Calcium Phosphate Complex Research Articles

Nature’s remedy to phosphate woes: calciprotein particles regulate systemic mineral metabolism

Calciprotein particles, colloidal complexes of calcium phosphate and plasma protein fetuin-A, have emerged as mediators of multiple biological effects attributed to phosphate. A new study postulates that circulating calciprotein particles, not phosphate, regulate the production of the major bone-derived phosphatonin, fibroblast growth factor-23. The signaling events coupling calciprotein particles sensing in bone to fibroblast growth factor-23 secretion remain to be established.

Comparative Evaluation of Casein Phosphopeptide - Amorphous Calcium Phosphate and Fluoride in Managing Early Caries Lesions

The aim of the present study was to assess the remineralizing potential of 10% casein phosphopeptide-amorphous calcium phosphate complex (CPP-ACP) -Recaldent used alone or with fluoride (CPP-ACFP) as compared to fluoride mouthrinse for the non-invasive treatment of occlusal non-cavitary caries lesions in vivo.A total of eighty subjects were randomly assigned to four groups, i.e. group 1 - 10% CPP-ACP, group 2- 10% CPP-ACP + 0.2% NaF, group 3- 0.05% NaF mouthrinse and group 4 �control. The lesions were assessed using visual examination and DIAGNOdent measurements. After 12 weeks of using the corresponding product, the results showed that the highest decrease in fluorescence was produced by CPP-ACFP (from 16.28�1.40 to 6.53�0.59, p=0.002), followed by CPP-ACP (from 15.70�1.82 to 8.18�0.70, p=0.004) and fluoride mouthrinse (from 14.32�1.72 to 7.88�0.68, p=0.005), significantly higher than in the control group (p[0.05). No significant differences were found between the three treatment groups.

Comparative Evaluation of Casein Phosphopeptide - Amorphous Calcium Phosphate and Fluoride in Managing Early Caries Lesions

The aim of the present study was to assess the remineralizing potential of 10% casein phosphopeptide-amorphous calcium phosphate complex (CPP-ACP) -Recaldent used alone or with fluoride (CPP-ACFP) as compared to fluoride mouthrinse for the non-invasive treatment of occlusal non-cavitary caries lesions in vivo.A total of eighty subjects were randomly assigned to four groups, i.e. group 1 - 10% CPP-ACP, group 2- 10% CPP-ACP + 0.2% NaF, group 3- 0.05% NaF mouthrinse and group 4 -control. The lesions were assessed using visual examination and DIAGNOdent measurements. After 12 weeks of using the corresponding product, the results showed that the highest decrease in fluorescence was produced by CPP-ACFP (from 16.28±1.40 to 6.53±0.59, p=0.002), followed by CPP-ACP (from 15.70±1.82 to 8.18±0.70, p=0.004) and fluoride mouthrinse (from 14.32±1.72 to 7.88±0.68, p=0.005), significantly higher than in the control group (p<0.05). No significant differences were found between the three treatment groups. Keywords: CPP-ACP, CPP-ACFP, fluoride mouthrinse, remineralization

Surface treatment with condensed phosphates reduced efflorescence formation on dry fermented sausages with alginate casings

The effect of a phosphate surface treatment on dry-fermented sausages with co-extruded alginate casings was investigated to reduce or inhibit the formation of white efflorescences on the surface of the sausages. Furthermore, with the co-extrusion process a continuous sausage production is possible with increased product volume compared to batch processing. Moreover, the production of meat snack products with very small diameters are possible. Solutions (5%) of different commercially available phosphates, mixture of di- and polyphosphates and two polyphosphates, were applied to the surfaces of the sausages after drying. The area amount of efflorescence was reduced from ∼65% on the control to 4–7%, depending on the phosphates used. The migration of magnesium and lactate to the surface of the sausage was inhibited by the diffusion of free phosphates and calcium phosphate complexes from the surface layer into the sausage. This study illustrated an easy application to prevent efflorescence formation.

Involvement of Vitamin K-Dependent Proteins in Vascular Calcification.

Vascular calcification results from an imbalance of promoters and inhibitors of mineralization in the vascular wall, culminating in the creation of an organized extracellular matrix deposition. It is characterized by the accumulation of calcium phosphate complex and crystallization of hydroxyapatite in the tunica media, leading to vessel stiffening. The underlying initiators of dysregulated calcification maintenance are diverse. These range from the expression of bone-associated proteins, to the osteogenic transdifferentiation of smooth muscle cells to osteoblast-like cells, to the release of fragmented apoptotic bodies and mineralization competent extracellular vesicles by smooth muscle cells, which act as a nucleation site for the deposition of hydroxyapatite crystals. The process involves a complex interplay between vitamin K-dependent calcification-inhibitory proteins, such as matrix γ-carboxyglutamate acid (Gla) protein, Gla-rich protein and growth arrest-specific gene 6 protein, and stimulatory mediators, such as osteocalcin. Vitamin K plays an important role as a cofactor for posttranslational γ-carboxylation of matrix Gla proteins in converting to a biologically active conformation. Drugs that inhibit vitamin K, such as warfarin, impair γ-carboxylation of Gla proteins, resulting in the accumulation of uncarboxylated proteins lacking calcification-inhibitory capacity. This article overviews the involvement of systemically and locally expressed vitamin K-dependent proteins in vascular calcification and their potential as biomarkers of calcification.

Ex vivo detection of calcium phosphate and calcium carbonate in rat blood serum

The total calcium (tCa) in blood serum comprises free Ca2+ ions (fCa), protein-bound calcium (prCa), and complexed calcium by small anions (cCa). The cCa fraction, in addition to fCa, has been indicated to have some physiological activity. However, there is little evidence for the structure of its constituents. Here we report an ex vivo detection of the cCa constituents by synchrotron X-ray absorption near-edge structure spectroscopy. We collected the data directly on rat blood serum and, by making use of the reference samples, derived a spectrum that exhibits the features of cCa constituents. Among the features are those of the complexes of calcium phosphate and calcium carbonate. The detected complexes in the cCa fraction are mainly Ca(η2-HPO4)(H2O)4 and Ca(η1-HCO3)(H2O)5+, in which HPO42− and HCO3− serve as bidentate and unidentate ligands, respectively. The remained H2O molecules on the coordination sphere of Ca2+ enable these complexes to behave partially like aquated Ca2+ ions in protein-binding. Besides, as the dominant part of prCa, albumin-bound calcium (albCa) exhibits a spectrum that closely resembles that of fCa, indicating weak interactions between the protein carboxyl groups and calcium. The weak-bound cCa and albCa, along with fCa and the relevant anions, compose a local chemical system that could play a role in maintaining the calcium level in blood.

Phosphate, Calcification in Blood, and Mineral Stress: The Physiologic Blood Mineral Buffering System and Its Association with Cardiovascular Risk.

Phosphate is an important cardiovascular risk factor and lowering elevated blood phosphate concentrations is a main therapeutic target in kidney patients. Phosphate is subject to the blood mineral buffering system which controls the precipitation of calcium and phosphate. Calciprotein particles (CPP), self-assembling complexes of calcium phosphate and serum proteins, are the nanomorphological correlates of this system. CPP1 are spherical, 50-100 nm in diameter, and contain amorphous mineral. CPP2 are oblongated, 100-200nm in the long axis, and they contain a crystalline mineral core. The relative abundance and biological activity of these particles are a matter of intense research, because they can cause oxidative stress, inflammation, and calcification in cellular assay. Therapeutically reducing this endogenous stressor by prolonging crystal formation time might improve patient outcome. This concise review article summarizes our current knowledge about the blood mineral buffering system and proposes Mineral Stress as a novel modifiable cardiovascular risk factor. It furthermore outlines possible implications this might have for improving patient care.

Performance and mechanisms of emerging animal-derived biochars for immobilization of heavy metals

Animal-derived biochars (ADBs) are a series of newly emerging biochars from the pyrolysis of animal carcasses. Their elemental composition and structural properties would be quite different to those of plant-based biochars and/or manure-derived biochars (MDBs). In this work, ADBs derived from cattle and swine carcasses were applied to investigate their sorption potentials and mechanisms for immobilization of Pb(II), Cd(II), Cu(II), and Hg(II). ADBs showed high calcium (Ca; >27%) and phosphorus (P; >15%) contents and relatively low carbon (C; <8%) content. Ca and P were present in the skeleton as calcium–phosphate complexes, dominantly as hydroxyapatite (HAP), which was identified by CP–MAS 31P NMR analysis. By contrast, the reference biochars derived from cattle, swine, and poultry manures possessed low Ca (<13%) and P (<4%) contents and relatively high C (>30%) content. Sorption experiments showed that ADBs were more effective in immobilizing Pb(II), Cd(II), and Cu(II) than MDBs. However, the affinities of ADBs for Hg(II) were lower than those of MDBs. Characterization analyses indicated that Pb(II) was effectively immobilized via precipitation, whereas ion exchange was the dominant mechanism in combining Cd(II) or Cu(II) with ADBs. ADBs are unsuitable for immobilizing Hg(II) because of the lack of oxygen-containing functional groups on the C surface, which can induce the reduction of Hg(II) to form Hg2Cl2. The immobilized heavy metals were difficult to be rereleased from ADBs in simulated acid rain environment. Hence, ADBs are promising biochars for immobilizing Pb(II), Cd(II), and Cu(II).

Calcium-phosphate complex increased during subchondral bone remodeling affects earlystage osteoarthritis

An activation of osteoclasts and subchondral bone remodeling is a major histologic feature of early-stage osteoarthritis (OA), which can be accompanied by an increase of calcium (Ca) and phosphate (Pi) level in the subchondral milieu. Considering articular cartilage gets most of nutrition from subchondral bone by diffusion, these micro-environmental changes in subchondral bone can affect the physiology of articular chondrocytes. Here, we have shown that Ca is increased and co-localized with Pi in articular cartilage of early-stage OA. The Ca-Pi complex increased the production of MMP-3 and MMP-13 in the hypertrophic chondrocytes, which was dependent on nuclear factor-kappa B (NF-kB), p38 and extracellular signal-regulated kinase (Erk) 1/2 mitogen-activated protein (MAP) kinase and Signal transducer and activator of transcription 3 (STAT3) signaling. The Ca-Pi complexes increased the expression of endocytosis markers, and the inhibition of the formation of the Ca-Pi complex ameliorated the Ca-Pi complex-mediated increases of MMPs expression in hypertrophic chondrocytes. Our data provide insight regarding the Ca-Pi complex as a potential catabolic mediator in the subchondral milieu and support the pathogenic role of subchondral bone in the early stages of cartilage degeneration.

Effect of fluoride dentifrice and casein phosphopeptide-amorphous calcium phosphate cream with and without fluoride in preventing enamel demineralization in a pH cyclic study.

Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) complexes are anticariogenic and capable of remineralizing the early stages of enamel lesions. The use of fluoride prevents dental decay and the association of CPP-ACP with fluoride can increase remineralization.Objective:To evaluate the effect of CPP-ACP and CPP-ACPF creams associated with a fluoride dentifrice to prevent enamel demineralization in a pH cyclic model.Material and Methods:Previously selected by surface microhardness (SH) analysis, human enamel blocks (n = 56) were submitted to daily treatment with dentifrice in a pH-cycling model. The enamel blocks were divided into four groups; G1: Crest™ Cavity Protection - Procter & Gamble (1,100 ppmF of NaF); G2: Crest™ +MI Paste (MP) - Recaldent™ GC Corporation Tokyo, Japan); G3: Crest™ + MI Paste Plus (MPP) - Recaldent™ 900 ppm as NaF, GC Corporation Tokyo, Japan), and G4: control, saliva. Specimens were soaked alternatively in a demineralizing solution and in artificial saliva for 5 d. The fluoride dentifrice, with proportion of 1:3 (w/w), was applied three times for 60 s after the remineralization period. The undiluted MP and MPP creams were applied for 3 m/d. After cycling, SH was re-measured and cross section microhardness measurements were taken.Results:The SH values observed for the groups G3 (257±70), G1 (205±70), and G2 (208±84) differed from the G4 group (98±110) (one-way ANOVA and Tukey's post hoc test). There were no differences between the groups G1xG2, G2xG3, and G1xG3 for demineralization inhibition. The percentage of volume mineral showed that, when applied with fluoride dentifrice, MPP was the most effective in preventing enamel demineralization at 50 µ from the outer enamel surface (Kruskal-Wallis and Mann Whitney p<0.05).Conclusion:Fluoride dentifrice associated with CPP-ACPF inhibited subsurface enamel demineralization.

Enantio- and Diastereoselective Cyclopropanation of β,γ-Unsaturated α-Ketoester by a Chiral Phosphate/Indium(III) Complex.

We report herein an enantioselective cyclopropanation of β,γ-unsaturated α-ketoesters with diazoesters by the complexes of InBr3 and chiral calcium phosphate. The reaction proceeds via a Michael addition-cyclization pathway to afford highly functionalized chiral cyclopropanes with excellent enantioselectivities (up to >99% ee) as a single diastereoisomer.

The addition of synthesized hydroxyapatite and fluorapatite nanoparticles to a glass-ionomer cement for dental restoration and its effects on mechanical properties

The objective of this study was to evaluate the effect of the addition of synthesized hydroxyapatite (HA) and fluorapatite (FA) nanoparticles to a glass-ionomer cement (GIC) on the mechanical properties, while preserving their unique and potent clinical properties. Bioceramics, such as HA and FA, have been recognized as restorative materials (e.g. GICs) in dentistry due to their chemical and biological compatibility with human hard tissues, which are considered calcium phosphate complexes. In this study, both of these inorganic nanoparticles (HA and FA) were synthesized via a wet-chemical precipitation method. The obtained nanoparticles were characterized with X-ray diffraction (XRD), inductively coupled plasma atomic emission spectroscopy (ICP-AES), Fourier transformed infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Brunauer-Emmett-Teller (BET) Theory. Then, HA and FA were incorporated into the powder component of the resin-modified cement (Fuji II, GC gold label, GC international, Tokyo, Japan) at 5% and 8wt%, and unblended powder was employed as control. Compressive strength (CS) and diametral tensile strength (DTA) before and after 1, 7 and 28 days of storage in distilled water were evaluated using a universal testing machine. Surface microhardness after 1 and 7 days of storage in distilled water was determined using Vickers microhardness tester. Setting and working time was measured as specified in the ASTM standard. The surface morphology of the modified GICs was examined using SEM observations. The morphology of the synthesized HA and FA nanoparticles was hexagonal, and their average sizes were about 25nm and 30nm, respectively. The mechanical results of the modified GICs ascertained addition of HA and FA (5 and 8wt%) into the glass ionomer cement after 7 days of storage in distilled water exhibited statistically higher CS of about 107–113.6MPa and 111–117.8MPa, respectively, and also higher DTS, 13–16MPa and 14–19MPa, respectively. The hardness of the glass ionomers containing HA and FA nanoparticles (5wt%) were increased by 2.21% and 11.77%, respectively. In addition, working time and setting time by adding the 5% nanoparticles were reduced about 8.5% and 13.23% for HA and 10.63% and 19.11% for FA, respectively. It was concluded that glass ionomer cements containing nanobioceramics (HA and FA) are promising restorative dental materials with improved mechanical properties. These experimental GICs may be potentially employed for higher stress-bearing site restorations, such as Class I and II restorations.

DEVELOPMENT OF THE NEW METHOD OF THE MELTED CHEESE PRODUCTS WITHOUT SALT-MELTERS USING CRYOMECHANOLYSIS

The aim of the work is elaboration of the principally new method of deep processing of rennet cheeses to the melting using the complex action of freezing and cryomechanolysis on the raw material that gives a possibility to destruct the hardly soluble biopolymers and to transform them into soluble form. The principally new method of the deep processing of rennet cheeses for receiving the melt cheese products without salts-smelters was elaborated. It differs from the traditional ones by the complete exclusion of the salts-smelters. This method is based on the use of the influence of freezing and fine-dispersed comminution on the raw material. It allows open biological potential of the rennet cheeses more fully and to extract the hidden (bound) protein forms from nanocomplexes of lipids and mineral substances. It allows destruct the proteins of rennet cheeses to the separate polymers and dipeptides and tripeptides. The used technological methods gave a possibility to exclude the salts-smelters at the rennet cheeses manufacturing. They favor the transformation of lipid-proteins paracaseinate calcium phosphate complexes to the separate amino acids and peptides and allow receive homogenous plastic mass. It was established, that at the complex action of freezing and fine-dispersed comminution on the rennet cheese the destruction of hardly soluble lipid-protein nanocomplexes and release of protein from the bound state into free one – nanoform (by 33,5…35 % more) takes place. The mechanisms of this process, connected with cryomechanodestruction of connections between lipids and proteins and non-fermented catalysis of nanocomplexes were described. It was established, that cryomechanodestruction and non-fermented catalysis of protein to the separate monomers – α-amino acids (by 55…60 %) takes place at freezing and fine-dispersed comminution of rennet cheese before melting. The mechanism of freezing and non-fermented analysis, connected with cryomechanocracking of protein molecules at the expanse of peptide protein connections destruction to the separate α-amino acids and their transformation into the free form was described. It was also demonstrated, that the conformational changes of protein molecules take place synchronously. The offered and elaborated nanotechnology of melt cheese products on the base of rennet cheeses without salts-smelters includes complex action of freezing and fine-dispersed comminution. The mechanisms of processes, connected with cryomechanodestruction of connections between lipids and protein to the separate α-amino acids are described. The cheese fillings for “Pancake” confectionary and cheese snacks – falafels were manufactured on the base of cheese mass, received using the new method and enriching vegetable nanoadditives. They exceed the well-known analogs by chemical composition and are remarkable for the storage life, increased in 2 times. At the same time the significant part of substances (BAS and biopolymers) in cheese filings is in nanodimensional form (55…60 % of protein), especially, free α-amino acids, easily assimilated by the human organism. The recipes and technologies of sauces-dressings, sauces-deeps, cheese snacks and so on are also elaborated on the base of cheese mass, received by the new method.

The Interactions of CPP-ACP with Saliva.

The repair of early dental caries lesions has been demonstrated by the application of the remineralisation technology based on casein phosphopeptide-stabilised amorphous calcium phosphate complexes (CPP–ACP). These complexes consist of an amorphous calcium phosphate mineral phase stabilised and encapsulated by the self-assembly of milk-derived phosphopeptides. During topical application of CPP–ACP complexes in the oral cavity, the CPP encounters the enamel pellicle consisting of salivary proteins and peptides. However the interactions of the CPP with the enamel salivary pellicle are not known. The studies presented here reveal that the predominant peptides of CPP–ACP complexes do interact with specific salivary proteins and peptides of the enamel pellicle, and provide a mechanism by which the CPP–ACP complexes are localised at the tooth surface to promote remineralisation.

Research Progress in Pseudoxanthoma Elasticum and Related Ectopic Mineralization Disorders

Heritable ectopic mineralization disorders represent a phenotypically diverse group of conditions characterized by deposition of calcium phosphate complexes in soft connective tissues. The prototype of such conditions is pseudoxanthoma elasticum (PXE), and related conditions with overlapping clinical features include generalized arterial calcification of infancy (GACI) and arterial calcification due to CD73 deficiency (ACDC). Molecular genetic investigations have revealed mutations in the genes physiologically involved in generation of inorganic pyrophosphate (PPi) and phosphate (Pi), and the findings suggest a unifying pathomechanism relating to reduced PPi/Pi ratio. This hypothesis is based on the notion that PPi serves as a powerful inhibitor of mineralization while Pi is a pro-mineralization factor, and an appropriate PPi/Pi ratio is critical for prevention of ectopic mineralization under homeostatic conditions.PXE International, the premiere patient support organization, advocating on behalf of patients and families with PXE, sponsors regular research meetings evaluating the progress in this and related ectopic mineralization disorders. The latest meetings were held in September 2014 in Bethesda, MD and in September 2015 in Budapest, Hungary. This report summarizes the latest progress in research on PXE and related ectopic mineralization disorders, based on presentations and discussions in these meetings, with pharmacologic implications for currently intractable disorders.

Comparative evaluation of the efficacy of fluoride varnish and casein phosphopeptide - Amorphous calcium phosphate in reducing Streptococcus mutans counts in dental plaque of children: An in vivo study.

Aim:To assess the comparative efficacy of fluoride varnish and casein phosphopeptide–amorphous calcium phosphate (CPP–ACP) complex visa viz. Streptococcus mutans in plaque, and thereby the role that these two agents could play in the prevention of dental caries.Materials and Methods:A cluster sample of 120 caries inactive individuals belonging to moderate and high caries risk group were selected from 3–5-year-old age group based on the criteria given by Krassee and were randomized to four groups, namely, fluoride varnish – Group I, CPP–ACP complex – Group II, mixture of CPP–ACP complex –Gourp III, and fluoride and routine oral hygiene procedures as control – Group IV. The results thus obtained were analyzed using Statistical Package for the Social Sciences (SPSS) version 16.Results:A statistically significant difference in the pre and post-application scores of S. mutans (P < 0.01) count was observed in all the groups with CPP–ACP plus fluoride group being the most proficient.Conclusion:Materials such as fluoride varnish, CPP–ACP, and CPP–ACP plus fluoride protects the tooth structure, preserving the integrity of primary dentition, with the most encouraging results being with CPP–ACP plus fluoride.

Properties of Hydrochloric Chitosan Solutions Modified With Nano- Calcium Phosphate Complex.

Properties of Hydrochloric Chitosan Solutions Modified With Nano- Calcium Phosphate Complex.

Effects of Treatment with Various Remineralizing Agents on the Microhardness of Demineralized Enamel Surface.

Background and aims. Remineralization of incipient caries is one of the goals in dental health care. The present study aimed at comparing the effects of casein phosphopeptide-amorphous calcium phosphate complex (CPP-ACP), Remin Pro®, and 5% sodium fluoride varnish on remineralization of enamel lesions.Materials and methods. In this in vitro study, 60 enamel samples were randomly allocated to six groups of 10. After four days of immersion in demineralizing solution, microhardness of all samples was measured. Afterward, groups 1-3 underwent one-time treatment with fluoride varnish, CPP-ACP, and Remin Pro®, respectively. Microhardness of groups 4-6 was measured not only after one-month treatment with the above-mentioned materials (for eight hours a day), but also after re-exposing to the demineralizing solution. The results were analyzed by one-way analysis of variance (ANOVA), repeated measures ANOVA, and Fisher’s least significant difference (LSD) test.Results. None of the regimens could increase microhardness in groups 1-3. However, one-month treatment regimens in groups 4-6 caused a significant increase in microhardness. The greatest microhardness was detected in the group treated with CPP-ACP (P = 0.001). In addition, although microhardness reduced following re-demineralization in all three groups, the mean reduction was minimum in the CPP-ACP-treated group (P < 0.001).Conclusion. While long-term repeated application of all compounds improved microhardness, the remineralization potential of CPP-ACP was significantly higher than that of Remin Pro® and sodium fluoride varnish.

Raw and treated marble wastes reuse as low cost materials for phosphorus removal from aqueous solutions: Efficiencies and mechanisms

Abstract Phosphorus removal from synthetic solutions by raw and calcined powdered marble wastes (RPMW and CPMW) has been investigated in batch mode under different experimental conditions. The results showed that RPMW and CPMW have high removal efficiencies, especially in acidic media. The maximum phosphorus removal capacities were evaluated to 103.9 and 181.2 mg·g−1 at an initial pH and an aqueous concentration of 5 and 350 mg·L−1, respectively. Phosphorus removal by RPMW occurred mainly through adsorption. However, for CPMW, phosphorus was removed not only by adsorption, but also by precipitation as calcium phosphate complexes. Specific analyses using scanning electron microscopy, energy dispersive spectroscopy, X-ray diffraction and Fourier transform infrared spectroscopy confirmed that this precipitate is most probably hydroxyapatite. On the other hand, CPMW have demonstrated an important ability in removing phosphorus from highly concentrated dairy wastewater (C0,P = 1000 mg·L−1) since only one dosage of 10 g·L−1 was enough to ensure more than 97% of phosphorus removal.

Remineralizing Efficacy of a CPP-ACP Cream on Enamel Caries Lesions in situ

The aim of this double-blind, randomized, cross-over in situ study was to compare the remineralizing effects induced by the application of casein phosphopeptide-stabilized amorphous calcium phosphate complexes (CPP-ACP)-containing cream (without fluoride) after the use of fluoride toothpaste with the prolonged use of fluoride toothpaste on enamel caries lesions in situ. During each of three experimental legs of 4 weeks, 13 participants wore intra-oral mandibular appliances with 8 pre-demineralized bovine enamel specimens in the vestibular flanges mimicking either ‘easily cleanable' or ‘proximal' surfaces (n = 312). The three randomly allocated treatments were as follows: (1) application of CPP-ACP-containing cream (GC Tooth Mouse, non-fluoride) after the use of fluoride toothpaste (1,400 ppm NaF; TM), (2) prolonged application of fluoride toothpaste (1,400 ppm NaF; positive control, PC) and (3) prolonged application of fluoride-free toothpaste (negative control, NC). Additionally, one of each of the two flanges was brushed twice daily with the respective toothpaste. The differences in integrated mineral loss as assessed by transversal microradiography were calculated between values before and after the in situ period. Changes in mineral loss were analysed for those pairs of subgroups differing in only one of the three factors (intervention, brushing and position). The PC treatment induced a significantly higher mineral gain compared with the TM and NC treatments. No significant differences between TM and NC for both positions were observed. In conclusion, the additional use of a CPP-ACP-containing cream seems to be less efficacious in remineralizing caries lesions than the prolonged application of fluoride toothpaste. © 2014 S. Karger AG, Basel