Spherical Calcium Phosphate Research Articles

Branched Polymeric Prenucleation Assemblies Initiate Calcium Phosphate Precipitation.

The formation of crystalline calcium phosphate (CaP) has recently gained ample attention as it does not follow the classic nucleation-and-growth mechanism of solid formation. Instead, the precipitation mechanisms can involve numerous intermediates, including soluble prenucleation species. However, structural features, stability, and transformation of such solution-state precursors remain largely undisclosed. Herein, we report a detailed and comprehensive characterization of the sequential events involved in calcium phosphate crystallization starting from the very early prenucleation stage. We integrated an extensive set of time-resolved methods, including NMR, turbidimetry, SAXS, cryo-TEM, and calcium-potentiometry to show that CaP nucleation is initiated by the transformation of "branched" polymeric prenucleation assemblies into amorphous calcium phosphate spheres. Such a mineralization process starts with the spontaneous formation of so-called nanometric prenucleation clusters (PNCs) that later assemble into those branched polymeric assemblies without calcium ion uptake from the solution. Importantly, the branched macromolecular species are invisible to many techniques (NMR, turbidity, calcium-potentiometry) but can readily be evidenced by time-resolved SAXS. We find that these polymeric assemblies constitute the origin of amorphous calcium phosphate (ACP) precipitation through an unexpected process: spontaneous dissolution is followed by local densification of 100-200 nm wide domains leading to ACP spheres of similar size. Finally, we demonstrate that the timing of the successive events involved in the CaP mineralization pathway can be kinetically controlled by the Ca2+/Pi molar ratio, such that the lifetime of the soluble transient species can be increased up to hours when decreasing it.

Pulmonary alveolar microlithiasis: A case report from Morocco with review of literature

Background: Pulmonary alveolar microlithiasis (PAM) is a rare infiltrative chronic lung disease characterized by deposition of spherical calcium phosphate microliths called calcospherites within the alveoli with predominance in lower and mid zones. Etiology and pathogenesis is not fully understood. Case presentation: We report a case of 58 year old female patient presenting with progressive shortness of breath and chronic bronchitis over a period of 4 years. Physical examination revealed she had signs of chronic respiratory failure. Chest radiograph showed dense micronodular opacities giving classical sandstorm appearance. High resolution computed tomography showed diffuse microcalcifications, bilateral ground glass attenuation and septal thickening with calcification along the interlobar septa and subpleural regions. Conclusion: According to our research this is the 15th case of PAM that has been declared in Morocco. Given the rarity of this entity, the purpose of this study was to increase knowledge of this disease among pulmonologists, in order to provide timely diagnosis.

Calcium phosphate cements comprising spherical porous calcium phosphate granules: synthesis, structure, and properties

ABSTRACT Calcium phosphate cements (CPCs) are used as artificial bone materials. The bone regeneration ability of CPCs can be improved by controlling their composition, porosity, and pore size. This study aims to design novel CPCs with high bone regeneration ability by controlling their microstructure. CPCs with macropores and micropores were prepared by incorporating spherical porous calcium phosphate granules composed of rod-shaped, calcium-deficient hydroxyapatite (CDHA) or plate-shaped octacalcium phosphate (OCP) particles. The granules were mixed with a binder (cement powder) composed primarily of α-tricalcium phosphate. The structure, morphology, compressive strength, porosity, specific surface area, pore size distribution, dissolution characteristics, and effects on cell viabilities were studied for the synthesized samples. The CPCs composed of porous granules had high porosity (~80%) and both macropores and micropores, which are expected to contribute to bone regeneration. The CPCs composed of porous granules showed a smaller specific surface area but a larger dissolution rate than the granule-free samples. The CPC composed of OCP granules showed a higher dissolution rate than the CPCs containing CDHA granules. In the cell culture experiments, the preosteoblasts proliferated on the CPCs, indicating that these CPCs could function as scaffolds for bone regeneration.

Spheroid co-culture of BMSCs with osteocytes yields ring-shaped bone-like tissue that enhances alveolar bone regeneration

Oral and maxillofacial bone defects severely impair appearance and function, and bioactive materials are urgently needed for bone regeneration. Here, we spheroid co-cultured green fluorescent protein (GFP)-labeled bone marrow stromal cells (BMSCs) and osteocyte-like MLO-Y4 cells in different ratios (3:1, 2:1, 1:1, 1:2, 1:3) or as monoculture. Bone-like tissue was formed in the 3:1, 2:1, and 1:1 co-cultures and MLO-Y4 monoculture. We found a continuous dense calcium phosphate structure and spherical calcium phosphate similar to mouse femur with the 3:1, 2:1, and 1:1 co-cultures, along with GFP-positive osteocyte-like cells encircled by an osteoid-like matrix similar to cortical bone. Flake-like calcium phosphate, which is more mature than spherical calcium phosphate, was found with the 3:1 and 2:1 co-cultures. Phosphorus and calcium signals were highest with 3:1 co-culture, and this bone-like tissue was ring-shaped. In a murine tooth extraction model, implantation of the ring-shaped bone-like tissue yielded more bone mass, osteoid and mineralized bone, and collagen versus no implantation. This tissue fabricated by spheroid co-culturing BMSCs with osteocytes yields an internal structure and mineral composition similar to mouse femur and could promote bone formation and maturation, accelerating regeneration. These findings open the way to new strategies in bone tissue engineering.

Сравнение токсического действия сферических и игольчатых кальций-фосфатных бионов на поврежденную интиму брюшной аорты крыс

Цель исследования - оценка токсического действия сферических кальций-фосфатных бионов и игольчатых кальций-фосфатных бионов на предварительно поврежденную интиму аорты крыс. Методика. Токсическое действие сферических кальций-фосфатных бионов и игольчатых кальций-фосфатных бионов на поврежденную интиму брюшной аорты крыс линии Wistar (n = 10 на группу) оценивали путем их однократного внутривенного введения после баллонной ангиопластики с эксплантацией поврежденного участка аорты через 5 нед. Биоптаты анализировали: 1) классическими гистологическими методами (окрашивание гематоксилин-эозином, ализариновым красным, по Вейгерту-ван Гизону и по Расселлу-Мовату); 2) иммунофлюоресцентным окрашиванием криосрезов (сочетанное окрашивание на CD31 и CD34, на CD31 и α-гладкомышечный актин (α-ГМА), на виментин и α-ГМА, на коллаген IV типа и α-ГМА). Для оценки влияния системного воспаления на КФБ-индуцированную эндотелиотоксичность определяли содержание моноцитарного хемоаттрактантного белка (МСР-1/CCL2) и церулоплазмина в сыворотке крови прооперированных крыс посредством иммуноферментного анализа. Результаты. Сферические кальций-фосфатные бионы и игольчатые кальций-фосфатные бионы вызывали выраженную гипертрофию интимы брюшной аорты в 90% (9 из 10 крыс) и 80% случаев (8 из 10 крыс) соответственно, в то время как частота гипертрофии в группе контрольных крыс составила лишь 10% (1 из 10 крыс). Неоинтима при экспозиции интимы брюшной аорты обоим типам бионов характеризовалась переходом фенотипа клеток мезенхимального ряда с контрактильного (α-ГМА-положительные и виментин-отрицательные гладкомышечные клетки) и неактивного (α-ГМА-отрицательные и виментин-положительные фибробласты) на активный синтетический (α-ГМА- и виментин-положительные клетки), что приводило к формированию значительных количеств экстрацеллюлярного матрикса. Внутривенное введение сферических кальций-фосфатных бионов и игольчатых кальций-фосфатных бионов не приводило к изменению уровней МСР-1/CCL2 и церулоплазмина в сыворотке крови, что свидетельствовало об отсутствии их возможного влияния на развитие системного воспалительного ответа. Заключение. Внутривенное введение кальций-фосфатных бионов после повреждения интимы брюшной аорты крыс путем баллонной ангиопластики вызывает развитие гипертрофии интимы, частота и выраженность которой не зависит от формы кальций-фосфатных бионов и которая характеризуется переходом фенотипа клеток мезенхимального ряда из контрактильного/неактивного на активный синтетический. При этом эндотелиотоксическое действие кальций-фосфатных бионов обусловлено их непосредственным воздействием на эндотелий, а не развитием системного воспаления. Purpose. To compare toxicity of spherical calcium phosphate bions (SCPB) and needle-shaped calcium phosphate bions (NCPB) to injured intima of rat aortas. Methods. Toxicity of SCPB and NCPB to injured abdominal aortas of Wistar rats (n = 10 per group) was evaluated using intravenous administration of the bions after balloon angioplasty. Rats were sacrificed five weeks postoperation, and an injured aortic segment was excised. Tissue preparations were stained with hematoxylin and eosin, alizarin red S, Weigert-van Gieson, and Movat’s pentachrome stains. Selected tissue samples were then examined using combined immunofluorescence staining (CD31/CD34, CD31/α-smooth muscle actin (α-SMA), α-SMA/vimentin, and α-SMA/collagen IV). Possible influence of systemic inflammation on CPB-induced endothelial toxicity was assessed by measuring monocyte chemoattractant protein-1 and ceruloplasmin in rat serum using the enzyme-linked immunosorbent assay. Results. Intravenous administration of SCPB or NCPB provoked intimal hyperplasia in 90% (9 of 10) and 80% (8 of 10) of rats vs. 10% (1 of 10) in the control group. The neointima was characterized by a phenotypic switch of mesenchymal cells, i.e. transition of a contractile (α-SMA-positive, vimentin-negative vascular smooth muscle cells) and quiescent (α-SMA-negative vimentin-positive fibroblasts) to an active synthetic phenotype (double-positive cells), which resulted in deposition of the extracellular matrix. Neither SCPB nor NCPB changed serum levels of pro-inflammatory molecules, МСР-1/CCL2, and ceruloplasmin. Conclusions. Intravenous administration of CPB upon balloon-induced vascular injury caused intimal hyperplasia regardless of the CPB shape. Hyperplasia foci were characterized by a switch of mesenchymal cells from a contractile/quiescent to an active synthetic phenotype. Endothelial toxicity of CPBs was defined by their direct cytotoxic action rather than induction of systemic inflammation.

MORPHOLOGICAL AND CHEMICAL PROPERTIES OF SPHERICAL AND NEEDLE CALCIUM PHOSPHATE BIONS

Aim. To compare morphological properties, mineral, and organic profile of spherical calcium phosphate bions (SCPB) and needle calcium phosphate bions (NCPB) for the assessment of the CPB-specific endothelial toxicity in models of mild or severe hypercalcemia/hyperphosphatemia in the further studies.Methods. Both SCPB and NCPB were artificially synthesized employing blood-mimetic medium either moderately or significantly supersaturated of calcium and phosphorus salts. Size and shape of SCPB and NCPB were investigated by scanning and transmission electron microscopy and atomic force microscopy. Elemental analysis was performed utilizing energy-dispersive X-ray spectroscopy, atomic emission spectroscopy, and CHNSO analysis, functional groups were examined using Fourier-transform infrared spectroscopy and Raman spectroscopy while chemical formula was identified by X-ray powder diffraction analysis. Protein profile of SCPB and NCPB was screened employing sodium dodecyl sulfate polyacrylamide gel electrophoresis following silver staining.Results. SCPB were visualized as crystalline spherical spongeous particles of 80-200 nm diameter and mean diameter of around 120 nm while NCPB represented needle crystals of a similar diameter. Both SCPB and NCPB had similar crystallinity, surface charge and tended to form clusters of several particles. Furthermore, both SCPB and NCPB were composed of carbon, oxygen, hydrogen, nitrogen, calcium, and phosphorus, contained phosphate (PO4 3-), carbonate (CO3 2-), and hydroxyl (OH- ) functional groups, and consisted of hydroxyapatite (Ca10(PO4 )6 (OH)2 ) and carbonate-hydroxyapatite (Ca10(PO4)3 (CO3)3 (OH)2 ). In addition, protein profile of SCPB and NCPB was similar and notable for the abundant albumin and fetuin A levels.Conclusion. Having similar size, surface charge, extent of crystallinity, and chemical composition, SCPB and NCPB possess a different shape.

Enhanced bleaching effect of bovine tooth by hydrogen peroxide via calcium phosphate spheres

In the study, calcium phosphate spheres (CPS) were prepared using phosphate buffer solutions by hydrothermal method and were used to manufacture a lab-made bleaching paste. The obtained paste was used to whiten bovine teeth in order to investigate whitening effect with CPS in the paste. The results showed that the bleaching paste made by CPS does not affect bleaching effect of hydrogen peroxide (HP). On the contrary, it can prolong the bleaching effect compared the pure HP.

The concentration of calcium phosphate spheres on micro hardness of dentin

Calcium phosphate spheres was synthesized by hydrothermal method and used to make anti-sensitivity Toothpaste. The toothpaste was used to apply on dentin surface daily. The calcium phosphate facilitated more remineralization effect as the applied time was 7 days compared to fewer days. The new remineralized layer leads to approximately 22% decrease of micro hardness of dentin.

Controllable synthesis of poly(acrylic acid)-stabilized nano-hydroxyapatite suspension by an ultrasound-assisted precipitation method

An improved precipitation method was developed to prepare stable nano-hydroxyapatite (HAP) suspension with the aid of steric effect of poly(acrylic acid) (PAA) and acoustic cavitation effect. The effects of reaction temperature and PAA concentration on the formation of nano-HAP suspension were studied. The rising temperature led to the transformation of spherical amorphous calcium phosphate (ACP) to needle-like nano-HAP crystals with the increase in size. The increasing PAA concentration mightily hindered the crystallization of HAP instead of decreasing the size. PAA provided steric effect to stabilize nano-HAP in solution due to the electrostatic interaction between –COO- of PAA and Ca2+ on the surface of HAP. The obtained PAA-stabilized nano-HAP suspension showed good stability in different media (water, PBS, RPMI 1640) and noncytotoxicity. This study provides a potential technology for the controllable synthesis of stable nano-HAP suspension.

Fabrication and Properties of Ca-P Bioceramic Spherical Granules with Interconnected Porous Structure

Spherical calcium phosphate (Ca-P) granules show advantages in filling bone cavity defects. In this study, gelatinizing technology combined with microsphere-sintering and gas-foaming methods was applied to fabricate porous spherical Ca-P bioceramic granules. The obtained three kinds of Ca-P granules (i.e., hydroxyapatite, HA-G; biphasic calcium phosphate, BCP-G; and tricalcium phosphate, TCP-G) exhibited uniformly spherical shapes and interconnected pore structures with controlled macropores (about 600 μm), and abundant minor- (10-100 μm) to microsized (<10 μm) pores. The obtained Ca-P granules contained only the HA and β-TCP phases, but their phase ratios (HA/β-TCP) had some changes in the sintering process. All of the Ca-P granules were favorable for serum protein adsorption and bonelike apatite formation, and TCP-G was superior to the other two. Cell-culturing results showed that these Ca-P granules could promote cell adhesion, proliferation, and expression of a series of osteogenic genes. These findings demonstrated that these Ca-P granules had good bioactivity and pro-osteogenic ability, especially for BCP-G and TCP-G. Therefore, the porous spherical Ca-P bioceramic granules fabricated by the novel method show great potential to serve as good candidates for bone defect filling materials.

A novel method to synthesize low-cost phosphate-based particles from natural water

Calcium phosphate (CaP) and magnesium phosphate (MgP) are widely used as biomaterials. A novel method for low-cost manufacturing of calcium phosphate spheres and magnesium phosphate micro particles was studied. The method focuses on novel strategies of utilizing seawater and lake water. In the natural water, the molar ratios of Mg/Ca are constant. The morphologies of the particles are determined by ratio of Ca/P. This simple method provides a prototype to synthesize low-cost inorganic spheres with natural water, which facilitate large-scale production.

Template-free synthesis of phosphate-based spheres via modified supersaturated phosphate buffer solutions.

Modified supersaturated phosphate buffer solutions were used to synthesize phosphate-based spheres, including calcium phosphate (CaP), strontium phosphate (SrP) and barium phosphate (BaP). A series of ions concentrations in the modified phosphate buffer solutions were investigated in order to study their effects in precipitates morphologies. During synthesis, it was found that magnesium was the key factor in sphere formation. The morphologies of calcium phosphate, strontium phosphate and barium phosphate precipitates varied as the concentration of magnesium ions varied. When sufficient magnesium was provided, the precipitates appeared spherical, and the diameter was in range of 0.5-2 μm. The morphologies, compositions and structure of spheres were characterized by x-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and N2 adsorption analysis. Moreover, the application of magnesium substituted calcium phosphate spheres in dentin tubules occlusion was investigated.

Selective preparation and characterization of nano-hydroxyapatite/collagen coatings with three-dimensional network structure

Nano hydroxyapatite/collagen (HA/COL) coatings were prepared on the surface of carbon/carbon (C/C) composites by a two-step process, i.e. electrochemically assisted co-deposition of calcium phosphate/collagen (CaP/COL) coatings and subsequent soaking CaP/COL coated C/C composites in Ca(OH)2 aqueous solution. Both CaP/COL and HA/COL coatings on the C/C composites exhibit three-dimensional network structure consisting of collagen fibers and uniform CaP covered on the surface of collagen fibers. CaP in the composite coatings was deposited into different forms depending on the concentration, Ca/P molar ratio and pH value of initial electrolyte. The concentration and pH value of initial electrolyte have little impact on the shape of CaP though they significantly influence the size of CaP. Higher Ca/P molar ratio of initial electrolyte leads to spherical amorphous calcium phosphate (ACP)/COL coatings. Plate-like octacalcium phosphate (OCP)/COL coatings were prone to form with low Ca/P molar ratios in the electrolyte. After Ca(OH)2-treatment as the second processing step, OCP/COL coatings on C/C were converted to HA/COL coatings. Their structure and compositions are similar to those of human bone tissue. SBF immersion tests indicate that HA/COL coated C/C composites have better in vitro bioactivity. In vitro cellular biocompatibility tests indicate OCP/COL and HA/COL coated C/C composites have almost the same biocompatibility.

Aortic calcified particles modulate valvular endothelial and interstitial cells.

Normal and calcified human valve cusps, coronary arteries, and aortae harbor spherical calcium phosphate microparticles of identical composition and crystallinity, and their role remains unknown. The objective was to examine the direct effects of isolated calcified particles on human valvular cells. Calcified particles were isolated from healthy and diseased aortae, characterized, quantitated, and applied to valvular endothelial cells (VECs) and interstitial cells (VICs). Cell differentiation, viability, and proliferation were analyzed. Particles were heterogeneous, differing in size and shape, and were crystallized as calcium phosphate. Diseased donors had significantly more calcified particles compared to healthy donors (P<.05), but there were no differences between the composition of the particles from healthy and diseased donors. VECs treated with calcified particles showed a significant decrease in CD31 and VE-cadherin and an increase in von Willebrand factor expression, P<.05. There were significantly increased α-SMA and osteopontin in treated VICs (P<.05), significantly decreased VEC and VIC viability (P<.05), and significantly increased number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive VECs (P<.05) indicating apoptosis when treated with the calcified particles. Isolated calcified particles from human aortae are not innocent bystanders but induce a phenotypical and pathological change of VECs and VICs characteristic of activated and pathological cells. Therapy tailored to reduce these calcified particles should be investigated.

A Study for Tooth Bleaching via Carbamide Peroxide-Loaded Hollow Calcium Phosphate Spheres.

The objective of this study was to investigate if a prolonged bleaching effect of carbamide peroxide-loaded hollow calcium phosphate spheres (HCPS) can be achieved. HCPS was synthesized via a hydrothermal reaction method. Carbamide peroxide (CP) was-loaded into HCPS by mixing with distilled water as solvent. We developed two bleaching gels containing CP-loaded HCPS: one gel with low HP concentration as at-home bleaching gel, and one with high HP concentration as in-office gel. Their bleaching effects on stained human permanent posterior teeth were investigated by measuring the color difference before and after bleaching. The effect of gels on rhodamine B degradation was also studied. To investigate the potential effect of remineralization of using HCPS, bleached teeth were soaked in phosphate buffer solution (PBS) containing calcium and magnesium ions. Both bleaching gels had a prolonged whitening effect, and showed a strong ability to degrade rhodamine B. After soaking in PBS for 3 days, remineralization was observed at the sites where HCPS attached to the teeth surface. CP-loaded HCPS could prolong the HP release behavior and improve the bleaching effect. HCPS was effective in increasing the whitening effect of carbamide peroxide and improving remineralization after bleaching process.

Towards understanding the distribution and tumor targeting of sericin regulated spherical calcium phosphate nanoparticles.

The study of calcium phosphate (CaP) nanoparticles in vivo is still incomplete, which has limited their applications for biomedical delivery. Herein, we synthesized amorphous spherical calcium phosphate (S-CaP) nanoparticles with an average size of 80 nm via a co-precipitation method in the presence of silk sericin as the regulation template. S-CaP was labeled by the near-infrared dye reagent DiR, and then, the labeled nanoparticles (S-CaP@DiR) were used to investigate the distribution and degradation in healthy mice by IVIS and TEM. The results showed that the S-CaP nanoparticles were mainly distributed in the liver, and ∼90% of them (500 μg) could be degraded by the liver within 2 weeks. Tumor-bearing mice were then prepared, and the S-CaP was injected intravenously. Strikingly, the nanoparticles can effectively target solid tumors in cancer cell-bearing mice, indicating that the solid tumor was a foundation for the enrichment of the nanoparticles by the EPR effect, which showed the important potential of biodegradable inorganic nanoparticles in clinical drugs for tumor therapy.

Optimizing the Water-Oil Emulsification Process for Developing CPC Microspheres

Spherical calcium phosphate cement carriers were synthesized by water/oil emulsion. A 23 factorial design was employed in order to optimize the size and form of the microspheres. Stirring speed, liquid-to-powder ratio and cement paste-to-oil ratio were varied in two different levels and the response variable was the microsphere's sphericity. The results showed that cement's setting reaction was responsible for stabilizing the microspheres within the oil phase resulting in a low crystalline hydroxyapatite. The sizes varied from 6.29 to 64.23 µm. From the factors studied, the stirring velocity and the interaction between the L/P ratio with the P/O ratio are the ones that are more effective in controlling microspheres' sphericity. The results suggest that the methodology studied may provide microspheres with appropriate properties to deliver cells and/or drugs without inducing inflammatory reactions.

Pulmonary Alveolar Microlithiasis “Stone Lungs”: A Case of Clinico-Radiological Dissociation

Pulmonary alveolar microlithiasis (PAM) is a rare infiltrative lung disease characterized by deposition of spherical calcium phosphate microliths called calcospherites within the alveoli. PAM was first described by Friedrich in 1856 and then by Harbitz in 1918. The disease pathogenesis is based on mutations in the SLC34A2 gene that encodes for the Type IIb sodium-phosphate cotransporter. The majority of the patients are diagnosed at an early age, usually between the ages of 20 and 40 years. The hallmark of this disease is a striking dissociation between the radiological findings and the mild clinical symptoms. We report a case of 35-year-old woman who presented post-motor vehicle accident with back pain and with minimal dyspnea on exertion. The final diagnosis was made after computed tomography and lung biopsy. The present case exhibits the remarkable clinico-radiological dissociation with complete calcification of the lungs on radiographic images with a relatively mild clinical presentation.

Biomimetic mineralization of nano-sized, needle-like hydroxyapatite with ultrahigh capacity for lysozyme adsorption

Because of its superior biocompatibility, hydroxyapatite (HA) has been widely exploited as a promising vehicle to deliver a broad range of therapeutics in a variety of biological systems. Herein, we report a biomimetic process to prepare nano-sized, colloidal stable HA with needle-like morphology by using carboxymethyl cellulose (CMC) as the template. It was revealed that the needle-like HA was transformed from the spherical amorphous calcium phosphate (ACP) nanoparticles after a 14-day period of aging under ambient conditions. The needle-like HA/CMC exhibited an ultra-high lysozyme adsorption capacity up to 930–940mg/g. Moreover, a sustained and pH-sensitive release of adsorbed lysozyme from HA/CMC was evidenced. Therefore, our biomimetic needle-like HA/CMC nanoparticles hold great potential in serving as an efficient carrier for the delivery and controlled release of lysozyme.

Apatite precipitation on a novel fast-setting calcium silicate cement containing fluoride

Aim: Calcium silicate cements are widely used in endodontics. Novel fast-setting calcium silicate cement with fluoride (Protooth) has been developed for potential applications in teeth crowns including cavity lining and cementation.Objective: To evaluate the surface apatite-forming ability of Protooth compositions as a function of fluoride content and immersion time in phosphate-buffered saline (PBS).Material and methods: Three cement compositions were tested: Protooth (3.5% fluoride and 10% radiocontrast), ultrafast Protooth (3.5% fluoride and 20% radiocontrast), and high fluoride Protooth (15% fluoride and 25% radiocontrast). Powders were cap-mixed with liquid, filled to the molds and immersed in PBS. Scanning electron microscopy, energy dispersive X-ray analysis, and Raman spectroscopy were used to characterize the precipitations morphology and composition after 1, 7, 28, and 56 days. Apatite/belite Raman peak height indicated the apatite thickness.Results: Spherical calcium phosphate precipitations with acicular crystallites were formed after 1-day immersion in PBS and Raman spectra disclosed the phosphate band at 965 cm−1, supporting the apatite formation over Protooth compositions. The apatite deposition continued and more voluminous precipitations were observed after 56 days over the surface of all cements. Raman bands suggested the formation of β-type carbonated apatite over Protooth compositions. High fluoride Protooth showed the most compact deposition with significantly higher apatite/belite ratio compared to Protooth and ultrafast Protooth after 28 and 56 days.Conclusions: Calcium phosphate precipitations (apatite) were formed over Protooth compositions after immersion in PBS with increasing apatite formation as a function of time. High fluoride Protooth exhibited thicker apatite deposition.