The Role of Process-Directing Agents on Enamel Lesion Remineralization: Fluoride Boosters.

The aim of this study was to investigate the effects of two process-directing agents (polyaspartic acid and osteopontin) used in a polymer-induced liquid-precursor (PILP) process on the remineralization of bacteria-induced enamel demineralization. Enamel demineralization lesions (depths of about 180-200 µm) were created and exposed to Streptococcus mutans, cultured with a 10% sucrose solution for 21 days, and remineralized using a PILP process (pH = 7.4, 14 days) with a calcium phosphate solution containing either polyaspartic acid or osteopontin in the presence or absence of fluoride (0.5 ppm). The specimens were examined under scanning electron microscopy. The fluoride was successfully incorporated into the PILP remineralization process for both polyaspartic acid and osteopontin. When the fluoride was added to the PILP remineralization solution, there was more uniform remineralization throughout the lesion than with either polyaspartic acid or osteopontin alone. However, in the absence of these process-directing agents, fluoride alone showed less remineralization with the formation of a predominantly surface-only layer. The PILP remineralization process relies on the ability of process-directing agents to stabilize calcium phosphate ions and holds promise for enamel lesion remineralization, and these agents, in the presence of fluoride, seem to play an important role as a booster or supplement in the continuation of remineralization by reducing the mineral gains at the surface layer.

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.

The Academy of Dental Materials: Providing roots and wings.

OBJECTIVES: The long history of the Academy of Dental Materials (ADM) is documented with its strategies (a) to rapidly communicate science among its members, (b) to establish special awards to stimulate new science, and (c) to develop new dental materials scientists. METHODS: We searched the history of the last 35 years of the ADM newsletters, transactions, journals, and officer notes. We document the (a) presidents, (b) meeting history, (c) membership growth, and (d) development of special awards through 2019 with the recent creation of the ADM Marshall Post-Doctoral Award. RESULTS: There are 36 years of recent ADM history, 42 international meetings, membership growth to 400 individuals from 15 countries, service of 19 presidents, Paffenbarger annual Awardees since 1989, induction of >200 fellows, and recognition of the first winner of Marshall Post-Doctoral Award in 2018. New directions for recruiting members are suggested. Three potential new thrusts for the organization are presented: artificial intelligence, genetic engineering, and intensive member mentoring. SIGNIFICANCE: These suggestions for the ADM provide a path for the ADM to continue to adapt to the ever changing scientific landscape.

Remineralization of demineralized dentin using a dual analog system.

OBJECTIVE: Improved methods are needed to remineralize dentin caries in order to promote conservation of dentin tissue and minimize the surgical interventions that are currently required for clinical treatment. Here, we test the hypothesis that bulk substrates can be effectively mineralized via a dual analog system proposed by others, using a tripolyphosphate (TPP) "templating analog" and a poly(acrylic acid) (PAA) or poly(aspartic acid) (pAsp) "sequestration analog," the latter of which generates the polymer-induced liquid-precursor (PILP) mineralization process studied in our laboratory. MATERIAL & METHODS: Demineralized human dentin slices were remineralized with and without pre-treatment with TPP, using either PAA or pAsp as the PILP process-directing agent. A control experiment with no polymer present was used for comparison. RESULTS: No mineralization was observed in any of the PAA groups. In both the pAsp and no polymer groups, TPP inhibited mineralization on the surfaces of the specimens but promoted mineralization within the interiors. Pre-treatment with TPP enhanced overall mineralization of the pAsp group. However, when analysed via TEM, regions with little mineral were still present. CONCLUSION: Poly(acrylic acid) was unable to remineralize demineralized dentin slices under the conditions employed, even when pre-treated with TPP. However, pre-treatment with TPP enhanced overall mineralization of specimens that were PILP-remineralized using pAsp.

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.

Influence of fluoride on the mineralization of collagen via the polymer-induced liquid-precursor (PILP) process.

OBJECTIVE: The polymer-induced liquid-precursor (PILP) mineralization process has been shown to remineralize artificial dentin lesions to levels consistent with those of native dentin. However, nanoindentation revealed that the moduli of those remineralized lesions were only ∼50% that of native dentin. We hypothesize that this may be due to the PILP process having been previously optimized to obtain high amounts (∼70wt%) of intrafibrillar crystals, but without sufficient interfibrillar mineral, another significant component of dentin. METHODS: Fluoride was added to the PILP-mineralization of collagen from rat tail tendon at varying concentrations to determine if a better balance of intra- versus inter-fibrillar mineralization could be obtained, as determined by electron microscopy. Nanoindentation was used to determine if fluoridated apatite could improve the mechanical properties of the composites. RESULTS: Fluoride was successfully incorporated into the PILP-mineralization of rat tail tendon and resulted in collagen-mineral composite systems with the mineral phase of hydroxyapatite containing various levels of fluoridation. As the fluoride concentration increased, the crystals became larger and more rod-like, with an increasing tendency to form on the fibril surfaces rather than the interior. Nanomechanical testing of the mineralized tendons revealed that fluoride addition did not increase modulus over PILP mineralization alone. This likely resulted from the separated nature of collagen fibrils that comprise tendon, which does not provide lateral reinforcement and therefore may not be suited for the compressive loads of nanoindentation. SIGNIFICANCE: This work contributes to the development of minimally invasive approaches to caries treatment by determining if collagen can be functionally mineralized.

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.

A novel approach for effective integration of new faculty leadership.

PURPOSE: We report on an accelerated and effective way of assimilating a new leader into a team at a large academic dental school department. METHODS: At University of California, San Francisco (UCSF), a new Chair was recruited through a national search to lead its largest department in the School of Dentistry. Two months after arrival, the new Chair embarked on a process of leadership assimilation among her executive team, facilitated by a professional consultant. Within four weeks, team members participated in one-on-one interviews with the professional facilitator consultant and then completed the leadership assimilation questionnaire and returned it electronically to the facilitator. The facilitator then summarized all answers into themes and met with the team members without the Chair to debrief. Thereafter, the facilitator met with the Chair to discuss the major themes. Next, the Chair met with the team members in a facilitated session to discuss the results and negotiate a path forward. RESULTS: Approximately half of the feedback described the "how" of leadership: comments on communication, building relationships, building trust, and understanding UCSF history. The remaining half described the "what": comments on vision, strategy, and operations. Team members indicated that the first debriefing session was helpful to alleviate initial anxiety and to start building team spirit. The session with the Chair was perceived as open and fruitful in which team members were able to express their concerns and hopes for the Department, while the Chair showed commitment to the team and the communication process. CONCLUSION: Leader assimilation allows teams to share their expectations and anxieties with the new leader early in the relationship in an open way, before new habits and beliefs are formed. Conversely, for the leader, it effectively and efficiently allows a window into the team members' thinking at a critical time period when otherwise first impressions occur. With a safe space created for open communication, the process allowed siloed individual division leaders to move toward a cohesive group while at the same time solidifying a commitment to the success of the new leader.

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.

Effect of proteoglycans at interfaces as related to location, architecture, and mechanical cues.

INTRODUCTION: Covalently bound functional GAGs orchestrate tissue mechanics through time-dependent characteristics. OBJECTIVE: The role of specific glycosaminoglycans (GAGs) at the ligament-cementum and cementum-dentin interfaces within a human periodontal complex were examined. Matrix swelling and resistance to compression under health and modeled diseased states was investigated. MATERIALS AND METHODS: The presence of keratin sulfate (KS) and chondroitin sulfate (CS) GAGs at the ligament-cementum and cementum-dentin interfaces in human molars (N=5) was illustrated by using enzymes, atomic force microscopy (AFM), and AFM-based nanoindentation. The change in physical characteristics of modeled diseased states through sequential digestion of keratin sulfate (KS) and chondroitin sulfate (CS) GAGs was investigated. One-way ANOVA tests with P<0.05 were performed to determine significant differences between groups. Additionally, the presence of mineral within the seemingly hygroscopic interfaces was investigated using transmission electron microscopy. RESULTS: Immunohistochemistry (N=3) indicated presence of biglycan and fibromodulin small leucine rich proteoglycans at the interfaces. Digestion of matrices with enzymes confirmed the presence of KS and CS GAGs at the interfaces by illustrating a change in tissue architecture and mechanics. A significant increase in height (nm), decrease in elastic modulus (GPa), and tissue deformation rate (nm/s) of the PDL-C attachment site (215±63-424±94nm; 1.5±0.7-0.4±0.2GPa; 21±7-48±22nm/s), and cementum-dentin interface (122±69-360±159nm; 2.9±1.3-0.7±0.3GPa; 18±4-30±6nm/s) was observed. CONCLUSIONS: The sequential removal of GAGs indicated loss in intricate structural hierarchy of hygroscopic interfaces. From a mechanics perspective, GAGs provide tissue recovery/resilience. The results of this study provide insights into the role of GAGs toward conserved tooth movement in the socket in response to mechanical loads, and modulation of potentially deleterious strain at tissue interfaces.

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.

Mineral density volume gradients in normal and diseased human tissues.

Clinical computed tomography provides a single mineral density (MD) value for heterogeneous calcified tissues containing early and late stage pathologic formations. The novel aspect of this study is that, it extends current quantitative methods of mapping mineral density gradients to three dimensions, discretizes early and late mineralized stages, identifies elemental distribution in discretized volumes, and correlates measured MD with respective calcium (Ca) to phosphorus (P) and Ca to zinc (Zn) elemental ratios. To accomplish this, MD variations identified using polychromatic radiation from a high resolution micro-computed tomography (micro-CT) benchtop unit were correlated with elemental mapping obtained from a microprobe X-ray fluorescence (XRF) using synchrotron monochromatic radiation. Digital segmentation of tomograms from normal and diseased tissues (N=5 per group; 40-60 year old males) contained significant mineral density variations (enamel: 2820-3095 mg/cc, bone: 570-1415 mg/cc, cementum: 1240-1340 mg/cc, dentin: 1480-1590 mg/cc, cementum affected by periodontitis: 1100-1220 mg/cc, hypomineralized carious dentin: 345-1450 mg/cc, hypermineralized carious dentin: 1815-2740 mg/cc, and dental calculus: 1290-1770 mg/cc). A plausible linear correlation between segmented MD volumes and elemental ratios within these volumes was established, and Ca/P ratios for dentin (1.49), hypomineralized dentin (0.32-0.46), cementum (1.51), and bone (1.68) were observed. Furthermore, varying Ca/Zn ratios were distinguished in adapted compared to normal tissues, such as in bone (855-2765) and in cementum (595-990), highlighting Zn as an influential element in prompting observed adaptive properties. Hence, results provide insights on mineral density gradients with elemental concentrations and elemental footprints that in turn could aid in elucidating mechanistic processes for pathologic formations.

The plastic nature of the human bone-periodontal ligament-tooth fibrous joint.

This study investigates bony protrusions within a narrowed periodontal ligament space (PDL-space) of a human bone-PDL-tooth fibrous joint by mapping structural, biochemical, and mechanical heterogeneity. Higher resolution structural characterization was achieved via complementary atomic force microscopy (AFM), nano-transmission X-ray microscopy (nano-TXM), and microtomography (MicroXCT™). Structural heterogeneity was correlated to biochemical and elemental composition, illustrated via histochemistry and microprobe X-ray fluorescence analysis (µ-XRF), and mechanical heterogeneity evaluated by AFM-based nanoindentation. Results demonstrated that the narrowed PDL-space was due to invasion of bundle bone (BB) into PDL-space. Protruded BB had a wider range with higher elastic modulus values (2-8GPa) compared to lamellar bone (0.8-6GPa), and increased quantities of Ca, P and Zn as revealed by µ-XRF. Interestingly, the hygroscopic 10-30µm interface between protruded BB and lamellar bone exhibited higher X-ray attenuation similar to cement lines and lamellae within bone. Localization of the small leucine rich proteoglycan biglycan (BGN) responsible for mineralization was observed at the PDL-bone interface and around the osteocyte lacunae. Based on these results, it can be argued that the LB-BB interface was the original site of PDL attachment, and that the genesis of protruded BB identified as protrusions occurred as a result of shift in strain. We emphasize the importance of bony protrusions within the context of organ function and that additional study is warranted.

Combinatorial effect of Si4+, Ca2+, and Mg2+ released from bioactive glasses on osteoblast osteocalcin expression and biomineralization.

Osteocalcin (OCN) expression is an essential osteogenic marker of successful bone regeneration therapies. This study hypothesizes that Si(4+) and Ca(2+) combinatorial released by bioactive glass enhance osteoblast biomineralization through up-regulation of OCN expression; and Mg(2+) release delays such enhancement. Osteoblasts (MC3T3-E1) were treated with ionic products of bioactive glass dissolution (6P53-b experimental bioactive glass and 45S5 commercial Bioglass™). Results showed that gene expressions, including OCN and its up-stream transcription factors (Runx2, ATF4, MSX1, SP7/OSX), growth factors and signaling proteins (BMP2, BMP6, SMAD3), were enhanced in both 45S5 and 6P53-b glass conditioned mediums (GCMs). This up-regulation led to enhanced mineral formation by 45S5 glass conditioned mediums ([GCM], Si(4+)+Ca(2+)) after 20 days, and by 45S5 GCM and 6P53-b GCM (Si(4+)+Ca(2+)+Mg(2+)) after 30 days. In examining the extracellular matrix generated by cells when exposed to each GCM, it was found that 45S5 GCM had slightly elevated levels of mineral content within ECM as compared to 6P53-b GCM after 30 days while control treatments exhibited no mineral content. The formation of well-defined mineralized nodules (distinct PO4(3-) [960 cm(-1)] and CO3(2-) [1072 cm(-1)] peaks from Raman Spectra) was observed for each GCM as the soluble glass content increased. In examining the individual and combined ion effects between Si(4+), Ca(2+), and Mg(2+), it was found Mg(2+) down-regulates OCN expression. Thus, ions released from both 45S5 and 6P53-b bioactive glasses up-regulate OCN expression and biomineralization while 6P53-b GCM Mg(2+) release down-regulated OCN expression and delayed osteoblast biomineralization. These results indicate that Si(4+), Ca(2+), and Mg(2+) combinatorially regulate osteoblast OCN expression and biomineralization.

Analysis of interfacial structure and bond strength of self-etch adhesives.

PURPOSE: To determine the bond strength, nanoleakage and interfacial morphology of four self-etch adhesives bonded to superficial dentin. METHODS: Microtensile (MT) (n= 15) and single plane shear (SP) (n= 8) bond tests were performed using human dentin polished through 320-grit SiC paper. Clearfil Protect Bond (PB), Clearfil S3 Bond (S3), Prompt L-Pop (PLP) and G-Bond (GB) were used according to their manufacturers' instructions. Composite was applied as cylinders with a thickness of 4 mm with a 1 mm diameter and stored in water at 370C for 24 hours. Specimens were debonded with a testing machine at a cross-head speed of 1 mm/minute. Means and standard deviations of bond strength were calculated. Data were analyzed using ANOVA. Fisher's PLSD intervals were calculated at the 0.05 level of significance. Failure modes were determined at x100. The hybrid layer was revealed by treatment with 5N HC1/5% NaOCl or fractured perpendicular to the interface and sputter coated with gold. Specimens were viewed at x1,000, x2,500, and x5,000 in a field emission SEM at 15 kV. Teeth (n=2) sectioned into 0.9 mm-thick slabs were immersed in ammoniacal silver nitrate solution for 24 hours, rinsed and immersed in photo-developing solution for 8 hours. Specimens were sectioned (90 nm-thick) and observed under TEM. RESULTS: Means ranged from 25.0 to 73.1 MPa for MT and from 15.5 to 56.4 MPa for SP. MT values were greater than SP, but were highly correlated (R2 = 0.99, P= 0.003) and provided the same order for the systems studied. Fisher's PLSD intervals (P< 0.05) for bond strength techniques and adhesives results were 1.7 and 2.3 MPa, respectively. Failures sites were mixed. TEM showed that hybrid layers were -0.5 pm for PB, GB and S3 and approximately 5 microm for PLP. SEM showed morphologic differences among adhesives. Silver nitrate deposits were observed within the interfaces for all adhesive systems.

Si and Ca individually and combinatorially target enhanced MC3T3-E1 subclone 4 early osteogenic marker expression.

This study tests the hypothesis that silicon and calcium ions combinatorially target gene expression during osteoblast differentiation. MC3T3-E1 subclone 4 osteoblast progenitors (transformed mouse calvarial osteoblasts) were exposed to Si(4+) (from Na(2)SiO(3)) and Ca(2+) (from CaCl(2):H(2)O) ion treatments both individually (0.4 mM each + control treatment) and combinatorially (0.4 mM Si(4+) + 0.4 mM Ca(2+) + control treatment) and compared to control treated (α-minimum essential medium, 10% fetal bovine serum, and 1% penicillin-streptomycin) cells. Cell proliferation studies showed no significant increase in cell density between treatments over 5 days of culture. Cellular differentiation studies involved addition of ascorbic acid (50 mg/L) for all treatments. Relative gene expression was determined for collagen type 1 (Col(I)α1/Col(I)α2), core-binding factor a (cbfa1/Runx2), and osteocalcin (OCN), which indicated osteoblast progenitor differentiation into a mineralizing phenotype. Increased Si(4+) or Ca(2+) ion treatments enhanced Col(I)α1, Col(I)α2, Runx2, and OCN expression, while increased Si(4+) + Ca(2+) ion treatments enhanced OCN expression. Moreover, it was found that a Si(4+)/Ca(2+) ratio of unity was optimal for maximal expression of OCN. Collagen fiber bundles were dense, elongated, and thick within extracellular matrices (ECM) exposed to Si(4+) and Si(4+) + Ca(2+) treatments, while collagen fiber bundles were sparse, short, and thin within Ca(2+) and control treated ECM. These results indicated that individual ions enhance multiple osteogenic gene expression, while combined ion treatments enhance individual gene expression. In addition, these results indicated that Si(4+) enhanced osteoblast gene expression and ECM formation at higher levels than Ca(2+). These results support the larger concept that ions (possibly released from bioactive glasses) could control bone formation by targeting osteoblast marker expression.

Functional remineralization of dentin lesions using polymer-induced liquid-precursor process.

It was hypothesized that applying the polymer-induced liquid-precursor (PILP) system to artificial lesions would result in time-dependent functional remineralization of carious dentin lesions that restores the mechanical properties of demineralized dentin matrix. 140 µm deep artificial caries lesions were remineralized via the PILP process for 7-28 days at 37°C to determine temporal remineralization characteristics. Poly-L-aspartic acid (27 KDa) was used as the polymeric process-directing agent and was added to the remineralization solution at a calcium-to-phosphate ratio of 2.14 (mol/mol). Nanomechanical properties of hydrated artificial lesions had a low reduced elastic modulus (E(R) = 0.2 GPa) region extending about 70 µm into the lesion, with a sloped region to about 140 µm where values reached normal dentin (18-20 GPa). After 7 days specimens recovered mechanical properties in the sloped region by 51% compared to the artificial lesion. Between 7-14 days, recovery of the outer portion of the lesion continued to a level of about 10 GPa with 74% improvement. 28 days of PILP mineralization resulted in 91% improvement of E(R) compared to the artificial lesion. These differences were statistically significant as determined from change-point diagrams. Mineral profiles determined by micro x-ray computed tomography were shallower than those determined by nanoindentation, and showed similar changes over time, but full mineral recovery occurred after 14 days in both the outer and sloped portions of the lesion. Scanning electron microscopy and energy dispersive x-ray analysis showed similar morphologies that were distinct from normal dentin with a clear line of demarcation between the outer and sloped portions of the lesion. Transmission electron microscopy and selected area electron diffraction showed that the starting lesions contained some residual mineral in the outer portions, which exhibited poor crystallinity. During remineralization, intrafibrillar mineral increased and crystallinity improved with intrafibrillar mineral exhibiting the orientation found in normal dentin or bone.

Mechanical heterogeneity of dentin at different length scales as determined by AFM phase contrast.

In this study we sought to gain insights of the structural and mechanical heterogeneity of dentin at different length scales. We compared four distinct demineralization protocols with respect to their ability to expose the periodic pattern of dentin collagen. Additionally, we analyzed the phase contrast resulting from AFM images obtained in tapping mode to interrogate the viscoelastic behavior and surface adhesion properties of peritubular and intertubular dentin, and partially demineralized dentin collagen fibrils, particularly with respect to their gap and overlap regions. Results demonstrated that all demineralization protocols exposed the gap and overlap zones of dentin collagen fibrils. Phase contrast analyses suggested that the intertubular dentin, where the organic matrix is concentrated, generated a higher phase contrast due a higher contribution of energy dissipation (damping) than the highly mineralized peritubular region. At increasing amplitudes, viscoelasticity appeared to play a more significant contribution to the phase contrast of the images of collagen fibrils. The overlap region yielded a greater phase contrast than the more elastic gap zones. In summary, our results contribute to the perspective that, at different length scales, dentin is constituted of structural features that retain heterogeneous mechanical properties contributing to overall mechanical performance of the tissue. Furthermore, the interpretation of phase contrast from images generated with AFM tapping mode appears to be an effective tool to gain an improved understanding of the structure and property relationship of biological tissues and biomaterials at the micro- and nano-scale.