Polymer-Induced Liquid Precursor (PILP) remineralization of artificial and natural dentin carious lesions evaluated by nanoindentation and microcomputed tomography.

OBJECTIVES: The study evaluates the efficacy to remineralize artificial and natural dentin lesions through restorative dental procedures that include the Polymer-Induced Liquid Precursor (PILP) method comprising polyaspartic acid (pAsp). METHODS: Novel ionomeric cement compositions based on bioglass 45S5 and pAsp mixtures, as well as conditioning solutions (conditioner) containing 5 mg/mL pAsp, were developed and tested on demineralized dentin blocks (3-4 mm thick) on shallow and deep lesions with the thickness of 140 µm ± 50 and 700 µm ± 50, respectively. In the first treatment group, 20 µL of conditioner was applied to demineralized shallow (n = 3) and deep (n = 3) lesion specimens for 20 s before restoration with glass ionomer cement (RMGIC). For the PILP cement treatment group, cement was applied onto the wet surface of the demineralized specimen for both shallow (n = 3) and deep (n = 3) artificial lesions after the application of the conditioner and before the final restoration. Sample groups were compared to RMGIC restoration, for both shallow and deep lesions (n = 3 each) and treatments in PILP-solution (n = 3 for deep lesions) without restoration for 4 weeks. All of the restored specimens were immersed in simulated body fluid (SBF) solution for 2 weeks and 4 weeks for shallow and deep lesions respectively to allow for remineralization. The artificial lesion specimens were evaluated for changes in the nanomechanical profile (E-modulus and hardness) using nanoindentation. Shallow lesions were analyzed by SEM under vacuum for changes in morphology caused by PILP treatments. Also, a pilot study on human third molars with moderate lesions in dentin (n = 3) was initiated to test the efficacy of treatments in natural lesions based on mineral densities using microcomputed tomography (µCT) at 0, 1, and 3 months. RESULTS: This study showed that functional remineralization of artificial lesions using PILP-releasing restoratives occurred, indicated by an increase of the elastic modulus in shallow lesions and in the middle zone of deep artificial lesions. The mechanical improvement was significant when compared to RMGIC restoration without pAsp (P < 0.05). Nonetheless, recovery across artificial lesions was most significant when specimens were immersed into PILP-solution with restorative (P < 0.01). Furthermore, natural lesions increased in mineral volume content to a higher degree when the restorative treatment included the PILP-method (P < 0.05). However, none of the natural lesions recovered to full mineral degree regardless of the treatments. CLINICAL SIGNIFICANCE/CONCLUSION: These findings indicate the benefit of PILP applications in the functional repair of dentin caries and illustrate the challenge to integrate the PILP-method into a restorative approach in minimally invasive dental procedures.

Enhanced silver diamine fluoride therapy using the PILP method -A nanoindentation study.

The aim of this study was to evaluate the feasibility of applying the polymer-induced liquid-precursor (PILP) method to enhance silver diamine fluoride (SDF) therapy. One hundred forty micrometer deep artificial caries lesions were treated with (A) 38% SDF solution and (B) 38% SDF containing poly-L-aspartic acid (pASP). Changes in the nanomechanical profile across the lesion were evaluated. Hydrated artificial lesions had a low reduced elastic modulus (0.3 GPa) and nanohardness (0.02 GPa) region extending about 100 µm into the lesion, with a gradual linear increase to about 168 µm where the values plateaued to around 18 GPa/1.0 GPa. Topical application of SDF resulted in significantly recovered properties (p<0.001). SDF containing pASP resulted in greater nanomechanical properties compared to SDF alone, showing similar sloped regions up to 96 µm, then SDF alone dropped while SDF containing pASP continued at a modest slope until reaching normal at 144 µm. This nanoindentation study shows enhanced SDF therapy using the PILP method.

Integrating the PILP-mineralization process into a restorative dental treatment.

The addition of charged polymers, like poly-aspartic acid (pAsp), to mineralizing solutions allows for transport of calcium and phosphate ions into the lumen of collagen fibrils and subsequent crystallization of oriented apatite crystals by the so-called Polymer-Induced Liquid Precursor (PILP) mineralization process, leading to the functional recovery of artificial dentin lesions by intrafibrillar mineralization of collagen. OBJECTIVE: To evaluate the feasibility of applying the PILP method as part of a restorative treatment and test for effectiveness to functionally remineralize artificial lesions in dentin. MATERIALS AND METHODS: Two methods of providing pAsp to standardized artificial lesions during a restorative procedure were applied: (A) pAsp was mixed into commercial RMGI (resin modified glass ionomer) cement formulations and (B) pAsp was added at high concentration (25mg/ml) in solution to rehydrate lesions before restoring with a RMGI cement. All specimens were immersed in simulated body fluid for two weeks to allow for remineralization and then analyzed for dehydration shrinkage, integrity of cement-dentin interface, degree of mineralization, and changes in the nanomechanical profile (E-modulus) across the lesion. RESULTS: After the remineralization treatment, lesion shrinkage was significantly reduced for all treatment groups compared to demineralized samples. Pores developed in RMGI when pAsp was added. A thin layer at the dentin-cement interface, rich in polymer formed possibly from a reaction between pAsp and the RMGI. When analyzed by SEM under vacuum, most lesions delaminated from the cement interface. EDS-analysis showed some but not full recovery of calcium and phosphorous levels for treatment groups that involved pAsp. Nanoindentations placed across the interface indicated improvement for RMGI containing 40% pAsp, and were significantly elevated when lesions were rehydrated with pAsp before being restored with RMGI. In particular the most demineralized outer zone recovered substantially in the elastic modulus, suggesting that functional remineralization has been initiated by pAsp delivery upon rehydration of air-dried demineralized dentin. In contrast, the effectiveness of the RMGI on functional remineralization of dentin was minimal when pAsp was absent. SIGNIFICANCE: Incorporation of pAsp into restorative treatments using RMGIs promises to be a feasible way to induce the PILP-mineralization process in a clinical setting and to repair the structure and properties of dentin damaged by the caries process.

The role of protease inhibitors on the remineralization of demineralized dentin using the PILP method.

Mineralized and sound dentin matrices contain inactive preforms of proteolytic enzymes that may be activated during the demineralization cycle. In this study, we tested the hypothesis that protease inhibitors (PI) preserve demineralized collagen fibrils and other constituents of the dentin matrix and thereby affect the potential for remineralization. Artificial carious lesions with lesion depths of 140 µm were created with acetate buffer (pH = 5.0, 66 hours), and remineralized using a polymer-induced-liquid-precursor (PILP) process (pH = 7.4, 14 days) containing poly(aspartic acid) (pAsp) as the process-directing agent. De- and remineralizing procedures were performed in the presence or absence of PI. Ultrastructure and mechanical recovery of demineralized dentin following PILP remineralization were examined and measured in water with atomic force microscopy (AFM) and nanoindentation. Nanomechanical properties of hydrated artificial lesions had a low elastic modulus (ER <0.4 GPa) extending about 100 µm into the lesion, followed by a sloped region of about 140 µm depth where values reached those of normal dentin (18.0-20.0 GPa). Mapping of mineral content by both micro-FTIR and micro x-ray computed tomography correlated well with modulus profiles obtained by nanoindentation. Tissue demineralized in the presence of PI exhibited higher elastic moduli (average 2.8 GPa) across the lesion and comprised a narrow zone in the outer lesion with strongly increased modulus (up to 8 GPa; p < 0.05), which might be related to the preservation of non-collagenous proteins that appear to induce calcium phosphate mineral formation even under demineralizing physical-chemical conditions. However, mechanical aspects of remineralization through the elastic modulus change, and the micromorphological aspects with SEM and TEM observation were almost identical with PILP treatments being conducted in the presence or absence of PI. Thus, the application of the protease inhibitors (PI) seemed to be less effective in promoting the remineralization of demineralized dentin.

Using biomimetic polymers in place of noncollagenous proteins to achieve functional remineralization of dentin tissues.

In calcified tissues such as bones and teeth, mineralization is regulated by an extracellular matrix, which includes non-collagenous proteins (NCP). This natural process has been adapted or mimicked to restore tissues following physical damage or demineralization by using polyanionic acids in place of NCPs, but the remineralized tissues fail to fully recover their mechanical properties. Here we show that pre-treatment with certain amphiphilic peptoids, a class of peptide-like polymers consisting of N-substituted glycines that have defined monomer sequences, enhances ordering and mineralization of collagen and induces functional remineralization of dentin lesions in vitro. In the vicinity of dentin tubules, the newly formed apatite nano-crystals are co-aligned with the c-axis parallel to the tubular periphery and recovery of tissue ultrastructure is accompanied by development of high mechanical strength. The observed effects are highly sequence-dependent with alternating polar and non-polar groups leading to positive outcomes while diblock sequences have no effect. The observations suggest aromatic groups interact with the collagen while the hydrophilic side chains bind the mineralizing constituents and highlight the potential of synthetic sequence-defined biomimetic polymers to serve as NCP mimics in tissue remineralization.

Strontium effects on root dentin tubule occlusion and nanomechanical properties.

OBJECTIVES: Dentin hypersensitivity often is treated by promotion of dentin tubule occlusion. In this in vitro study we evaluated nanomechanical properties and degree of tubule occlusion conferred to sound and demineralized human root dentin following treatment with a 10% (w/w) strontium acetate solution and its relation to the treatment duration and delivery method. METHODS: 24 human cervical root dentin disks (8 groups of 3) were polished through 0.25 µm. 12 disks were subjected to an acid challenge (1% citric acid, pH 3.8) for 2 min. The specimens were incubated in artificial saliva, treated by soaking or brushing with deionized (DI) water or a solution of 10% strontium acetate for 2 min twice a day for 28 days. The occlusion percent and nanomechanical properties were determined at the baseline, 5, 14 and 28 days. Cross-sectioned specimens were prepared to evaluate the depth affected by strontium acetate / dentin interaction by SEM. Statistical analysis was performed using linear mixed effects models. RESULTS: A 10% strontium acetate treatment over 5-28 days significantly increased tubule occlusion for normal root dentin and to a lesser extent for demineralized dentin and increased the AFM based nanomechanical properties of demineralized dentin. Brushing was more effective than soaking in recovery of properties of demineralized dentin when treated with strontium. No difference in tubuleocclusion was found between the two delivery methods. SIGNIFICANCE: Strontium acetate itself proved to have the ability to occlude dentin tubules and result in small changes in the mechanical properties of dentin.

In vitro evaluation of adhesive characteristics of 4-META/MMA-TBB resin with organic filler.

OBJECTIVES: A commercial restorative material, BondfillSB (BF), is a modification of 4-META/MMA-TBB resin cement. BF uses a self-etching primer and added pre-polymerized organic fillers. We compared BF with another self-etching system, EasyBond (EB), in shear bond strength, bonded interface characteristics to human dentin and contraction gap when used in bulk-filling. METHODS: Shear bond strength of BF and EB + Z100 (Z), bonded by different experience-level operators, was evaluated. Bonded interfaces were characterized by SEM, AFM, and AFM based nano-indentation. Contraction gaps (CG) at 0h and 24h after polymerization were evaluated for BF or EB bulk filled class I cavities. To meet the clinical recommendation, BF's powder was replaced by experimental radioopaque powder (BFO) for the CG study. EB was used with Z (EBZ) or with a resin marketed for bulk-fill base (SureFil-SDR-flow (EBSF)). RESULTS: Shear bond strengths (Mean ± Standard Deviation (S.D.)) of BF (37.4 ± 2.6 MPa; n=36) were higher and less variable than EBZ (18.2 ± 7.6 MPa; n=36) (p<0.0001, One-way ANOVA). Weibull characteristic strength (η) differed significantly between materials (p<0.0001) but not between operators (p=0.90). EBZ often had non-uniform interfaces and a wider band of reduced elastic modulus (E) of greater than 20 µm across the interface. BF had uniform interfaces and a smaller width of affected dentin under the interface (∼1 µm). There was a difference in dentin-E between EBZ and BF up to 9 µm from the interface (mixed-effects model; P=0.03). A stratified linear regression model used for CG. EBSF and BFO showed significantly smaller CG than EBZ at time 0. None of three combinations showed any significant change between 0h-CG and 24h-CG. SIGNIFICANCE: BF possessed bonding characteristics required to serve as a restorative.

Effect of sterilization by gamma radiation on nano-mechanical properties of teeth.

OBJECTIVES: Extracted teeth used in dental research need to be considered infective and hence be sterilized without the materials' properties being altered. This study examined the effect of gamma radiation on the nano-mechanical properties of dentin and enamel of extracted human third molars. METHODS: Whole teeth were sterilized using gamma radiation doses of 7 kGy and 35 kGy, respectively; teeth of the control group were not treated with gamma radiation. Crowns were sectioned occlusally and polished. Elastic modulus and hardness were tested using atomic force microscopy with nano-indentations under wet conditions. RESULTS: The authors found no significant dose-response relationship in elastic modulus or hardness in either dentin or enamel. SIGNIFICANCE: Nano-indentation is a common technique for the determination of local mechanical properties in biological hard tissues. Gamma radiation is an efficient way to sterilize extracted teeth while alteration of dentin and enamel mechanical properties are minimized.

Elevated TGF-beta2 signaling in dentin results in sex related enamel defects.

UNLABELLED: Initiation of enamel formation requires reciprocal signaling between epithelially and mesenchymally derived cells. OBJECTIVE: In this study, we used a transgenic mouse model which drives overexpression of an activated form of TGF-beta2 under control of the osteocalcin promoter, to investigate the role of TGF-beta2 in the dental mesenchyme, on enamel formation. DESIGN: Dentin and enamel were imaged by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Dentin mechanical properties were characterized for hardness and elasticity, following nanoindentation with a modified AFM. Pores found in enamel were quantified and compared using image analysis software (Scion Imagetrade mark). RESULTS: The elastic modulus of dentin was significantly reduced in the male TGF-beta2 overexpressor mice as compared to male wildtype mice, with no significant differences between female mice. Similarly, there were significantly more pores in enamel of the male transgenic mice as compared to male wildtype mice, with no significant differences between female mice. In situ hybridization of the continuously erupting incisor confirmed that osteocalcin expression was limited to the odontoblast cell layer at all stages of tooth formation. CONCLUSION: TGF-beta2 overexpression in the dentin matrix, results in sex-linked differences in dentin and enamel formation.