Application of impedance spectroscopy to evaluate the effect of different setting accelerators on the developed microstructures of calcium phosphate cements

The main goal of the present study was to evaluate the effect of different setting accelerator agents on the developed microstructures of calcium phosphate cements (CPCs) by employing the impedance spectroscopy (IS) technique. Six compositions of CPCs were prepared from mixtures of commercial dicalcium phosphate anhydrous (DCPA) and synthesized tetracalcium phosphate (TTCP) as the solid phases. Two TTCP/DCPA molar ratios (1/1 and 1/2) and three liquid phases (aqueous solutions of Na(2)HPO(4), tartaric acid (TA) and oxalic acid (OA), 5% volume fraction) were employed. Initial (I) and final (F) setting times of the cement pastes were determined with Gillmore needles (ASTM standard C266-99). The hardened samples were characterized by X-ray powder diffraction (XRD), Fourier transformed infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and apparent density measurements. The IS technique was employed as a non-destructive tool to obtain information related to porosity, tortuosity and homogeneity of the cement microstructures. The formulation prepared from a TTCP/DCPA equimolar mixture and OA as the liquid phase presented the shortest I and F (12 and 20 min, respectively) in comparison to the other studied systems. XRD analyses revealed the formation of low-crystallinity hydroxyapatite (HA) (as the main phase) as well as the presence of little amounts of unreacted DCPA and TTCP after 24 h hardening in 100% relative humidity. This was related to the proposed mechanisms of dissolution of the reactants. The bands observed by FTIR allowed identifying the presence of calcium tartrate and calcium oxalate in the samples prepared from TA and OA, in addition to the characteristic bands of HA. High degree of entanglement of the formed crystals was observed by SEM in samples containing OA. SEM images were also correlated to the apparent densities of the hardened cements. Changes in porosity, tortuosity and microstructural homogeneity were determined in all samples, from IS results, when the TTCP/DCPA ratio was changed from 1/1 to 1/2. The cement formulated from an equimolar mixture of TTCP/DCPA and OA as the liquid phase presented setting times, degree of conversion to low-crystallinity HA and microstructural features suitable to be used as potential bone cement in clinical applications. The IS technique was shown to be a very sensitive and non-destructive tool to relate the paste composition to the developed microstructures. This approach could be very useful to develop calcium phosphate bone cements for specific clinical demands.

In vivo biological behavior of calcium phosphate cements with different Ca/P ratio

Bioceramics with different Ca/P ratio were prepared from a mechanical mixture of NaPO3, CaCO3, Ca(OH)2 and phosphate buffer solution and implanted in rats subcutaneous tissues. The cements were characterized by Thermo gravimetric analysis (TG-TDA), X-ray diffraction and 31P-NMR. The implant sites were excised after 1, 4 and 16 weeks, fixed, dehydrated, included in paraffin wax for serial cutting and examined under the light transmitted microscope. They were biocompatible and biodegradable when implanted in rat subcutaneous. None of the materials induced ectopic osteogenesis. According to the results, the studied materials seem to be able for manufacturing reabsorbable bone implants.

Growth of calcium phosphate coating on Ti-7.5Mo alloy after anodic oxidation

Titanium and its alloys are widely used as biomaterials due to their mechanical, chemical and biological properties. To enhance the biocompatibility of titanium alloys, various surface treatments have been proposed. In particular, the formation of titanium oxide nanotubes layers has been extensively examined. Among the various materials for implants, calcium phosphates and hydroxyapatite are widely used clinically. In this work, titanium nanotubes were fabricated on the surface of Ti-7.5Mo alloy by anodization. The samples were anodized for 20 V in an electrolyte containing glycerol in combination with ammonium fluoride (NH4F, 0.25%), and the anodization time was 24 h. After being anodized, specimens were heat treated at 450 °C and 600°C for 1 h to crystallize the amorphous TiO2 nanotubes and then treated with NaOH solution to make them bioactive, to induce growth of calcium phosphate in a simulated body fluid. Surface morphology and coating chemistry were obtained respectively using, field-emission scanning electron microscopy (FEG-SEM), AFM and X-ray diffraction (XRD). It was shown that the presence of titanium nanotubes induces the growth of a sodium titanate nanolayer. During the subsequent invitro immersion in a simulated body fluid, the sodium titanate nanolayer induced the nucleation and growth of nano-dimensioned calcium phosphate. It was possible to observe the formation of TiO2 nanotubes on the surface of Ti-7.5Mo. Calcium phosphate coating was greater in the samples with larger nanotube diameter. These findings represent a simple surface treatment for Ti-7.5Mo alloy that has high potential for biomedical applications. © (2013) Trans Tech Publications, Switzerland.

Evaluation of metallic brackets adhesion after the use of bleaching gels with and without amorphous calcium phosphate (ACP): in vitro study

OBJETIVO: avaliar, in vitro, a influência do clareamento dentário com gel contendo fosfato de cálcio amorfo (ACP) na resistência da união adesiva de braquetes metálicos. MÉTODOS: trinta e seis dentes incisivos bovinos foram seccionados no limite coronorradicular e tiveram suas coroas incluídas em cilindros de PVC. Os corpos de prova foram divididos em três grupos (n = 12), de acordo com a realização do tratamento clareador e tipo de gel utilizado, sendo: G1 (controle) - sem clareamento; G2 - clareamento com gel sem ACP (Whiteness Perfect, FGM); G3 - clareamento com gel contendo ACP (Nite White ACP, Discus Dental). Os grupos G2 e G3 foram submetidos a 14 ciclos de clareamento, seguidos de intervalo de espera de 15 dias para a fixação adesiva dos braquetes metálicos. O ensaio mecânico de cisalhamento foi realizado em máquina universal Kratos, com velocidade de 0,5mm/min. Após o teste mecânico, os corpos de prova foram avaliados quanto ao índice de remanescente adesivo (ARI). Os resultados foram submetidos à ANOVA, ao teste de Tukey e ao de Kruskall-Wallis (α = 5%). RESULTADOS: diferenças significativas foram observadas entre os grupos testados. O grupo controle G1 (11,1MPa) mostrou uma resistência ao cisalhamento estatisticamente superior aos grupos submetidos ao clareamento (G2 = 5,40MPa; G3 = 3,73MPa), os quais não diferiram entre si. Não se observou diferença significativa para o ARI entre os grupos estudados. CONCLUSÃO: o clareamento dentário reduz a resistência da união adesiva de braquetes metálicos, enquanto a presença de ACP no gel clareador não influencia os resultados encontrados.

Biochemical and microbiological characteristics of in situ biofilm formed on materials containing fluoride or amorphous calcium phosphate

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The Influence of the Heat Treatment Temperatures in Calcium Phosphate Synthesis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Analysis of anticaries potential of pit and fissures sealants containing amorphous calcium phosphate using synchrotron microtomography

The aim of this study was to analyze the anticaries potential of pit and fissure sealants containing amorphous calcium phosphate (ACP) by synchrotron microtomography. Bovine enamel blocks (4x4 mm; n=50) were selected through surface hardness (Knoop) analysis. Slabs were obtained through cross-sections taken 1 mm from the border of the enamel. Five indentations, spaced 100 mu m apart, were made 300 mu m from the border. Ten specimens were prepared for each tested material (Ultraseal XT plus TM, Aegis, Embrace, Vitremer and Experimental Sealant). The materials were randomly attached to the sectioned surfaces of the enamel blocks and fixed with sticky wax. The specimens were submitted to pH cycling. After that, the surface hardness (SH1) was determined, and the blocks were submitted to synchrotron microcomputed tomography analysis to calculate the mineral concentration (Delta g(HAp) cm(-3)) at different areas of the enamel. The comparison between the SH1 and DgHAp cm(-3) showed a correlation for all groups (r=0.840; p<0.001). The fluoride groups presented positive values of DgHAp cm(-3), indicating a mineral gain that was observed mainly in the outer part of the enamel. The ACP showed mineral loss in the outer enamel compared with fluoride groups, although it inhibited the demineralization in the deeper areas of enamel. The combination of two remineralizing agents (fluoride and ACP) was highly effective in preventing demineralization.

Characterization of calcium phosphate coating produced by biomimetic method

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Natural rubber latex coated with calcium phosphate for biomedical application

Natural rubber latex (NRL) is a flexible biomembrane that possesses angiogenic properties and has recently been used for guided bone regeneration, enhancing healing without fibrous tissue, allergies or rejection. Calcium phosphate (Ca/P) ceramics have chemical, biological, and mechanical properties similar to mineral phase of bone, and ability to bond to the host tissue, although it can disperse from where it is applied. Therefore, to create a composite that could enhance the properties of both materials, NRL biomembranes were coated with Ca/P. NRL biomembranes were soaked in 1.5 times concentrated SBF solution for seven days, avoiding the use of high temperatures. SEM showed that Ca/P has been coated in NRL biomembrane, XRD showed low crystallinity and FTIR showed that is the carbonated type B. Furthermore, hemolysis of erythrocytes, quantified spectrophotometrically using materials (Ca/P, NRL, and NRL + Ca/P) showed no hemolytic effects up to 0.125 mg/mL (compounds and mixtures), indicating no detectable disturbance of the red blood cell membranes. The results show that the biomimetic is an appropriate method to coat NRL with Ca/P without using high temperatures, aiming a new biomembrane to improve guided bone regeneration.

Amorphous calcium phosphate sealants: remineralization of carious lesions in the enamel can be achieved with use of pit-and-fissure sealants containing amorphous calcium phosphate

New pit-and-fissure sealants with the capacity to release calcium and phosphate because of the presence of ACP have been introduced into the dental marketplace. With the continuous introduction of new dental materials, it is important not only to research and confirm their properties, but also to propose modifications or associations that may contribute to their improvement.

Biogenic calcium phosphate transformation in soils over millennial time scales

Changes in bioavailability of phosphorus (P) during pedogenesis and ecosystem development have been shown for geogenic calcium phosphate (Ca-P). However, very little is known about long-term changes of biogenic Ca-P in soil. Long-term transformation characteristics of biogenic Ca-P were examined using anthropogenic soils along a chronosequence from centennial to millennial time scales. Phosphorus fractionation of Anthrosols resulted in overall consistency with the Walker and Syers model of geogenic Ca-P transformation during pedogenesis. The biogenic Ca-P (e.g., animal and fish bones) disappeared to 3% of total P within the first ca. 2,000 years of soil development. This change concurred with increases in P adsorbed on metal-oxides surfaces, organic P, and occluded P at different pedogenic time. Phosphorus K-edge X-ray absorption near-edge structure (XANES) spectroscopy revealed that the crystalline and therefore thermodynamically most stable biogenic Ca-P was transformed into more soluble forms of Ca-P over time. While crystalline hydroxyapatite (34% of total P) dominated Ca-P species after about 600-1,000 years, beta-tricalcium phosphate increased to 16% of total P after 900-1,100 years, after which both Ca-P species disappeared. Iron-associated P was observable concurrently with Ca-P disappearance. Soluble P and organic P determined by XANES maintained relatively constant (58-65%) across the time scale studied. Disappearance of crystalline biogenic Ca-P on a time scale of a few thousand years appears to be ten times faster than that of geogenic Ca-P.

In vitro and in vivo evaluation of the biocompatibility of a calcium phosphate/poly(lactic-co-glycolic acid) composite

This study assess the effects of bioceramic and poly(lactic-co-glycolic acid) composite (BCP/PLGA) on the viability of cultured macrophages and human dental pulp fibroblasts, and we sought to elucidate the temporal profile of the reaction of pulp capping with a composite of bioceramic of calcium phosphate and biodegradable polymer in the progression of delayed dentine bridge after (30 and 60 days) in vivo. Histological evaluation of inflammatory infiltrate and dentin bridge formation were performed after 30 and 60 days. There was similar progressive fibroblast growth in all groups and the macrophages showed viability. The in vivo study showed that of the three experimental groups: BCP/PLGA composite, BCP and calcium hydroxide (Ca(OH)(2)) dentin bridging was the most prevalent (90 %) in the BCP/PLGA composite after 30 days, mild to moderate inflammatory response was present throughout the pulp after 30 days. After 60 days was observed dentine bridging in 60 % and necrosis in 40 %, in both groups. The results indicate that understanding BCP/PLGA composite is biocompatible and by the best tissue response as compared to calcium hydroxide in direct pulp capping may be important in the mechanism of delayed dentine bridge after 30 and 60 days.

Cytocompatibility of Porous Biphasic Calcium Phosphate Granules With Human Mesenchymal Cells by a Multiparametric Assay

This work aims to evaluate the cytocompatibility of injectable and moldable restorative biomaterials based on granules of dense or porous biphasic calcium phosphates (BCPs) with human primary mesenchymal cells, in order to validate them as tools for stem cell-induced bone regeneration. Porous hydroxyapatite (HA) and HA/beta-tricalcium phosphate (beta-TCP) (60: 40) granules were obtained by the addition of wax spheres and pressing at 20 MPa, while dense materials were compacted by pressing at 100 MPa, followed by thermal treatment (1100 degrees C), grinding, and sieving. Extracts were prepared by 24-h incubation of granules on culture media, with subsequent exposition of human primary mesenchymal cells. Three different cell viability parameters were evaluated on the same samples. Scanning electron microscopy analysis of the granules revealed distinct dense and porous surfaces. After cell exposition to extracts, no significant differences on mitochondrial activity (2,3-bis(2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) or cell density (Crystal Violet Dye Elution) were observed among groups. However, Neutral Red assay revealed that dense materials extracts induced lower levels of total viable cells to porous HA/beta-TCP (P < 0.01). Calcium ion content was also significantly lower on the extracts of dense samples. Porogenic treatments on BCP composites do not affect cytocompatibility, as measured by three different parameters, indicating that these ceramics are well suited for further studies on future bioengineering applications.

The Influence of Osteoblast Differentiation Stage on Bone Formation in Autogenously Implanted Cell-Based Poly(Lactide-Co-Glycolide) and Calcium Phosphate Constructs

We tested the hypothesis that the osteoblast differentiation status of bone marrow stem cells (BMSCs) combined with a three-dimensional (3D) structure modulates bone formation when autogenously implanted. Rat BMSCs were aspirated, expanded, and seeded into a 3D composite of poly(lactide-co-glycolide) and calcium phosphate (PLGA/CaP) to produce a hybrid biomaterial. Calvarial defects were implanted with (1) scaffold without cells (SC/NC), (2) scaffold and BMSCs (SC + BMSC), (3) scaffold and osteoblasts differentiated for 7 days (SC + OB7), and (4) for 14 days (SC + OB14). After 4 weeks, there was more bone formation in groups combining scaffold and cells, SC + BMSC and SC + OB7. A nonsignificant higher amount of bone formation was observed on SC + OB14 compared with SC/NC. Additionally, more blood vessels were counted within all hybrid biomaterials, without differences among them, than into SC/NC. These findings provide evidences that the cell differentiation status affects in vivo bone formation in autogenously implanted cell-based constructs. Undifferentiated BMSCs or osteoblasts in early stage of differentiation combined with PLGA/CaP scaffold favored bone formation compared with plain scaffold and that one associated with more mature osteoblasts.

Nanostructuring by templated synthesis of nanowires and controlled crystallization of calcium phosphate on self-assembled monolayers

The idea was to obtain nanowires in a chemical laboratory under convenient and simple conditions by employing templates. Thus it was possible to produce nanochains by interlinking of gold colloids synthesized by the two-phase-method of M. Brust with by making use of vanadiumoxide nanotubes as template. The length of the resulting nanowires is varying between 1100 nm and 200 nm with a diameter of about 16 nm. Due to a flexible linker the obtained nanowires are not completely rigid. These unique structural features could make them interesting objects for structuring and assembling in the nanoscale range. Another way to produce gold nanowires was realized by a two-step surface metallization procedure, using type I collagen fibres as a template. Gold colloids were used to label the collagen fibres by direct electrostatic interaction, followed by growth steps to enhance the size of the adsorbed colloidal gold crystals, resulting in a complete metallization of the template surface. The length of the resulting gold nanowires reaches several micrometers, with a diameter ~ 100 to 120 nm. To gain a deeper insight into the process of biomineralization the cooperative effect of self-assembled monolayers as substrate and a soluble counterpart on the nucleation and crystal growth of calcium phosphate was studied by diffusion techniques with a pH switch as initiator. As soluble component Perlucin and Nacrein were used. Both are proteins originally extracted from marine organisms, the first one from the Abalone shell and the second one from oyster pearls. Both are supposed to facilitate the calcium carbonate formation in vivo. Studies with Perlucin revealed that this protein shows a clear cooperative effect at a very low concentration with a hydrophobic surface promoting the calcium phosphate precipitation resulting in a sponge like structure of hydroxyapatite. The Perlucin molecule is very flexible and is unfolded by adsorbing to the hydrophobic surface and uncovers its active side. Hydrophilic surfaces did not have a deeper impact. Studies with Nacrein as additive have shown that the protein stabilizes octacalcium phosphate at room temperature on carboxylic self-assembled monolayer and at 34 °C on all other employed surfaces by interaction with the mineral. On the hydroxyl-, alkyl-, and amin-terminated self-assembled monolayers at room temperature the octacalcium phosphate get transformed to hydroxyapatite. Main analytical techniques which are used in this work are transmission electron microscopy, high resolution scanning electron microscopy, surface plasmon resonance spectroscopy, atomic force microscopy, Raman micro-spectroscopy and quartz crystal microbalance.