed to the formation of rodlike hydroxypyromorphite crystals on the surface of SCP via dissolution-precipitation and ion exchange reactions, accompanied by the presence of lead sulfide precipitates. High removal efficiency, fast removal kinetics and excellent selectivity toward Pb（II） made the obtained SCP material an ideal candidate for Pb（II） ions decontamination in practical application.

Effects of quaternary ammonium chain length on the antibacterial and remineralizing effects of a calcium phosphate nanocomposite

Composites containing nanoparticles of amorphous calcium phosphate （NACP） remineralize tooth lesions and inhibit caries. A recent study synthesized quaternary ammonium methacrylates （QAMs） with chain lengths （CLs） of 3-18 and determined their effects on a bonding agent. This study aimed to incorporate these QAMs into NACP nanocomposites for the first time to simultaneously endow the material with antibacterial and remineralizing capabilities and to investigate the effects of the CL on the mechanical and biofilm properties. Five QAMs were synthesized： DMAPM （CL3）, DMAHM （CL6）, DMADDM （CL12）, DMAHDM （CL16）, and DMAODM （CL18）. Each QAM was incorporated into a composite containing 20% NACP and 50% glass fillers. A dental plaque microcosm biofilm model was used to evaluate the antibacterial activity. The flexural strength and elastic modulus of nanocomposites with QAMs matched those of a commercial control composite （n = 6; P 〉 0.1）. Increasing the CL from 3 to 16 greatly enhanced the antibacterial activity of the NACP nanocomposite （P 〈 0.05）; further increasing the CL to 18 decreased the antibacterial potency. The NACP nanocomposite with a CL of 16 exhibited biofilm metabolic activity and acid production that were 10-fold lesser than those of the control composite. The NACP nanocomposite with a CL of 16 produced 2-log decreases in the colony-forming units （CFU） of total microorganisms, total streptococci, and mutans streptococci. In conclusion, QAMs with CLs of 3-18 were synthesized and incorporated into an NACP nanocomposite for the first time to simultaneously endow the material with antibacterial and remineralization capabilities. Increasing the C/reduced the metabolic activity and acid production of biofilms and caused a 2-log decrease in CFU without compromising the mechanical properties. Nanocomposites exhibiting strong anti-biofilm activity, remineralization effects, and mechanical properties are promising materials for tooth restorations that inhibit caries.

Effect of humic substances on the precipitation of calcium phosphate

For phosphorus （P） recovery from wastewater, the effect of humic substances （HS） on the precipitation of calcium phosphate was studied. Batch experiments of calcium phosphate precipitation were undertaken with synthetic water that contained 20 mg/L phosphate （as P） and 20 mg/L HS （as dissolved organic carbon, DOC） at a constant pH value in the range of 8.0-10.0. The concentration variations of phosphate, calcium （Ca） and HS were measured in the precipitation process; the crystalline state and compositions of the precipitates were analysed by powder X-ray diffraction （XRD） and chemical methods, respectively. It showed that at solution pH 8.0, the precipitation rate and removal efficiency of phosphate were greatly reduced by HS, but at solution pH ≥9.0, the effect of HS was very small. The Ca consumption for the precipitation of phosphate increased when HS was added; HS was also removed from solution with the precipitation of calcium phosphate. At solution pH 8.0 and HS concentrations ≤3.5 mg/L, and at pH ≥ 9.0 and HS concentrations ≤ 10 mg/L, the final precipitates were proved to be hydroxyapatite （HAP） by XRD. The increases of solution pH value and initial Ca/P ratio helped reduce the influence of HS on the precipitation of phosphate.

One-year water-ageing of calcium phosphate composite containing nano-silver and quaternary ammonium to inhibit biofilms

Dental composites are commonly used restorative materials; however, secondary caries due to biofilm acids remains a major problem. The objectives of this study were （1） to develop a composite containing quaternary ammonium dimethacrylate （QADM）, nanoparticles of silver （NAg）, and nanoparticles of amorphous calcium phosphate （NACP）, and （2） to conduct the first investigation of the mechanical properties, biofilm response and acid production vs water-ageing time from 1 day to 12 months. A 4 x 5 design was utilized, with four composites （NACP-QADM composite, NACP-NAg composite, NACP-QADM-NAg composite, and a commercial control composite）, and five water-ageing time periods （1 day, and 3, 6, 9, and 12 months）. After each water- ageing period, the mechanical properties of the resins were measured in a three-point flexure, and antibacterial properties were tested via a dental plaque biofilm model using human saliva as an inoculum. After 12 months of water-ageing, NACP-QADM- NAg had a flexural strength and elastic modulus matching those of the commercial control （P〉 0.1）. Incorporation of QADM or NAg into the NACP composite greatly reduced biofilm viability, metabolic activity and acid production. A composite containing both QADM and NAg possessed a stronger antibacterial capability than one with QADM or NAg alone （P〈0.05）. The anti-biofilm activity was maintained after 12 months of water-ageing and showed no significant decrease with increasing time （P〉0.1）. In conclusion, the NACP-QADM-NAg composite decreased biofilm viability and lactic acid production, while matching the load- bearing capability of a commercial composite. There was no decrease in its antibacterial properties after 1 year of water-ageing. The durable antibacterial and mechanical properties indicate that NACP-QADM-NAg composites may be useful in dental restorations to combat caries.

Calcium phosphate cements for bone engineering and their biological properties

Calcium phosphate cements （CPCs） are frequently used to repair bone defects. Since their discovery in the 1980s, extensive research has been conducted to improve their properties, and emerging evidence supports their increased application in bone tissue engineering. Much effort has been made to enhance the biological performance of CPCs, including their biocompatibility, osteoconductivity, osteoinductivity, biodegradability, bioactivity, and interactions with cells. This review article focuses on the major recent developments in CPCs, including 3D printing, injectability, stem cell delivery, growth factor and drug delivery, and pre- vascularization of CPC scaffolds via co-culture and tri-culture techniques to enhance angiogenesis and osteogenesis.

Preparation of calcium phosphate coating on pure titanium substrate by electrodeposition method

The influences of pH value, electrolyte temperature and loading time on depositing calcium phosphate coating on pure titanium substrate by electrodeposition process were investigated. The process was carried out with an electrochemical work-station supplying a direct current power at potential of -0.8V （vs SCE）. The electrolyte consists of 7 mmol·L-1 CaCl2·2H2O, 3 mmol·L-1 Ca（H2PO4）2·H2O and 2.5% H2O2. NaOH and HCl solutions were used to adjust pH value. The deposited samples were characterized by X-ray diffraction and scanning electron microscope. The comparison of the deposits obtained at lower and higher pH values demonstrates that the crystallization process at the interface is favoured by high pH value. With temperature increasing, the deposited hydroxyapatite is occasionally of plate-like shape, and the width and the length of the deposited calcium phosphates at 65 ℃ are larger than those at 55 ℃. Therefore, it is confirmed that the morphology and microstructure of electrochemically deposited calcium phosphates can be regulated. Additionally, the coating formed in electrolyte with H2O2 additive is homogeneous and the evolution of H2 bubble can be eliminated.

Surface characterization and corrosion behavior of calcium phosphate(Ca-P)base composite layer on Mg and its alloys using plasma electrolytic oxidation(PEO):A review

Magnesium has been known as an appropriate biological material on account of its good biocompatibility and biodegradability properties in addition to advantageous mechanical properties.Mg and its alloys are of poor corrosion resistance.Its high corrosion rate leads to its quick decomposition in the corrosive ambiance and as a result weakening its mechanical properties and before it is repaired,it will vanish.The corrosion and degradation rate must be controlled in the body to advance the usage of Mg and its alloys as implants.Different techniques have been utilized to boost biological properties.Plasma electrolytic oxidation(PEO)can provide porous and biocompatible coatings for implants among various techniques.Biodegradable implants are generally supposed to show enough corrosion resistance and mechanical integrity in the body environment.Much research has been carried out in order to produce PEO coatings containing calcium phosphate compounds.Calcium phosphates are really similar to bone mineral composition and present great biocompatibility.The present study deals with the usage of calcium phosphates as biocompatible coatings applied on Mg and its alloys to study the properties and control the corrosion rate.

Bone tissue engineering via nanostructured calcium phosphate biomaterials and stem cells

Tissue engineering is promising to meet the increasing need for bone regeneration. Nanostructured calcium phosphate （CAP） biomaterials/scaffolds are of special interest as they share chemical/crystallographic similarities to inorganic components of bone. Three applications of nano-CaP are discussed in this review： nanostructured calcium phosphate cement （CPC）; nano-CaP composites; and nano-CaP coatings. The interactions between stem cells and nano-CaP are highlighted, including cell attachment, orientation/ morphology, differentiation and in vivo bone regeneration. Several trends can be seen： （i） nano-CaP biomaterials support stem cell attachment/proliferation and induce osteogenic differentiation, in some cases even without osteogenic supplements; （ii） the influence of nano-CaP surface patterns on cell alignment is not prominent due to non-uniform distribution of nano-crystals; （iii） nano-CaP can achieve better bone regeneration than conventional CaP biomaterials; （iv） combining stem cells with nano-CaP accelerates bone regeneration, the effect of which can be further enhanced by growth factors; and （v） cell microencapsulation in nano-CaP scaffolds is promising for bone tissue engineering. These understandings would help researchers to further uncover the underlying mechanisms and interactions in nano-CaP stem cell constructs in vitro and in vivo, tailor nano-CaP composite construct design and stem cell type selection to enhance cell function and bone regeneration, and translate laboratory findings to clinical treatments.

Porous Calcium Phosphate Ceramic Scaffolds for Tissue Engineering

This study examined the biological response of two porous calcium phosphate ceramics, hydroxyapntite （ HA ） and hydroxyapaptite/β-tricalcium phosphate （ HA/β- TCP ） scaffolds. Three different cell types , a human osteoblastic cell line （ HOS ） , primary human osteoblasts （HOB） and human mesenehymal stem cells （MSCs）, were used to examine biocompatibility and osteoinductive capacity. The experimental results showed both materials were highly biocompatible and proliferation was significantly greater on pure HA （ P 〈 0.01 ）, with a peak in proliferation at day 7. Protein levels were significantly higher （ P 〈 0.05） than the control Thermanox（ TMX （ tm） ） for both test materials. Osteoinduction of MSCs was observed on both test materials, with cells seeded on HA/ β-TCP showing greater alkaline phosphatase activity compared to HA alone, indicating an enhancement in osteoinductive property. Both materials show good potential for use as tissue engineered scaffolds.

Calcium phosphate nanoparticles show an effective activation of the innate immune response in vitro and in vivo after functionalization with flagellin

For subunit vaccines,adjuvants play a key role in shaping the magnitude,persistence and form of targeted antigen-specific immune response.Flagellin is a potent immune activator by bridging innate inflammatory responses and adaptive immunity and an adjuvant candidate for clinical application.Calcium phosphate nanoparticles are efficient carriers for different biomolecules like DNA,RNA,peptides and proteins.Flagellin-functionalized calcium phosphate nanoparticles were prepared and their immunostimulatory effect on the innate immune system,i.e.the cytokine production,was studied.They induced the production of the proinflammatory cytokines IL-8 （Caco-2 cells） and IL-1β（bone marrow-derived macrophages; BMDM） in vitro and IL-6 in vivo after intraperitoneal injection in mice.The immunostimulation was more pronounced than with free flagellin.

Preparation of Laminin-apatite-polymer Composites Using Metastable Calcium Phosphate Solutions

A synthetic polymer with a laminin-apatite composite layer on its surface would be useful as a percutaneous device. The preparation of such a composite was attempted in the present study using poly（ ethylene terephthalate ） （PET） and polyethylene （PE） as the synthetic polymer. PET and PE plates and those pretreated with an oxygen plasma were alternately dipped in calcium and phosphate ion solutions, and then immersed in a metastable calcium phosphate solution supplemented with laminin （ LCP solution ）. The PET and PE plates pretreated with an oxygen plasma formed a uniform and continuous layer of a laminin-apatite composite on their surfaces. In contrast, the PET and PE plates that had not been pretreated with an oxygen plasma did not form a continuous layer of a laminin-apatite composite on their surfaces. The hydrophilic functional groups on the PET and PE surfaces introduced by the plasma treatment were responsible for the successful laminin-apatite coruposite coating.