Inhibitory effect of Salvadora persica extract (Miswak) on collagen degradation in demineralized dentin: In vitro study.

BACKGROUND/PURPOSE: Root dentin is vulnerable to acid attack, suggesting a higher risk of demineralization than coronal enamel. This study aimed to evaluate the inhibitory effect of Miswak extract on collagen degradation of demineralized dentin lesion. MATERIALS AND METHODS: Demineralized bovine root dentin specimens were treated for 1 h by 20% Miswak extract and 0.12% Chlorehexidine (CHX) as a positive control group, and then subjected to collagenolytic attack (clostridium histolyticum 0.5 CDU/mL, 16 h). These cyclic treatments were repeated for 3 days. After the cyclic treatment, the images of the specimens were captured with a light microscope and the lesion depth of degraded collagen layer of all specimens was measured. The mean lesion depth was calculated and compared between the groups using descriptive and One-way ANOVA followed by Post hoc Tukey's tests. Significant level was set at p < 0.05. RESULTS: The mean lesion depth of CHX (28.6 ±â€¯3.37 µm) had the least value, followed by Miswak (37.5 ±â€¯4.01 µm) then the control (78.4 ±â€¯18.43 µm) group. There was a significant difference in the mean lesion depth among the three groups (p = 0.000). CONCLUSION: Miswak aqueous extract from S. persica was found to preserve the dentin collagen matrix from collagenase enzyme. This could be due to the organic compounds like flavonoids, saponins, alkaloids, tannins, and others which have been reported in literature. Present finding suggests that Miswak might play a positive effect in dentin caries prevention.

Comparison of resin-based and glass ionomer sealants with regard to fluoride-release and anti-demineralization efficacy on adjacent unsealed enamel.

This study compared resin-based and glass ionomer sealants with regard to their fluoride-release behavior and anti-demineralization potential on adjacent unsealed enamel surfaces. Sealant cavities prepared on bovine enamel blocks were filled with fluoride-containing resin sealants [TeethmateF-1 (TF), ClinproTM (CP)], and glass ionomer sealant [Fuji VII (FVII)]. Specimens were then incubated in artificial saliva for 14 days to measure fluoride. Thereafter, demineralization was performed for 10 days, and the anti-demineralization efficacy was assessed by Swept Source Optical Coherence Tomography (SS-OCT), and cross-sectional nanohardness. All data were statistically analyzed by using ANOVA. FVII exhibited the highest fluoride release. SS-OCT and nanohardness findings indicated that anti-demineralization efficacy of TF was the greatest, whereas FVII was not significantly different from that of CP. Resin sealants released a lower amount of fluoride but exhibited anti-demineralization effects on the adjacent unsealed enamel surfaces that were comparable to that of a glass ionomer sealant.

Effect of a calcium phosphate and fluoride paste on prevention of enamel demineralization.

This study aimed to examine the anti-demineralization capacities of (a) tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA) and 950 ppm fluoride paste, (b) casein phosphopeptide amorphous calcium phosphate paste and (c) 950 ppm fluoride solution using optical coherence tomography (OCT). Enamel blocks were cut from the bovine incisors and treated using one of the above-mentioned three materials or deionized water as control (n=10). All samples were subjected to a demineralization gel for 1 h followed by a remineralization solution for 23 h. This experimental cycle was repeated for 28 days. The specimens were imaged using OCT at baseline and at four stages and measured lesion depth using image analysis software (ImageJ). Repeated measures ANOVA revealed that demineralization time, material and their interaction significantly affected the optical lesion depth (p<0.001). TTCP and DCPA and 950 ppm fluoride paste and 950 ppm fluoride solution showed significantly lower lesion progress compare to other groups (p<0.05).

Effects of experimental pastes containing surface pre-reacted glass ionomer fillers on inhibition of enamel demineralization.

This study aimed to evaluate the inhibitory effect of experimental pastes containing surface pre-reacted glass ionomer (S-PRG) fillers on enamel demineralization. Bovine blocks were treated twice a day for 4 days by 7 groups; experimental pastes containing 0-30 wt% S-PRG filler (S00, S01, S05, S10, and S30), deionized water (DW) as negative control, and NaF paste (MP) as positive control. The surfaces were demineralized by acetic acid for 3 days. Mineral loss (ML) was calculated by micro-computed X-ray tomography. The treated surface was finally investigated with scanning electron microscope (SEM) and micro-focused particle induced X-ray emission (micro-PIXE). S05, S10 and S30 demonstrated significantly lower ML than S00, S01 and DW (p<0.05). S10 showed the greatest inhibitory effect, which was significantly greater than MP. The S-PRG filler containing experimental pastes demonstrated a potential to inhibit enamel demineralization. Sr ion incorporation was confirmed on the enamel surface with the experimental pastes.

A pilot study to assess the morphology and progression of non-carious cervical lesions.

OBJECTIVE: This longitudinal pilot study aimed to morphologically and quantitatively investigate the progress of non-carious cervical lesions (NCCLs) by using swept-source optical coherence tomography (SS-OCT). METHODS: The samples examined comprised sets of NCCL epoxy resin replicas obtained from 10 lesions in 6 patients who attended annual dental visits over 4 or 5 years. SS-OCT images of the replicas were analyzed in terms of the maximum depth (Dmax) and corresponding vertical width (VW) - using an image analyzer to estimate progression of the NCCLs over time. RESULTS: It was found that differences between wedge- and saucer-shaped lesions were morphologically distinguished well by the OCT images. There were significant differences in dimensions among Dmax, VW and horizontal width (HW). HW was the largest and Dmax was the smallest. Although no significant differences in absolute values of annual progression rates were found among Dmax, VW and HW, the percentage increase in Dmax was significantly greater compared to VW and HW. The ratios of Dmax to corresponding VW ranged from 0.49 to 1.01 for the wedge-shaped lesions and from 0.13 to 0.44 for saucer-shaped lesions, respectively. CONCLUSIONS: The dimensional analysis demonstrated notable progression with large variations. The wedge-shaped lesions appeared to show greater Dmax values compared to the saucer-shaped lesions. CLINICAL SIGNIFICANCE: With respect to the depth, the wedge-shaped lesions may progress at a greater rate compared to the saucer-shaped lesions.

A comparative study of the susceptibility of cut and uncut enamel to erosive demineralization.

This study aimed to evaluate the susceptibility of cut and uncut enamel surfaces to an erosive challenge and to examine the resultant characteristics/morphological changes. Ten extracted human incisors were used for preparation of enamel samples, and samples were immersed in citric acid. After 3 (total 3 min) and 6 cycles (total 6 min) of erosive challenges, surface loss (SL) and morphological changes were measured using scanning microscopy and FIB-TEM. Ca release (CA) and surface hardness (SH) were measured using a calcium-sensitive electrode and hardness tester respectively. Mean values of all measurements were statistically analyzed by using a t-test. Uncut enamel samples had significantly lower SL and greater SH than cut enamel (p<0.01). Cut enamel samples after 3 cycles showed higher CA compared with those from uncut enamel samples (p<0.05). Cut enamel was shown to be more susceptible to acidic dissolution and deeper acid penetration than uncut enamel after erosive demineralization.

Effects of zinc fluoride on inhibiting dentin demineralization and collagen degradation in vitro: A comparison of various topical fluoride agents.

Root caries is developed because of demineralization followed by enzymatic collagen degradation. This in vitro study aimed to examine the inhibitory efficacy of ZnF2 on dentin demineralization and collagen degradation. Bovine dentin specimens were treated either with ZnF2 or HCl-acidified ZnF2 (ZnF2/HCl) and then demineralized. Anti-demineralization efficacy was assessed by TMR as mineral loss (ΔZ). The efficacy was compared with silver diammine fluoride (SDF), KF, and acidulated phosphate fluoride (APF). For evaluating anti-collagen degradation, EDTA-demineralized dentin specimens were treated by one of four fluoride agents [SDF, APF, ZnF2/HCl, NaF] followed by collagenase challenge. The eroded depth of collagen layer in the lesion was assessed using optical microscope. ΔZ of SDF, KF, ZnF2/HCl, and APF were significantly lower compared with ZnF2 and Control (no treatment). Regarding anti-collagen degradation, SDF and ZnF2/HCl demonstrated a significant difference in the eroded depth compared with Control. Although SDF possessed higher efficacy, ZnF2/HCl might be beneficial as a staining-free agent.

Effects of brushing timing after erosive challenge on enamel loss in situ: White light interferometer and nanoindentation study.

This in situ study aimed to evaluate effects of waiting periods after erosive challenge before toothbrushing on enamel abrasion and nanoindentation hardness. Ten subjects wore intraoral appliances each with a set of 4 bovine enamel blocks. The enamel blocks were subjected to 2 cycles a day for 3 days as follows; intraoral exposure to form acquired pellicle and extraoral erosion followed by either 0, 3, 30 or 60 min intraoral exposure and then brushing, which was performed using an automatic brushing machine. Abrasive loss was assessed by white light interferometry. Nanoindentation was performed to calculate relative hardness. Abrasion and relative hardness were statistically analyzed by ANOVA. Abrasive loss was significantly less in groups exposed to saliva compared with 0 min (p<0.05); there was no significant difference between 30 and 60 min (p>0.05). Relative hardness was statistically higher after intraoral exposure, but no differences existed among any intraoral exposure periods (p>0.05).

Effect of thermal cyclic stress on acid resistance of resin-infiltrated incipient enamel lesions in vitro.

This study aimed to examine the effect of thermal cycling on gap formation at the interface between infiltrated resin (ICON(®)) and enamel lesion and on the durability of anti-demineralization efficacy to predict the future performance. SS-OCT technique was examined to determine whether it has the potential to detect the gap. Bovine enamel lesions were prepared, and the infiltrated resin was applied to the lesion. Resin-infiltrated lesion specimens were thermal cycled 10,000 cycles and further demineralized in pH 4.5 buffer for 7 days. Released Ca (mg/cm(2)) was quantified by Ca electrode. The SS-OCT technique was applied to detect the gap, and SEM observation was performed to determine the presence of the gap. There was no significant difference in the amount of Ca release before and after the thermal cycling, suggesting long-lasting anti-demineralization efficacy of the resin. SS-OCT and SEM observations indicated no apparent gap formation after the thermal cycling.

Effects of coating materials on nanoindentation hardness of enamel and adjacent areas.

OBJECTIVES: Materials that can be applied as thin coatings and actively release fluoride or other bioavailable ions for reinforcing dental hard tissue deserve further investigation. In this study we assessed the potential of resin coating materials in protection of underlying and adjacent enamel against demineralization challenge using nanoindentation. METHODS: Enamel was coated using Giomer (PRG Barrier Coat, PBC), resin-modified glass-ionomer (Clinpro XT Varnish, CXT), two-step self-etch adhesive (Clearfil SE Protect, SEP) or no coating (control). After 5000 thermal cycles and one-week demineralization challenge, Martens hardness of enamel beneath the coating, uncoated area and intermediate areas was measured using a Berkovich tip under 2mN load up to 200µm depth. Integrated hardness and 10-µm surface zone hardness were compared among groups. RESULTS: Nanoindentation and scanning electron microscopy suggested that all materials effectively prevented demineralization in coated area. Uncoated areas presented different hardness trends; PBC showed a remarkable peak at the surface zone before reaching as low as the control, while CXT showed relatively high hardness values at all depths. SIGNIFICANCE: Ion-release from coating materials affects different layers of enamel. Coatings with fluoride-releasing glass fillers contributed to reinforcement of adjacent enamel. Surface prereacted glass filler-containing PBC superficially protected neighboring enamel against demineralization, while resin-modified glass-ionomer with calcium (CXT) improved in-depth protection. Cross-sectional hardness mapping of enamel on a wide range of locations revealed minute differences in its structure.

In vitro dentine remineralization with a potential salivary phosphoprotein homologue.

OBJECTIVE: Advantages of introducing a salivary phosphoprotein homologue under standardized in vitro conditions to simulate the mineral-stabilizing properties of saliva have been proposed. This study longitudinally investigates the effects of casein, incorporated as a potential salivary phosphoprotein homologue in artificial saliva (AS) solutions with/without fluoride (F) on in vitro dentine lesion remineralization. DESIGN: Thin sections of bovine root dentine were demineralized and allocated randomly into 6 groups (n=18) having equivalent mineral loss (ΔZ) after transverse microradiography (TMR). The specimens were remineralized using AS solutions containing casein 0µg/ml, F 0ppm (C0-F0); casein 0µg/ml, F 1ppm (C0-F1); casein 10µg/ml, F 0ppm (C10-F0); casein 10µg/ml, F 1ppm (C10-F1); casein 100µg/ml, F 0ppm (C100-F0) or casein 100µg/ml, F 1ppm (C100-F1) for 28days with TMR taken every 7 days. RESULTS: Surface mineral precipitation, evident in group C0-F1, was apparently inhibited in groups with casein incorporation. Repeated measures ANOVA with Bonferroni correction revealed higher ΔZ for non-F and non-casein groups than for their counterparts (p<0.001). Subsequent multiple comparisons showed that mineral gain was higher (p<0.001) with 10µg/ml casein than with 100µg/ml when F was present in the earlier stages of remineralization, with both groups achieving almost complete remineralization after 28 days. CONCLUSION: Casein is a potential salivary phosphoprotein homologue that could be employed for in vitro dentine remineralization studies. Concentration related effects may be clinically significant and thus must be further examined.

Adsorption behavior of methacryloyloxydecyl dihydrogen phosphate on an apatite surface at neutral pH.

This study aimed to quantify the adsorption affinity of neutralized 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP-N) toward hydroxyapatite (HA) and dicalcium phosphate dihydrate (DCPD) at pH 7.0 by employing the Langmuir isotherm model. Furthermore, the effects of inorganic phosphate (Pi) and fluoride (F(-) ) ions on the adsorption of 10-MDP-N onto HA and DCPD were examined. Fixed amounts of HA and DCPD powders were suspended in different concentrations of 10-MDP-N solutions and were incubated for 18 h. Equilibrated concentrations of 10-MDP-N were measured by spectrophotometry and the adsorption affinity was estimated using the Langmuir model. Moreover, the adsorption was examined by zeta-potential analysis. The results indicated that significant Langmuir correlation was noted in both substrates, along with an increasing negative zeta-potential; however, in DCPD the correlation was less strong. The addition of 1.0 mM Pi slightly delayed the adsorption of 10-MDP-N onto both substrates, whereas 3.0 mM Pi drastically delayed adsorption onto HA but completely inhibited adsorption onto DCPD. Up to 50 ppm, F(-) enhanced the adsorption onto HA, and the adsorption plateaued at higher concentrations of F(-) , whereas no obvious influence of F(-) on the adsorption onto DCPD was noted.

Comparative study of demineralized collagen degradation determined by hydroxyproline assay and microscopic depth measurement.

INTRODUCTION: Quantification of collagen degradation is an important parameter to evaluate dentin caries progression or the efficacy of caries prevention aid. The aim of this study was to validate the simple light microscopic technique (LM) to evaluate collagen degradation by comparing with hydroxyproline assay technique (HPN). MATERIALS AND METHODS: Bovine root dentin blocks were embedded in acrylic resin, polished and covered with nail varnish except a 1.5 × 2.5mm window. The specimens were demineralized in acetate buffer (pH 4.3) for 3 days to create incipient lesions and were exposed to collagenase enzyme for 6, 9 and 16 h. The specimens were sectioned into thin sections (200-220 µm) to measure the degraded depth of collagen matrix by LM. The enzyme solutions were allocated to HPN assay using the simplified chloramines-T method. Correlation between LM and HPN was evaluated by Pearson correlation analysis. Anti-collagen degradation efficacy of 0.12% chlorhexidine (CHX) was evaluated by LM. RESULT: The depths of the degraded collagen and amount of hydroxyproline in 3 exposure periods were 27.8 ± 3.8 µm and 28.7 ± 4.2 µg for 6h, 48.1 ± 8.6 µm and 45.3 ± 6.1 µg for 9h, and 74.2 ± 9.7 µm and 71.3 ± 8.0 µg for 16 h, respectively. A significantly positive correlation (r=0.94, CI: 0.88-0.97, p<0.0001) was observed between LM and HPN and incubation time showed a linear correlation with amount of collagen degradation (R(2)=0.92). The CHX group (28.6 ± 3.3 µm) showed significantly lower collagen degradation than that of control group (53.1 ± 7.8 µm: p<0.01). CONCLUSION: The LM might be a reliable and simplified method to evaluate collagen degradation.

Impact of toothpaste on abrasion of sound and eroded enamel: An in vitro white light interferometer study.

PURPOSE: To evaluate the influence of brushing using toothpastes marketed under different categories on abrasion of sound and eroded enamel in vitro at nanometer scale using a white light interferometer (WLI). METHODS: Enamel surface of resin-embedded bovine incisors were fine polished with diamond slurry and divided into testing area (approximately 2 mm x 4 mm) and reference area using a nail varnish. The enamel specimens were randomly assigned to 10 groups (n = 10 each); six of which were subjected to erosive challenge. The testing area in these eroded groups was exposed to 10 ml of Coca-Cola for 90 seconds and then rinsed for 10 seconds in deionized water (DW). Enamel specimens, except for those in one eroded group, were brushed by an automatic brushing machine with 120 linear motion strokes in 60 seconds under load of 250 g with/without toothpaste slurry. After the toothbrushing abrasion, each specimen was rinsed for 10 seconds with DW followed by immersion in artificial saliva for 2 hours. Toothpaste slurries were prepared containing one of the four toothpastes used and DW in a ratio of 1:2. The erosion-abrasion cycle was repeated three times. Then, the nail varnish was removed and enamel surface loss (SL) was measured by the WLI. Data were statistically analyzed by one-way ANOVA followed by Bonferroni's correction at significance level of 0.05. RESULTS: For eroded specimens, the mean SL values of groups not brushed and brushed with no toothpaste were not significantly different, but were significantly lower than those of whitening, anti-erosion and anti-caries toothpaste groups (P < 0.001). The whitening toothpaste group showed significantly higher SL than all other groups (P < 0.001). For sound enamel specimens, SL was not measured except for the whitening toothpaste group.

Study on the influence of leucine-rich amelogenin peptide (LRAP) on the remineralization of enamel defects via micro-focus x-ray computed tomography and nanoindentation.

Regeneration of severely damaged enamel (e.g. deep demineralized lesions) is currently not possible, because the structural units of enamel crystal construction are removed after its maturation. The aim of this in vitro study was to evaluate the effect of surface impregnation by leucine-rich amelogenin peptide (LRAP) on the remineralization of eroded enamel using micro-focus x-ray computed tomography (µCT). Fifteen bovine enamel blocks were embedded in resin and three zones (sound, demineralization, and remineralization) were defined on each specimen. Lesions were prepared by immersing the samples in demineralization solution for 7 d. The samples were soaked in distilled water or 60 or 120 µg mL(-1) solution of LRAP in water for 30 min. After the surface treatment, specimens were incubated in artificial saliva for either 5 or 10 d at 37 °C. The amount of mineral gain (dΔZ%) and the relative changes in the lesion depth (dLD%), obtained from µCT, were used to evaluate the effect of LRAP on the remineralization of lesions. The effects of LRAP on cross-sectional integrated hardness ΔINH were studied after 10 d using nanoindentation. ANOVA test was used to determine the effect of time and/or LRAP concentration on dΔZ%, dLD% and ΔINH mean values. Tukey's analysis was used for multiple comparison testing (α = 0.05). Analysis of µCT data showed significant effect of time and LRAP concentration on the dΔZ% (p = 0.013, p = 0.003) and the dLD% (p < 0.001, p = 0.002) mean values. The nanoindentation hardness was significantly improved by 120 µg mL(-1) LRAP (p = 0.02). Also, the peptide treatment affected the mineral distribution throughout the lesion by inhibiting of superficial deposition. This study showed that the treatment of eroded lesions in enamel by LRAP can improve and regulate the pattern of remineralization in vitro.

Inhibition of hydroxyapatite growth by casein, a potential salivary phosphoprotein homologue.

Salivary phosphoproteins are essential in tooth mineral regulation but are often overlooked in vitro. This study aimed to evaluate the effect of casein, as a salivary phosphoprotein homologue, on the deposition and growth of hydroxyapatite (HA) on tooth surfaces. Hydroxyapatite growth was quantified using seeded crystal systems. Artificial saliva (AS) containing HA powder and 0, 10, 20, 50, or 100 µg ml(-1) of casein, or 100 µg ml(-1) of dephosphorylated casein (Dcasein), was incubated for 0-8 h at 37°C, pH 7.2. Calcium concentrations were measured using atomic absorption spectroscopy (AAS). Surface precipitation of HA on bovine enamel and dentine blocks, incubated in similar conditions for 7 d, was examined using field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) with selected area electron diffraction (SAED). Casein adsorption was assessed using modified Lowry assays and zeta-potential measurements. The AAS results revealed a concentration-dependent inhibition of calcium consumption. Hydroxyapatite precipitation occurred when no casein was present, whereas precipitation of HA was apparently completely inhibited in casein-containing groups. Adsorption data demonstrated increasingly negative zeta-potential with increased casein concentration and an affinity constant similar to proline-rich proteins with Langmuir modelling. Casein inhibited the deposition and growth of HA primarily through the binding of esterized phosphate to HA active sites, indicating its potential as a mineral-regulating salivary phosphoprotein homologue in vitro.

Evaluation of a new hardness tester (Cariotester): Comparison with transverse microradiography for assessing the inhibitory effect of fluoride application on bovine root dentin demineralization.

The objective of this study was to investigate the correlation between CT depth, indentation depth determined by a new hardness tester (Cariotester), and the transverse microradiography (TMR) parameters, i.e., lesion depth and mineral loss. For that purpose, this study evaluated the feasibility of using Cariotester as a root caries diagnostic system and capability of Cariotester to detect effect of fluoride application on inhibiting dentin demineralization. Fluorides were applied to bovine root dentin specimens, which were subsequently demineralized for 1-21 days and then CT depth and TMR parameters were assessed. There were significant correlations between CT depth and TMR parameters in fluoride and non-fluoride groups. There were significant differences between fluoride and non-fluoride groups for CT depth and TMR parameters respectively. Current results suggested that Cariotester may be capable of providing an objective evaluation of root caries progression and the fluoride effect on inhibiting dentin demineralization.

Measurement of surface hardness of primary carious lesions in extracted human enamel -measurement of Knoop hardness using Cariotester.

The clinical feasibility of a novel device called a Cariotester was investigated by measuring the Knoop hardness (KHN) of white spot lesions diagnosed as ICDAS code 1, 2 or 3. To obtain an equation for converting the Cariotester indentation depth into the KHN, a regression analysis was performed between the depth and measured KHN for human enamel. The Cariotester was then used to measure the indentation depth for white spots (ICDAS code 1, 2 or 3) in extracted teeth, and the KHN values were determined using the above equation. The KHN was 219.9±19.7, 162.4±24.0 and 31.7±17.5 for code 1, 2 and 3 lesions, respectively, which was 30, 49 and 90% lower than that for healthy enamel. Using the formula reported in the literature, the mineral density was calculated to be 87.7 vol.% for healthy enamel, and 75.1, 66.1 and 35.5 vol.% for code 1, 2 and 3 lesions, respectively.

Optical coherence tomography for evaluation of enamel and protective coatings.

Optical coherence tomography (OCT) is an interferometric imaging technique. This study aimed to employ OCT to evaluate four different resin-based materials including a coating containing glass-ionomer filler and calcium, a giomer, and two fluoride-releasing self-etch resins. The coating and its underlying and adjacent enamel were monitored using swept-source OCT (center wavelength: 1330 nm) at baseline, after 5,000 thermal cycles, and after 1, 4 and 7 days of demineralization (pH 4.5). The coatings showed different thicknesses (60-250 micrometers) and various levels of structural and interfacial integrity. OCT could detect a demineralization inhibition zone adjacent to the edge of the fluoride- and calcium-releasing material. Localized demineralization was occasionally observed under thinner coatings. Protection of susceptible enamel surfaces by thin resin-based bioactive coatings provides protection from demineralization. OCT can be used to non-destructively monitor the integrity of such coatings, as well as enamel changes beneath and adjacent to them.

Immobilization of phosphate monomers on collagen induces biomimetic mineralization.

BACKGROUND: Immobilization of phosphoproteins on type-I collagen via covalent binding may induce extra- and intrafibrillar mineralization. OBJECTIVE: This study tested the hypothesis that methacrylate phosphate esters immobilized on reconstituted type-I collagen can mimic the nucleating role of phosphoproteins. METHODS: Three functional monomers (MDP, GPDM and Phenyl-P) that differed in chemical structure and steric hindrances around the phosphate moiety were evaluated. Reconstituted type-I collagen was either left untouched (control) or treated by 5% monomer/ethanol for 20 s. All samples were incubated in simulated dentinal fluid as mineralizing medium at 37°C for 7 or 14 days. The extra- and intrafibrillar mineralization were examined by SEM and TEM/SAED crystallography, respectively. RESULTS: FT-IR spectroscopy showed that the phosphate groups were incorporated on reconstituted collagen, irrespective of their chemical structure. MDP immobilization induced dense growth of extrafibrillar mineral over time, while with GPDM- and Phenyl-P-immobilized collagen, mineralization was moderate and sparse, respectively. TEM/SAED evidence disclosed that intrafibrillar minerals exclusively occurred in MDP-immobilized collagen. CONCLUSIONS: Immobilization of MDP, which had the lowest steric hindrance, could induce significant biomimetic extra- and intrafibrillar mineralization; resembling the lowest level of hierarchy organization of dentin.