STMP and PVPA as Templating Analogs of Noncollagenous Proteins Induce Intrafibrillar Mineralization of Type I Collagen via PCCP Process.

The phosphorylated noncollagenous proteins (NCPs) play a vital role in manipulating biomineralization, while the mechanism of phosphorylation of NCPs in intrafibrillar mineralization of collagen fibril has not been completely deciphered. Poly(vinylphosphonic acid) (PVPA) and sodium trimetaphosphate (STMP) as templating analogs of NCPs induce hierarchical mineralization in cooperation with indispensable sequestration analogs such as polyacrylic acid (PAA) via polymer-induced liquid-like precursor (PILP) process. Herein, STMP-Ca and PVPA-Ca complexes are proposed to achieve rapid intrafibrillar mineralization through polyelectrolyte-Ca complexes pre-precursor (PCCP) process. This strategy is further verified effectively for remineralization of demineralized dentin matrix both in vitro and in vivo. Although STMP micromolecule fails to stabilize amorphous calcium phosphate (ACP) precursor, STMP-Ca complexes facilely permeate into intrafibrillar interstices and trigger phase transition of ACP to hydroxyapatite within collagen. In contrast, PVPA-stabilized ACP precursors lack liquid-like characteristic and crystallize outside collagen due to rigid conformation of PVPA macromolecule, while PVPA-Ca complexes infiltrate into partial intrafibrillar intervals under electrostatic attraction and osmotic pressure as evidenced by intuitionistic 3D stochastic optical reconstruction microscopy (3D-STORM). The study not only extends the variety and size range of polyelectrolyte for PCCP process but also sheds light on the role of phosphorylation for NCPs in biomineralization.

Citrate Improves Biomimetic Mineralization Induced by Polyelectrolyte-Cation Complexes Using PAsp-Ca&Mg Complexes.

Magnesium ions are highly enriched in early stage of biological mineralization of hard tissues. Paradoxically, hydroxyapatite (HAp) crystallization is inhibited significantly by high concentration of magnesium ions. The mechanism to regulate magnesium-doped biomimetic mineralization of collagen fibrils has never been fully elucidated. Herein, it is revealed that citrate can bioinspire the magnesium-stabilized mineral precursors to generate magnesium-doped biomimetic mineralization as follows: Citrate can enhance the electronegativity of collagen fibrils by its absorption to fibrils via hydrogen bonds. Afterward, electronegative collagen fibrils can attract highly concentrated electropositive polyaspartic acid-Ca&Mg (PAsp-Ca&Mg) complexes followed by phosphate solution via strong electrostatic attraction. Meanwhile, citrate adsorbed in/on fibrils can eliminate mineralization inhibitory effects of magnesium ions by breaking hydration layer surrounding magnesium ions and thus reduce dehydration energy barrier for rapid fulfillment of biomimetic mineralization. The remineralized demineralized dentin with magnesium-doped HAp possesses antibacterial ability, and the mineralization mediums possess excellent biocompatibility via cytotoxicity and oral mucosa irritation tests. This strategy shall shed light on cationic ions-doped biomimetic mineralization with antibacterial ability via modifying collagen fibrils and eliminating mineralization inhibitory effects of some cationic ions, as well as can excite attention to the neglected multiple regulations of small biomolecules, such as citrate, during biomineralization process.

Reversion of ACP Nanoparticles into Prenucleation Clusters via Surfactant for Promoting Biomimetic Mineralization: A Physicochemical Understanding of Biosurfactant Role in Biomineralization Process.

Amphiphilic biomolecules are abundant in mineralization front of biological hard tissues, which play a vital role in osteogenesis and dental hard tissue formation. Amphiphilic biomolecules function as biosurfactants, however, their biosurfactant role in biomineralization process has never been investigated. This study, for the first time, demonstrates that aggregated amorphous calcium phosphate (ACP) nanoparticles can be reversed into dispersed ultrasmall prenucleation clusters (PNCs) via breakdown and dispersion of the ACP nanoparticles by a surfactant. The reduced surface energy of ACP@TPGS and the electrostatic interaction between calcium ions and the pair electrons on oxygen atoms of C-O-C of D-α-tocopheryl polyethylene glycol succinate (TPGS) provide driving force for breakdown and dispersion of ACP nanoparticles into ultrasmall PNCs which promote in vitro and in vivo biomimetic mineralization. The ACP@TPGS possesses excellent biocompatibility without any irritations to oral mucosa and dental pulp. This study not only introduces surfactant into biomimetic mineralization field, but also excites attention to the neglected biosurfactant role during biomineralization process.

Remineralization of Dentin with Cerium Oxide and Its Potential Use for Root Canal Disinfection.

Objective: This study was to investigate a novel antibacterial biomimetic mineralization strategy for exploring its potential application for root canal disinfection when stabilized cerium oxide was used. Material and Methods: A biomimetic mineralization solution (BMS) consisting of cerium nitrate and dextran was prepared. Single-layer collagen fibrils, collagen membranes, demineralized dentin, and root canal system were treated with the BMS for mineralization. The mineralized samples underwent comprehensive characterization using various techniques, including transmission electron microscopy (TEM), high-resolution TEM (HRTEM), Fourier transform infrared spectroscopy (FTIR), scanning transmission electron microscopy (STEM), selected-area electron diffraction (SAED), energy-dispersive X-ray spectroscopy (EDX), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and micro-CT. Additionally, the antimicrobial properties of the BMS and the remineralized dentin were also analyzed with broth microdilution method, live/dead staining, and SEM. Results: Cerium ions in the BMS underwent a transformation into cerium oxide nanoparticles, which were deposited in the inter- and intra-fibrillar collagen spaces through a meticulous bottom-up process. XPS analysis disclosed the presence of both Ce (III) and Ce (IV) of the generated cerium oxides. A comprehensive examination utilizing SEM and micro-CT identified the presence of cerium oxide nanoparticles deposited within the dentinal tubules and lateral canals of the root canal system. The BMS and remineralized dentin exhibited substantial antibacterial efficacy against E. faecalis, as substantiated by assessments involving the broth dilution method and live/dead staining technique. The SEM findings revealed the cell morphological changes of deceased E. faecalis. Conclusion: This study successfully demonstrated antibacterial biomimetic mineralization as well as sealing dentinal tubules and lateral branches of root canals using cerium nitrate and dextran. This novel biomimetic mineralization could be used as an alternative strategy for root canal disinfection.

Intrafibrillar mineralization of type I collagen with calcium carbonate and strontium carbonate induced by polyelectrolyte-cation complexes.

Calcium carbonate (CaCO3), possessing excellent biocompatibility, bioactivity, osteoconductivity and superior biodegradability, may serve as an alternative to hydroxyapatite (HAp), the natural inorganic component of bone and dentin. Intrafibrillar mineralization of collagen with CaCO3 was achieved through the polymer-induced liquid precursor (PILP) process for at least 2 days. This study aims to propose a novel pathway for rapid intrafibrillar mineralization with CaCO3 by sequential application of the carbonate-bicarbonate buffer and polyaspartic acid (pAsp)-Ca suspension. Fourier transform infrared (FTIR) spectroscopy, zeta potential measurements, atomic force microscopy/Kelvin probe force microscopy (AFM/KPFM), and three-dimensional stochastic optical reconstruction microscopy (3D STORM) demonstrated that the carbonate-bicarbonate buffer significantly decreased the surface potential of collagen and CO32-/HCO3- ions could attach to collagen fibrils via hydrogen bonds. The electropositive pAsp-Ca complexes and free Ca2+ ions are attracted to and interact with CO32-/HCO3- ions through electrostatic attractions to form amorphous calcium carbonate that crystallizes gradually. Moreover, like CaCO3, strontium carbonate (SrCO3) can deposit inside the collagen fibrils through this pathway. The CaCO3-mineralized collagen gels exhibited better biocompatibility and cell proliferation ability than SrCO3. This study provides a feasible strategy for rapid collagen mineralization with CaCO3 and SrCO3, as well as elucidating the tissue engineering of CaCO3-based biomineralized materials.

Remineralizing effects of hydroxypropyl methylcellulose film-loaded amorphous calcium phosphate nanoprecursors on enamel artificial caries lesions.

OBJECTIVES: This study was to investigate hydroxypropyl methylcellulose (HPMC) film as a carrier for amorphous fluorinated calcium phosphate (AFCP) nanoprecursors to continuously deliver biomimetic remineralization of enamel artificial caries lesions (ACL). MATERIALS AND METHODS: The AFCP/HPMC films were comprised of 25 wt% AFCP nanoparticles and 75 wt% HPMC. They were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and biocompatibility tests. Forty enamel ACL were prepared and randomly divided into four groups (n = 10): The enamel surfaces were covered with a pure HPMC film, Tooth Mousse Plus (contains 10% CPP-ACP and 0.2% NaF), and AFCP/HPMC film, or without any things (serving as negative control). Subsequently, all samples were alternatively kept in artificial saliva and a modified pH-cycling before they were characterized by Micro-CT, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), attenuated total reflectance (ATR)-FTIR, XRD, and nanoindentation. RESULTS: After the enamel ACL was challenged by pH cycling, Tooth Mousse Plus and AFCP/HPMC film groups exhibited less lesion depth and mineral loss than the negative control and pure HPMC film groups. Additionally, the AFCP/HPMC film group revealed a highest remineralization rate of 55.34 ± 3.10 % among the all groups (p < 0.001). The SEM findings showed that the enamel ACL were densely deposited with minerals in the AFCP/HPMC film group, and the EDX results suggested a higher content of fluorine in the remineralized tissues. In particular, the AFCP/HPMC film group exhibited the best nanomechanical performance after 2 weeks of pH cycling (p < 0.05), with the hardness (H) restored from 0.29 ± 0.19 to 2.69 ± 0.70 GPa, and elastic modulus (Er) restored from 10.77 ± 5.30 to 68.83 ± 12.72 GPa. CONCLUSION: The AFCP/HPMC film might be used as a promising strategy for arresting or reversing incipient enamel caries lesions.

Polyelectrolyte-Cation Complexes Using PAsp-Sr Complexes Induce Biomimetic Mineralization with Antibacterial Ability.

Remineralized dentin with an antibacterial ability is still a significant challenge in dentistry. Previously, a polyelectrolyte-calcium complexes pre-precursor (PCCP) process is proposed for rapid collagen mineralization. In the present study, the expansion concept of the PCCP process is explored by replacing the calcium with other cations, such as strontium. The results of transmission electron microscopy (TEM), 3D stochastic optical reconstruction microscopy, energy-dispersive X-ray analysis, Fourier transform infrared spectroscopy, and high-resolution TEM with selected area electron diffraction demonstrate that biomimetic mineralization of collagen fibrils and demineralized dentin could be fulfilled with Sr&F-codoped hydroxyapatite (HAp) after they are treated with poly-aspartic acid-strontium (PAsp-Sr) suspension followed by a phosphate&fluoride solution. Moreover, dentin remineralized with Sr&F-codoped HAp exhibits in vitro and in vivo antibacterial ability against Streptococcus mutans. The cytotoxicity and oral mucosa irritation tests reveal excellent biocompatibility of mineralization mediums (PAsp-Sr suspension and phosphate&fluoride solution). The demineralized dentin's mechanical properties (elastic modulus and microhardness) could be restored almost to that of the intact dentin. Hence, the expansion concept of the PCCP process that replaces calcium ions with some cationic ions along with fluorine opens up new horizons for generating antibacterial remineralized dentin containing ions-doped HAp with excellent biocompatibility via biomimetic mineralization technology.

In-Depth Occlusion of Dentinal Tubules and Rapid Remineralization of Demineralized Dentin Induced by Polyelectrolyte-Calcium Complexes.

Dentin hypersensitivity (DH) is triggered by external stimuli irking fluid flow through exposed dentinal tubules (DTs). Three commercially available desensitizing agents as control in this study only achieve limited occlusion depths of ≈10 µm in the DTs as well as scarce remineralization of demineralized dentin matrix. Herein, polyelectrolyte-calcium complexes pre-precursor (PCCP) process is proposed for managing DH that demineralized dentin with exposed DTs is rubbed with ultrahighly concentrated polyelectrolyte-calcium suspension (4 g L-1 -5.44 m) followed by phosphate solution (3.25 m), each 10 min, leading to heavy remineralization of demineralized dentin and compact occlusion of the DTs over 200 µm after 1 day of in vitro and in vivo incubation. For the first time, it is demonstrated that the PCCP process relies on the pH-dependent electrostatic attraction between electropositive polyelectrolyte-calcium complexes and electronegative inwalls of DTs comprised of collagen fibrils and hydroxyapatite crystals under alkaline condition. The PCCP process might shed light on a promising dentin desensitizing strategy for DH management via rapid in-depth DT occlusion and remineralization of demineralized dentin.

Hydroxypropylmethylcellulose as a film and hydrogel carrier for ACP nanoprecursors to deliver biomimetic mineralization.

Demineralization of hard tooth tissues leads to dental caries, which cause health problems and economic burdens throughout the world. A biomimetic mineralization strategy is expected to reverse early dental caries. Commercially available anti-carious mineralizing products lead to inconclusive clinical results because they cannot continuously replenish the required calcium and phosphate resources. Herein, we prepared a mineralizing film consisting of hydroxypropylmethylcellulose (HPMC) and polyaspartic acid-stabilized amorphous calcium phosphate (PAsp-ACP) nanoparticles. HPMC which contains multiple hydroxyl groups is a film-forming material that can be desiccated to form a dry film. In a moist environment, this film gradually changes into a gel. HPMC was used as the carrier of PAsp-ACP nanoparticles to deliver biomimetic mineralization. Our results indicated that the hydroxyl and methoxyl groups of HPMC could assist the stability of PAsp-ACP nanoparticles and maintain their biomimetic mineralization activity. The results further demonstrated that the bioinspired mineralizing film induced the early mineralization of demineralized dentin after 24 h with increasing mineralization of the whole demineralized dentin (3-4 µm) after 72-96 h. Furthermore, these results were achieved without any cytotoxicity or mucosa irritation. Therefore, this mineralizing film shows promise for use in preventive dentistry due to its efficient mineralization capability.

A Hydroxypropyl Methylcellulose Film Loaded with AFCP Nanoparticles for Inhibiting Formation of Enamel White Spot Lesions.

OBJECTIVE: This study investigated the effects of mineralizing film consisting of hydroxypropyl methylcellulose (HPMC) and amorphous fluorinated calcium phosphate (AFCP) nanoparticles on enamel white spot lesions (WSLs). MATERIAL AND METHODS: The AFCP nanoparticles and mineralizing film were prepared via nanoprecipitation and solvent evaporation, respectively. They were characterized with Fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), transmission electron microscopy (TEM), selected area electron diffraction (SAED), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), inductively coupled plasma atomic emission spectrometry (ICP-AES), and fluoride ion selective electrode. Thirty-two human enamel slices (4 mm × 4 mm × 1.5 mm) were highly polished and randomly assigned to four groups: negative control (no treatment); pure HPMC film; mineralizing film; GC Tooth Mousse Plus® (contains 10% CPP-ACP and 0.2% NaF). Subsequently, samples were challenged by a modified pH-cycling and characterized by color measurement, Micro-CT, SEM/EDX, and nanoindentation. RESULTS: The mineralizing film could sustain release of Ca, P and F ions over 24 h and maintain AFCP nanoparticles in metastable state over 8~12 h. During 4 weeks of pH cycling, the mineralizing film group exhibited least color change (∆E), mineral loss and lesion depth (120 ± 10 µm) among four groups (p < 0.05). SEM findings revealed that the porosities among enamel crystals increased in negative control and pure HPMC film groups after pH cycling, whereas in mineralizing film group, the original microstructure of enamel was well conserved and mineral deposits were detected between enamel prisms. Mineralizing film group demonstrated a least reduction of nanomechanical properties such as elastic modulus of 77.02 ± 6.84 GPa and hardness of 3.62 ± 0.57 GPa (p < 0.05). CONCLUSION: The mineralizing film might be a promising strategy for prevention and management of WSLs via inhibiting enamel demineralization and promoting enamel remineralization.

Separated root tip formation associated with a fractured tubercle of dens evaginatus: A case report.

BACKGROUND: Several previous studies have reported an unusual root formation in which a fractured apical fragment of an immature root continued to develop independent of the main root after trauma to an immature tooth. To date, there have been only rare reports of the continuing apical formation of the fractured root associated with dens evaginatus (DE). This paper presents a case of a separated root tip formation associated with a fractured tubercle of DE. CASE SUMMARY: An 11-year-old boy was referred for gingival sinus on the buccal side of the right mandibular second premolar (tooth # 45). Clinically, tooth # 45 was free of caries, but there was a sign of a fractured tubercle of DE on the occlusal surface. Radiography showed that the root canal of tooth # 45 was widely radiolucent. A separated root apex was found apically under the main root and was nearly completely formed with an apical orifice at the apical tip. Tooth # 45 was diagnosed as tubular fracture of DE with chronic apical periodontitis. A revascularization technique was recommended to treat the tooth. At 3-mo and 1-yr follow-up, the patient remained asymptomatic. Periapical radiography revealed that the separated root tip distally drifted with closure of the apex. However, the root length and thickness of the main root did not increased. CONCLUSION: Clinicians should be aware that even if tubercle of DE is fractured in an immature tooth, the root tip may be separated from the main root and completely formed.

The effect of prime-and-rinse approach using MDP micellar solutions on extrafibrillar demineralization and dentin bond performance.

OBJECTIVE: This study investigated the effects of prime-and-rinse approach using 10-methacryloyloxydecyl dihydrogen phosphate (MDP) micellar solutions on extrafibrillar demineralization and dentin bond performance of etch-and-rinse adhesive. METHODS: The micellar solutions were prepared by adding 15% MDP in two ethanol-aqueous (75:25, 55:45 V/V%) solutions, referring to MDP/EtOH75 and MDP/EtOH55. After mid-coronal dentin surfaces were either etched (control) or conditioned with MDP/EtOH75 and MDP/EtOH55 and rinsed, they were applied with adhesive (Adpter Single Bond 2) in dry- or wet-bonding mode and placed with composite resin (Filtek Z350 XT). They were prepared into multiple micro-beams for micro-tensile bond strengths (MTBS) testing after storage in water for 24 h or subjecting to thermocycling. The other pretreated dentin surfaces were analyzed by TF-XRD, ATR-FTIR, HRTEM, FE-SEM, contact angle measurement and nanoindentation testing. The MTBS data was analyzed with two-way ANOVA followed by LSD post-hoc test. RESULTS: MDP/EtOH75 produced significantly greater MTBS values than MDP/EtOH55 and control after thermocycling aging in dry- or wet-bonding mode (P < 0.05). The ATR-FTIR spectrums shows that ratios of phosphate/monomer (1,034 cm-1/1,716 cm-1) on MDP/EtOH75-, MDP/EtOH55-treated dentin surfaces are 0.51 and 0.23, respectively. This is confirmed by HRTEM images and SAED pattern that intrafibrillar minerals were mostly preserved after treatment with MDP/EtOH75. MDP/EtOH75 produced significantly higher elastic modulus and nanohardness on pretreated dentin surface than MDP/EtOH55 (P < 0.05). TF-XRD pattern shows some MDP-Ca salts remained on the primed dentin surface. SIGNIFICANCE: Prime-and-rinse approach using MDP/EtOH75 micellar solution could produce mostly extrafibrillar demineralization, and greatly increase dentin bond durability in dry- or wet-bonding mode.

Biomimetic fabrication and application of fibrous-like nanotubes.

AIMS: To investigate the biomimetic fabrication of fibrous-like organic-inorganic hybrid structures via a simple bottom-up approach, viz. self-assembly of simple molecules, and apply fibrous-like composites as a novel primer to improve dentin bond strengths of self-etch adhesives. MATERIALS AND METHODS: The resultants of commercial amorphous calcium phosphate (ACP) nanoparticles and 10-methacryloyloxydecyl dihydrogen phosphate (MDP) ethanol-aqueous solution were analyzed by TEM, SEM, XRD, DLS and AFM. The acid and alkali resistance of abovementioned self-assembled composites were analyzed with TEM. Micro-tensile bond strengths (MTBS) tests were performed after polished dentin surfaces were pretreated with self-assembled composites. The pretreated dentin surfaces and dentin-resin interfaces were characterized by SEM/TEM. KEY FINDINGS: ACP nanoparticles in MDP solution could self-assemble into fibrous-like nanotube structures in 8 nm diameter. Self-assembly and self-proliferation process went from ACP nanoparticles, dissolved ACP nanoparticles (less than 50 nm), twig-like structures and fibrous-like nanotubes to cellular networks. The fibrous-like nanotubes were only detected when the amount of ACP in reaction system were more than 0.01 g. The more ACP interacted with MDP, the more fibrous-like nanotubes were formed. After the dentin surfaces were treated with fibrous-like nanotube composites, MTBS could be significantly improved. Moreover, the fibrous-like nanotube structures could be resistant to acidic challenge, and were stable at least for 3 months. SIGNIFICANCE: The fibrous-like nanotube structures could be self-assembled via a bottom-up approach at certain ratio of MDP and commercial ACP nanoparticles. The application of fibrous-like nanotube composites as a novel primer prior to self-etch adhesives greatly improved dentin bond strengths.

Polyelectrolyte-calcium complexes as a pre-precursor induce biomimetic mineralization of collagen.

Polyelectrolytes such as polyaspartic acid (PAsp) are critical in biomimetic mineralization as stabilizers of amorphous calcium phosphate (ACP) precursors and as nucleation inhibitors similar to non-collagenous proteins (NCPs). Nevertheless, the application of polyelectrolyte-calcium complexes as a pre-precursor, such as PAsp-Ca complexes, in the mineralization of collagen is unexplored. Herein, we propose a polyelectrolyte-Ca complex pre-precursor (PCCP) process for collagen mineralization. By combining three-dimensional (3D) STORM, potential measurements, and cryogenic transmission electron microscopy with molecular dynamics simulations, we show that liquid-like electropositive PAsp-Ca complexes along with free calcium ions infiltrate electronegative collagen fibrils. The PAsp-Ca complexes are immobilized within the fibrils via chelation and hydrogen bonds, and outward movement of free calcium ions is prevented while phosphate and hydroxide are recruited through electrostatic attractions. Afterwards, ACP instantly forms and gradually crystallizes. The PCCP process not only unites two distinct crystallization pathways (classical (free Ca/P ions) and non-classical (polyelectrolyte-Ca complexes)), but also provides a novel strategy for rapid biomimetic mineralization of collagen.

A novel fluorescent adhesive-assisted biomimetic mineralization.

We propose a novel fluorescent adhesive-assisted biomimetic mineralization strategy, based on which 1 wt% of sodium fluorescein and 25 wt% of polyacrylic acid stabilized amorphous calcium phosphate (PAA-ACP) nanoparticles were incorporated into a mild self-etch adhesive (Clearfil S3 Bond) as a fluorescent mineralizing adhesive. The characterization of the PAA-ACP nanoparticles indicates that they were spherical particles clustered together, each particle with a diameter of approximately 20-50 nm, in a metastable phase with two characteristic absorption peaks (1050 cm-1 and 580 cm-1). Our results suggest that the fluorescent mineralizing adhesive was non-cytotoxic with minimal esthetic interference and its fluorescence intensity did not significantly decrease within 6 months. Our data reveal that the fluorescent mineralizing adhesive could induce the extra- and intra-fibrillar remineralization of the reconstituted type I collagen, the demineralized enamel and dentin substrate. Our data demonstrate that a novel fluorescent adhesive-assisted biomimetic mineralization strategy will pave the way to design and produce anti-carious materials for the prevention of dental caries.

Effects of regional enamel and prism orientations on bovine enamel bond strength and cohesive strength.

This study investigated the regional microtensile bond strength (MTBS) and cohesive strength of bovine enamel. The crowns of bovine incisors were sectioned, either horizontally along incisal and cervical thirds to produce horizontal and tangential segments, or longitudinally along the midline to produce longitudinal segments. Half of the horizontal and longitudinal segments were prepared using a 45° bevel. Then, the differently sectioned enamel surfaces were treated with one- or two-step self-etch adhesives (Clearfil SE Bond or Clearfil S3 Bond) and a composite resin (Clearfil Majesty) was placed. Resin-bonded enamel samples were cut into beams for use in the MTBS tests. Labial horizontal and longitudinal segments of pure enamel beams were prepared for cohesive strength tests. Enamel microstructures were analyzed by scanning electron microscopy. Three-way anova followed by Tukey's post-hoc HSD multiple comparisons procedure showed that a 45° bevel cut did not statistically significantly improve enamel MTBS, which varied with the different regions. The longitudinally sectioned resin-bonded enamel samples had the lowest MTBS, and the horizontal enamel cohesive strength was weaker than that of the longitudinal enamel. The scanning electron microscopy fractographs indicated that rows of parallel prisms were detached from the fractured surfaces. In conclusion, the regional enamel MTBS and the cohesive strength are strongly related to the enamel microstructures and prism orientations.