Comparison of calcium-based technologies to remineralise enamel subsurface lesions using microradiography and microhardness.

Assessment of enamel subsurface lesion remineralisation is essential for the evaluation of novel remineralisation technologies. The gold standard to assess subsurface mineral gain of enamel lesions is transverse microradiography (TMR). However, some studies have utilised surface microhardness (SMH) to evaluate efficacy of remineralisation agents. The aim of this study was to assess remineralisation of enamel subsurface lesions using TMR and SMH after in vitro treatment with calcium-containing technologies, and to test correlation between the TMR and SMH measurements. The parameters obtained from the TMR and SMH analyses of enamel subsurface remineralisation were not significantly correlated. Furthermore, the enamel subsurface remineralisation as measured by TMR was significantly correlated with the water-soluble calcium concentration of the remineralisation products. Scanning electron microscopy revealed surface precipitates formed by specific remineralisation treatments obfuscated accurate assessment of remineralisation by SMH. It was concluded that TMR is a more appropriate method for analysis of enamel subsurface remineralisation, and that SMH values of remineralised enamel should be interpreted with caution. Using TMR the level of remineralisation (%R) by the different technologies was CPP-ACP/F (31.3 ± 1.4%); CPP-ACP (24.2 ± 1.4%); CaSO4/K2HPO4/F (21.3 ± 1.4%); f-TCP/F (20.9 ± 1.0%); Nano-HA/F (16.3 ± 0.3%); Nano-HA (15.3 ± 0.6%) and F alone control (15.4 ± 1.3%).

Acceleration of Enamel Subsurface Lesion Remineralisation by Intralesion pH Modulation.

Remineralisation of demineralised enamel subsurface lesions can be enhanced by pretreatment of the lesions with base (NaOH). The aim of this study was to test the effect of intralesion pH modulation on remineralisation of demineralised enamel subsurface lesions by casein phosphopeptide-stabilised amorphous calcium fluoride phosphate (CPP-ACFP) in vitro. Two remineralisation models were utilised, the first involving 60-min cyclic pH modulation for 105 h and the second involved short-term cyclic pH modulation (12-min cycle, 240 min total duration) compared with the equivalent time of continuous treatment (200 min total duration). The intralesion pH modulation was achieved by cyclic exposure to a pH 12.9 NaOH solution and a CPP-ACFP remineralisation solution at pH 5.5. Percent remineralisation was assessed using transverse microradiography with data statistically analysed using a 2-sample Student t test. For the first model, the intralesion pH modulation group had significantly (p < 0.001) higher remineralisation (43.8 ± 6.9%) than the control group (28.2 ± 5.8%) cycled with water. For the second model, the intralesion pH modulation group had significantly (p < 0.001) higher remineralisation (23.1 ± 3.4%) than the group with continuous equivalent CPP-ACFP treatment time (1.9 ± 1.3%). In both models, intralesion pH modulation significantly accelerated remineralisation, and this was attributed to the effect pH modulation had on the diffusion gradients of ions/ion pairs and the degree of saturation with respect to apatite phases within the lesion fluid.

Bioavailable fluoride in calcium-containing dentifrices.

Calcium added to dentifrices can complex with fluoride ions to reduce intra-oral bioavailability and therefore efficacy in preventing dental caries. Six commercially available dentifrices containing different types of calcium and fluoride were analyzed for total and bioavailable fluoride levels by adding 10 g of dentifrice to 30 mL of distilled deionized water and mixing vigorously for 1 min to simulate toothbrushing. One milliliter of the dentifrice/water slurry was immediately centrifuged and the supernatant removed for bioavailable fluoride analysis and the mixed slurry prior to centrifugation used for total fluoride analysis using a modified microdiffusion method. The concentration of fluoride was determined using a fluoride ion-selective electrode calibrated with internal fluoride standards. All the dentifrices had similar total fluoride concentrations to those indicated on their labels (94% to 105%). However, only one dentifrice that contained calcium in the form of casein phosphopeptide amorphous calcium phosphate (CPP-ACP) had almost 100% (97%) of fluoride in bioavailable form. The other dentifrices contained calcium carbonate and they exhibited significantly (p < 0.001) lower bioavailable fluoride levels (27% to 61%), through the generation of poorly soluble fluoride phases. The saliva biomimetic CPP, as CPP-ACP, in a dentifrice stabilised calcium and fluoride ions to maintain fluoride's bioavailability.

Recharge and increase in hardness of GIC with CPP-ACP/F.

OBJECTIVE: To assess the effect of CPP-ACP/F recharging on ion release and hardness of GIC Fuji-Triage (VII) and Fuji-Triage-EP (VII-EP) containing CPP-ACP/F. METHODS: CPP-ACP distribution in Fuji-Triage-EP was determined using immunofluorescence. Thirty blocks of Fuji-Triage and Fuji-Triage-EP with the same surface area were placed individually in 5mL of 50mM lactic acid (pH 5) for three days. Every 12h ten Fuji-Triage and ten Fuji-Triage-EP blocks were treated with 2mL of either MI Paste Plus (CPP-ACP/F) solution (1g paste+4mL water), Placebo MI paste solution (no CPP-ACP/F), or distilled water for 2min. After each 2min treatment the blocks were rinsed with distilled water and placed back into the acid. Calcium, inorganic phosphate and fluoride levels in the acid solution were measured using atomic absorption spectrophotometry, colorimetry and ion specific electrode respectively. Vickers surface hardness of the GIC was also determined. Data were analysed using a two-sample t-test and one-way ANOVA with a Bonferroni-Holm correction for multiple comparisons. RESULTS: CPP-ACP was distributed throughout Fuji-Triage-EP. Significantly (p<0.001) higher calcium, inorganic phosphate and fluoride ion release and greater surface hardness (acid resistance) was observed in both GIC's treated with the CPP-ACP/F paste. Fuji-Triage-EP released higher ion levels and exhibited greater surface hardness (acid resistance) than Fuji-Triage. SIGNIFICANCE: Topical application of CPP-ACP/F paste to GIC Fuji-Triage-EP recharged ion release and increased surface hardness (acid resistance) which may help improve properties and resistance to degradation as well as improve ion release for caries control.

Addition of CPP-ACP to yogurt inhibits enamel subsurface demineralization.

OBJECTIVES: The aim of this study was to investigate the capacity of an approved food additive with anticariogenic properties, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), when added to a processed, sugar-containing yogurt with added lactic-acid bacteria (probiotics), to prevent demineralization of enamel subsurface lesions in vitro. METHODS: Enamel subsurface demineralised lesions were created in thirty extracted human third molars. These were then exposed to artificial saliva (AS) with: 1) Danone yogurt alone; 2) Danone yogurt with 0.2 % w/w CPP-ACP; or 3) Danone yogurt with 0.5 % w/w CPP-ACP at 37 °C for two weeks. The yogurt/AS was replaced with fresh preparations each day. At the completion of each treatment the enamel slabs were embedded, sectioned and analyzed using transverse microradiography to measure changes in enamel lesion depths and subsurface mineral content. Yogurt samples were analysed for soluble calcium (Ca) and inorganic phosphate (Pi) levels and pH. RESULTS: Yogurt alone demineralized enamel subsurface lesions to produce significantly larger lesions. However, the addition of 0.2 % CPP-ACP to the yogurt resulted in significant reduction in demineralization compared with yogurt alone (p < 0.0001). The addition of 0.5 % CPP-ACP to the yogurt produced a net remineralization effect with a significant increase in lesion mineral content (p < 0.0001). The addition of CPP-ACP resulted in a significant (p < 0.0001) dose-related increase in Ca, Pi and pH. CONCLUSIONS: The addition of CPP-ACP to a commercial yogurt exhibited a dose related protective effect with 0.5 % CPP-ACP producing remineralization of existing enamel subsurface lesions under the in vitro experimental conditions. CLINICAL SIGNIfiCANCE: The results of this study suggest that some processed yogurts with added sugar could result in enamel demineralization when frequently consumed by individuals with poor oral hygiene. The addition of CPP-ACP to these yogurts may help prevent demineralization and promote enamel subsurface lesion remineralization, and therefore, make them safer for teeth.

Effects of Bovine Serum Albumin and High pH Pre-Treatment on the Remineralisation of Enamel Subsurface Lesions in vitro.

Accumulated intra-lesion protein such as serum albumin has been speculated to impede remineralisation of carious enamel lesions. The aim of this study was to assess whether intra-lesion bovine serum albumin (BSA) affected subsequent remineralisation of enamel subsurface lesions. Confocal microscopy was used to confirm localisation of BSA in artificial enamel subsurface lesions and its subsequent degradation by a high pH sodium hypochlorite treatment. An in vitro remineralisation experiment tested the effect of intra-lesion BSA, and its degradation by sodium hypochlorite, on remineralisation of subsurface lesions by casein phosphopeptide stabilised amorphous calcium fluoride phosphate. In addition, lesions without BSA were pre-treated with one of 2 high pH solutions (sodium hypochlorite or sodium hydroxide) prior to remineralisation to test whether the high pH pre-treatment influenced remineralisation. Data were obtained on remineralisation using transverse microradiography and were analysed with a one-way ANOVA. Intra-lesion BSA had no significant effect on remineralisation compared with that of control lesions. Pre-treatment of BSA-containing lesions with sodium hypochlorite significantly increased remineralisation. The lesions without BSA that were pre-treated with either sodium hypochlorite or sodium hydroxide also showed the same level of remineralisation as the BSA-containing lesions pre-treated with sodium hypochlorite indicating that the increased remineralisation was pH related. Hence, it was concluded that intra-lesion BSA did not affect remineralisation of artificial enamel subsurface lesions in this model system and that a high pH pre-treatment enhanced remineralisation.

The prebiotic effect of CPP-ACP sugar-free chewing gum.

OBJECTIVES: To determine if chewing gum containing casein phosphopeptide stabilised amorphous calcium phosphate (CPP-ACP) promoted an increase in the abundance of Streptococcus sanguinis and other species associated with dental health in supragingival plaque in a clinical study. MATERIALS AND METHODS: Nineteen participants were recruited for a three-leg cross-over, randomised, controlled clinical trial. Participants chewed a sugar-free gum with or without CPP-ACP six times daily for 20 min over two weeks. The study also involved no gum chewing (no gum) for the same two week period. Participants were randomly assigned to one of the test gums or no gum for each intervention period. Participants abstained from oral hygiene and had washout periods of two weeks between intervention periods. After each intervention period, supragingival plaque was collected and analysed for bacterial composition by sequencing the V4 variable region of the 16S rRNA gene. Data were analysed using a linear mixed model. RESULTS: The CPP-ACP gum intervention produced a significant (p < 0.01) increase in the proportions of S. sanguinis (112%), as well as the commensal species Rothia dentocariosa (127%), Corynebacterium durum (80%) and Streptococcus mitis (55%) when compared with the no gum intervention. All the species that were promoted by the CPP-ACP gum are known to possess one or both of the alkali-producing enzymes arginine deiminase and nitrate reductase. CONCLUSION: This clinical study demonstrated that chewing a sugar-free gum containing CPP-ACP promoted prebiosis by significantly increasing the proportion of S. sanguinis and other health-associated bacterial species in supragingival plaque. CLINICAL SIGNIFICANCE: Regular chewing of CPP-ACP sugar-free gum increases the proportions of health-associated commensal species in supragingival plaque to promote prebiosis and oral homeostasis.

Effects of soy and bovine milk beverages on enamel mineral content in a randomized, double-blind in situ clinical study.

Soy beverages are promoted as healthy alternatives to bovine milk even though they can contain added sugar. OBJECTIVES: To compare enamel mineral content after consumption of bovine milk or a soy beverage in a double-blind, randomized, cross-over in situ clinical study. MATERIALS AND METHODS: Human enamel slabs with subsurface lesions were prepared and inserted into intra-oral appliances worn by volunteers who consumed 200 ml of either bovine milk or a soy beverage over a 60 s period once a day for 15 days. Enamel lesion depth and mineral content were measured using transverse microradiography. Saliva samples were collected immediately after consuming the beverages and calcium, inorganic phosphate and fluoride levels analysed. Data were statistically analysed using a linear mixed model. RESULTS: Depth of the enamel subsurface lesions increased by 7.1 ±â€¯2.0 µm and mineral content decreased by 47 ±â€¯22 vol% min.µm after consumption of the soy beverage indicating demineralization. However, after consumption of bovine milk the depth of the lesions decreased by 7.6 ±â€¯3.5 µm and mineral content increased by 202 ±â€¯43 vol% min.µm indicating remineralization. The changes were significantly different (p < 0.001) between the two beverages. Fluoride levels were similar in the saliva samples for both beverages, however the calcium and inorganic phosphate levels for the bovine milk group were significantly higher (p < 0.02) than those for the soy beverage group. CONCLUSIONS: In this randomized, double-blind in situ clinical trial consumption of a soy beverage demineralized enamel whereas bovine milk produced remineralization. CLINICAL SIGNIFICANCE: Although soy beverages are promoted as healthy alternatives to bovine milk the added sugar and low calcium bioavailability of the soy drink makes frequent consumption a caries risk. (Trial registration no. ISRCTN19137849).

Self-assembly of dental surface nanofilaments and remineralisation by SnF2 and CPP-ACP nanocomplexes.

Dental caries, erosion and hypersensitivity are major public health problems. SnF2 is used widely in oral care products to help prevent/treat these conditions. Casein phosphopeptide-stabilised amorphous calcium phosphate nanocomplexes (CPP-ACP) are a biomimetic nanotechnology of salivary phosphopeptide-ACP complexes that deliver bioavailable calcium and phosphate ions to promote dental remineralisation (repair). We show here using in vitro studies and a double-blind, randomised controlled, cross-over design in situ clinical trial that SnF2 and CPP-ACP interact to form a nanofilament coating on the tooth surface and that together they are superior in their ability to promote dental remineralisation. Sn(II) by cross-linking the CPP-ACP helps to stabilise the complexes which improves delivery to the tooth surface and enhances binding and ion incorporation into tooth mineral. The combination of SnF2 and CPP-ACP in oral care products may significantly improve their efficacy in prevention/treatment of dental caries/erosion and hypersensitivity.

Porphyromonas gingivalis in Alzheimer's disease brains: Evidence for disease causation and treatment with small-molecule inhibitors.

Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, was identified in the brain of Alzheimer's disease patients. Toxic proteases from the bacterium called gingipains were also identified in the brain of Alzheimer's patients, and levels correlated with tau and ubiquitin pathology. Oral P. gingivalis infection in mice resulted in brain colonization and increased production of Aß1-42, a component of amyloid plaques. Further, gingipains were neurotoxic in vivo and in vitro, exerting detrimental effects on tau, a protein needed for normal neuronal function. To block this neurotoxicity, we designed and synthesized small-molecule inhibitors targeting gingipains. Gingipain inhibition reduced the bacterial load of an established P. gingivalis brain infection, blocked Aß1-42 production, reduced neuroinflammation, and rescued neurons in the hippocampus. These data suggest that gingipain inhibitors could be valuable for treating P. gingivalis brain colonization and neurodegeneration in Alzheimer's disease.

Importance of bioavailable calcium in fluoride dentifrices for enamel remineralization.

OBJECTIVES: To compare remineralization of enamel subsurface lesions by fluoride dentifrices with added calcium in a double-blind, randomized, cross-over, in situ study. METHODS: Human enamel with subsurface lesions were prepared and inserted into intra-oral appliances worn by volunteers. A slurry (1 g toothpaste/4 ml H2O) was rinsed for 60 s, 4 times per day for 14 days. Seven toothpastes were tested: (i) 1450 ppm F (NaF), (ii) 5000 ppm F (NaF), (iii) 1450 ppm F (MFP) with calcium sodium phosphosilicate (CSP), (iv) 1450 ppm F (MFP) with CaCO3/Arg, (v) 1150 ppm F (SnF2) with amorphous calcium phosphate (ACP), (vi) 1100 ppm F (NaF) with casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and (vii) 5000 ppm F (NaF) with functionalized tri-calcium phosphate (TCP). Total (acid soluble) and bioavailable (water soluble) calcium, inorganic phosphate and fluoride levels of the dentifrices were measured using ion chromatography (F/MFP) and spectrophotometry (Ca and inorganic phosphate). Enamel lesion mineral content was measured using transverse microradiography. Data were statistically analysed using a linear mixed model. RESULTS: All calcium and fluoride containing toothpastes released > 90% of bioavailable fluoride and were superior to the respective fluoride alone toothpastes in remineralization of enamel subsurface lesions. The level of remineralization followed the order: CPP-ACP/1l00 ppm F > ACP/1150 ppm F = TCP/5000 ppm F > 5000 ppm F = CaCO3/Arg/1450 ppm F = CSP/1450 ppm F > 1450 ppm F. Bioavailable calcium levels significantly correlated with enhanced remineralization of enamel subsurface lesions. CONCLUSIONS: Bioavailable calcium in fluoride dentifrices enhanced remineralization of enamel subsurface lesions.

Polyols and remineralisation of enamel subsurface lesions.

Sugar-free chewing gum containing polyols has been demonstrated to reduce caries experience in randomised controlled clinical trials. A range of polyols (mannitol, sorbitol, xylitol and maltitol) can be found in sugar-free gums and it has been claimed that they can facilitate calcium uptake into enamel subsurface lesions promoting remineralisation. OBJECTIVES: The aim of this study was to compare the effect of polyols on remineralisation of enamel subsurface lesions in vitro by artificial saliva (AS) and by AS containing the salivary biomimetic casein phosphopeptide amorphous calcium phosphate (CPP-ACP). METHODS: The polyols (12.6% w/v) and CPP-ACP (0.376% w/v) were used at physiologically relevant concentrations approximating those released into saliva during chewing a CPP-ACP/polyol chewing gum. Enamel subsurface lesions were exposed to one of the polyols (xylitol, sorbitol, maltitol, mannitol) in AS or AS containing CPP-ACP for 7days at 37°C with a change of solution each day. Remineralisation of the enamel subsurface lesions was measured by transverse microradiography. RESULTS: A statistical test for equivalence showed there was no difference in remineralisation between the AS solutions with or without any of the polyols. The AS+CPP-ACP solution substantially promoted remineralisation over AS alone independently of any polyol added. CONCLUSION: This controlled in vitro study showed that polyols at physiologically relevant concentrations did not promote remineralisation of enamel subsurface lesions by facilitating calcium uptake into the lesion.

Comparative study of the measurement of enamel demineralization and remineralization using transverse microradiography and electron probe microanalysis.

Transverse microradiography (TMR) and electron probe microanalysis (EPMA) are commonly used for characterizing dental tissues. TMR utilizes an approximately monochromatic X-ray beam to determine the mass attenuation of the sample, which is converted to volume percent mineral (vol%min). An EPMA stimulates the emission of characteristic X-rays from a variable volume of sample (dependent on density) to provide compositional information. The aim of this study was to compare the assessment of sound, demineralized, and remineralized enamel using both techniques. Human enamel samples were demineralized and a part of each was subsequently remineralized. The same line profile through each demineralized lesion was analyzed using TMR and EPMA to determine vol%min and wt% elemental composition and atomic concentration ratio information, respectively. The vol%min and wt% values determined by each technique were significantly correlated but the absolute values were not similar. This was attributable to the complex ultrastructural composition, the variable density of the samples analyzed, and the nonlinear interaction of the EPMA-generated X-rays. EPMA remains an important technique for obtaining atomic ratio information, but its limitations in determining absolute mineral content indicate that it should not be used in place of TMR for determining the mineral density of dental hard tissues.

Effect of added calcium phosphate on enamel remineralization by fluoride in a randomized controlled in situ trial.

UNLABELLED: Dental products containing calcium phosphate and fluoride are claimed to enhance enamel remineralization over fluoride products. OBJECTIVES: To compare remineralization of enamel subsurface lesions by dental products with added calcium phosphate in a double-blind, randomized, cross-over in situ study. METHODS: Human enamel specimens with subsurface lesions were prepared and inserted into intra-oral appliances worn by volunteers. A slurry (1g product plus 4 ml H(2)O) of each product was rinsed for 60s, 4 times per day for 10 days. Six products were tested (i) placebo, (ii) 1000 ppm F, (iii) 5000 ppm F, (iv) Tooth Mousse (TM), (v) TM plus 900 ppm F (TMP) and (vi) Clinpro with 950 ppm F. Calcium, inorganic phosphate and fluoride levels were measured in post-rinse/saliva samples using ion chromatography. Mineral content was measured using transverse microradiography. RESULTS: Only TM and TMP significantly increased salivary calcium and phosphate levels. The products produced remineralization in the following order from lowest to highest: placebo<1000 ppm F=Clinpro<5000 ppm F

Remineralization of enamel subsurface lesions by chewing gum with added calcium.

OBJECTIVES: Chewing sugar-free gum has been shown to promote enamel remineralization. Manufacturers are now adding calcium to the gum in an approach to further promote enamel remineralization. The aim of this study was to compare the remineralization efficacy of four sugar-free chewing gums, two containing added calcium, utilizing a double-blind, randomized, crossover in situ model. METHODS: The sugar-free gums were: Trident Xtra Care, Orbit Professional, Orbit and Extra. Ten subjects wore removable palatal appliances with four human-enamel half-slab insets containing subsurface demineralized lesions. For four times a day for 14 consecutive days subjects chewed one of the chewing gums for 20min. After each treatment the enamel slabs were removed, paired with their respective demineralized control slabs, embedded, sectioned and mineral level determined by microradiography. After 1-week rest the subjects chewed another of the four gums and this was repeated until each subject had used the four gum products. RESULTS: Chewing with Trident Xtra Care resulted in significantly higher remineralization (20.67+/-1.05%) than chewing with Orbit Professional (12.43+/-0.64%), Orbit (9.27+/-0.59%) or Extra (9.32+/-0.35%). The form of added calcium in Trident Xtra Care was CPP-ACP and that in Orbit Professional calcium carbonate with added citric acid/citrate for increased calcium solubility. CONCLUSIONS: Although saliva analysis confirmed release of the citrate and calcium from the Orbit Professional gum the released calcium did not result in increased enamel remineralization over the normal sugar-free gums. These results highlight the importance of calcium ion bioavailability in the remineralization of enamel subsurface lesions in situ.

Porphyromonas gingivalis RgpA-Kgp proteinase-adhesin complexes penetrate gingival tissue and induce proinflammatory cytokines or apoptosis in a concentration-dependent manner.

The RgpA-Kgp proteinase-adhesin complexes of Porphyromonas gingivalis were observed, using immunostaining, in human gingival tissue associated with periodontitis but not in healthy tissue. The staining pattern suggested a concentration gradient from the subgingival plaque into the subjacent gingival connective tissue. Intense immunostaining was observed in areas displaying gross disturbance of tissue architecture. P. gingivalis cells and the RgpA-Kgp complexes at low concentrations were shown to stimulate secretory intercellular adhesion molecule 1, interleukin-8 (IL-8), IL-6, and macrophage chemoattractant protein secretion from cultured human epithelial (KB) and fibroblast (MRC-5) cells. However, at high concentrations a reduction in the level of these mediators was observed. In contrast, macrophage inflammatory protein 1alpha and IL-1alpha were stimulated only at high P. gingivalis cell concentrations. P. gingivalis cells and the RgpA-Kgp complexes were shown to induce apoptosis in KB and MRC-5 cells in a time- and dose-dependent manner. These data suggest that the RgpA-Kgp complexes penetrate the gingival connective tissue; at low concentrations distal from the plaque the complexes stimulate the secretion of proinflammatory mediators, while at high concentrations proximal to the plaque they induce apoptosis and attenuate the secretion of proinflammatory mediators.

Remineralization of enamel subsurface lesions in situ by the use of three commercially available sugar-free gums.

BACKGROUND: Commercially available sugar-free chewing gums have been claimed to provide oral health benefits. AIM: The aim of this randomized, double-blind crossover in situ study was to compare the efficacy of three commercially available sugar-free chewing gums: Trident White, Orbit, and Orbit Professional, in remineralizing enamel subsurface lesions in situ. DESIGN: Specimens containing enamel subsurface lesions were sectioned into test and control half-slabs with the test half-slabs inserted into removable palatal appliances. For each test chewing period, subjects were randomly allocated one of three test gums. Subjects (n = 10) chewed the randomly allocated gum for a 20-min period four times per day for 14 days. Each subject chewed all three test gums, with a 7-day washout period between crossovers. After each 14-day cycle, test and control half-slabs were paired, embedded in resin, sectioned, and subjected to microradiography to determine remineralization. RESULTS: The gum TW produced significantly greater remineralization (18.4 +/- 0.9%) than Orbit (8.9 +/- 0.5%) and Orbit Professional (10.5 +/- 0.9%). CONCLUSION: The superior remineralization activity of the TW gum in situ was attributed to the presence of casein phosphopeptide-amorphous calcium phosphate nanocomplexes.