NANOG priming before full reprogramming may generate germ cell tumours.

Reprogramming somatic cells into a pluripotent state brings patient-tailored, ethical controversy-free cellular therapy closer to reality. However, stem cells and cancer cells share many common characteristics; therefore, it is crucial to be able to discriminate between them. We generated two induced pluripotent stem cell (iPSC) lines, with NANOG pre-transduction followed by OCT3/4, SOX2, and LIN28 overexpression. One of the cell lines, CHiPS W, showed normal pluripotent stem cell characteristics, while the other, CHiPS A, though expressing pluripotency markers, failed to differentiate and gave rise to germ cell-like tumours in vivo. Comparative genomic hybridisation analysis of the generated iPS lines revealed that they were genetically more stable than human embryonic stem cell counterparts. This analysis proved to be predictive for the differentiation potential of analysed cells. Moreover, the CHiPS A line expressed a lower ratio of p53/p21 when compared to CHiPS W. NANOG pre-induction followed by OCT3/4, SOX2, MYC, and KLF4 induction resulted in the same tumour-inducing phenotype. These results underline the importance of a re-examination of the role of NANOG during reprogramming. Moreover, this reprogramming method may provide insights into primordial cell tumour formation and cancer stem cell transformation.

Ex vivo assessment of irrigant penetration and renewal during the cleaning and shaping of root canals: a digital subtraction radiographic study.

AIM: To assess radiographically irrigant penetration in the root canal system of curved roots during root canal shaping procedures ex vivo. METHODOLOGY: Thirty extracted mandibular molar teeth with moderate to severe curvature were used. A special aiming device was used to guarantee that each successive radiograph was taken with the same positioning. The mesiolingual canal of each tooth was instrumented using the ProTaper system. For each step of the shaping procedure, two irrigation modalities were repeated in the same order. Active irrigation consisted of a 0.5-mL flush of sodium diatrizoate solution (Hypaque 50%) immediately followed by agitation with a size 08 K-file. Passive irrigation consisted of a 0.5-mL flush of sodium hypochlorite solution delivered with a syringe through a 27-gauge notched tip needle. A digital radiograph was taken after each modality and stored on computer for subsequent digital subtraction and measures of the depths of irrigant penetration. Comparisons were performed within an analysis-of-variance framework in a repeated-measures approach. RESULTS: The penetration of irrigants was significantly greater for each successive step of the shaping procedure when the two modalities were analysed separately (P < 0.001). The difference between the two modalities was statistically significant for each step of the shaping procedure (P < 0.0001). CONCLUSIONS: Shaping root canals improved both penetration and exchange of irrigant inside the root canal system. Complete renewal of the solution was impossible to achieve with a conventional syringe delivery system and a limited volume of solution. Recapitulation with a K-file after flushing improved irrigant penetration.

Ex vivo assessment of irrigant penetration and renewal during the final irrigation regimen.

AIM: To assess irrigant penetration in curved canals after shaping procedures ex vivo. METHODOLOGY: Thirty extracted mandibular molars with moderate to severe root canal curvature were included. A special aiming device was used to guarantee that each successive radiograph was taken with the same positioning. The mesiolingual canal of each tooth was instrumented with ProTaper rotary files. Apical third preparation was completed with an F1 instrument before additional step-back enlargement using F2 then F3 instruments. For each apical taper, the teeth were submitted successively to active irrigation (AI) then to passive irrigation (PI). AI consisted of a 0.5 mL flush of sodium diatrizoate (Hypaque 50%) immediately followed by manual mechanical activation with a gutta-percha point. PI consisted in flushing the canals with sodium hypochlorite passively delivered with a syringe. A digital radiograph was taken after each modality. The influence of needle tip design, needle tip insertion level, irrigant volume, root canal taper and solution activation was assessed by using digital subtraction radiography and measures of the depths of irrigant penetration. Comparisons were performed within an analysis of variance framework in a repeated-measures approach. RESULTS: For PI, all the four explanatory variables 'apical taper', 'volume of irrigant used', 'corono-apical level of needle tip placement', 'needle tip design' had a significant (P < 0.005) influence on outcome of irrigation penetration. CONCLUSIONS: Only active irrigation allowed complete penetration and exchange of irrigating solution. For syringe irrigation alone, the level of needle tip placement in the canal was the most dominating factor.

[Cognitive impact of mitoxantrone and methylprednisolone in multiple sclerosis: an open label study]. / Impact cognitif d'un traitement associant mitoxantrone et méthylprednisolone dans la sclérose en plaques.

Interferons beta have shown some positive effects on cognitive function in multiple sclerosis (MS). The potential immunosuppressive impact of mitoxantrone on cognitive dysfunction in MS has never been evaluated. We assessed changes in cognitive dysfunction in patients with very active MS treated with mitoxantrone combined with methylprednisolone. We assessed a non randomized controlled trial including successively 15 consecutive MS patients. Very active MS was defined by a progression of at least two EDSS points or more than two relapses during the previous year and at least one enhanced lesion after gadolinium infusion on MRI. All patients received a monthly intravenous pulse of mitoxantrone (20mg) for six months with methylprednisolone (1g). Global cognitive efficiency, memory and executive function were assessed before treatment (M0) and after six months (M6) and 12 months (M12) of treatment. To evaluate the learning effect, 15 healthy subjects also participated. A significant improvement in global cognitive efficiency was observed at M6 and was sustained at M12, as a few parameters on memory and executive functions. We suggest that mitoxantrone combined with methylprednisolone has a potential positive effect on cognitive functions.

Cytotoxicity of endodontic materials over 6-weeks ex vivo.

AIM: To test the hypothesis that extending the time of a traditional ex vivo cytotoxicity test helps to identify trends in the behaviour of root core materials and sealers, which could ultimately aid in predicting their clinical safety and performance. METHODOLOGY: Endodontic sealers and core specimens were initially tested in direct contact with L929 fibroblasts for 72 h. Cell response was estimated by measuring cellular succinate dehydrogenase activity relative to Teflon controls. Cytotoxicity (% of more active cells) was reassessed after 1, 3, 4 and 6 weeks, with the specimens stored in a physiologically balanced salt-solution between tests. RESULTS: Distinct trends in cytotoxicity among both core materials and sealers were observed over the 6-week test. Four of the six sealers and two of the three core materials showed cell viabilities of <30% of Teflon after 6 weeks (>70% cytotoxicity). CONCLUSIONS: The current results suggest that some endodontic materials have an elevated biological risk for extended intervals.

Long-term sealing ability of Pulp Canal Sealer, AH-Plus, GuttaFlow and Epiphany.

AIM: To evaluate the long-term sealing ability of four contemporary endodontic sealers [Pulp Canal Sealer (PCS), AH-Plus, GuttaFlow and Epiphany] using a fluid filtration technique. METHODOLOGY: The palatal roots of 40 human maxillary molar teeth were selected and the root canal was prepared using a crown-down technique (apical size 40, 6% taper). Roots were irrigated with 3% NaOCl, 17% EDTA solution and rinsed with distilled water. Canals were filled with either PCS, AH-Plus, GuttaFlow or Epiphany using a single-cone technique (n = 8). Twenty-four hours after filling, the roots were connected to an automatic flow-recording device (Flodec System) filled with double-distilled water under pressure (0.2 bar) to measure leakage. Flow rates were assessed at 6, 12 or 24-h and after 1-year of storage. RESULTS: None of the materials fully prevented fluid flow. Fluid flow decreased after 6 h and decreased further after 12 h. After 24 h, PCS and AH-Plus allowed significantly more fluid flow than GuttaFlow and Epiphany. After 1 year, PCS allowed significantly more fluid flow than the other materials. No significant changes in leakage occurred between 24 h and 1 year. CONCLUSIONS: GuttaFlow and Epiphany allowed less fluid movement along filled straight roots.

In vitro cytotoxicity of dental composites based on new and traditional polymerization chemistries.

UNLABELLED: The biological response to dental restorative polymer composites is mediated by the release of unpolymerized residual monomers. Several new composite formulations claim to reduce unpolymerized residual mass. The current study assessed the cytotoxic responses to several of these new formations and compared them with more traditional formulations. Our hypothesis predicted that if these new polymerization chemistries reduce unpolymerized residual mass, the cytotoxicity of these materials also should be reduced relative to traditional formulations. METHODS: Materials (HerculiteXRV, Premise, Filtek Supreme, CeramxDuo, Hermes, and Quixfil) were tested in vitro in direct contact with Balb mouse fibroblasts, initially, then after aging in artificial saliva for 0, 1, 3, 5, or 8 weeks. The toxicity was determined by using the MTT assay to the estimate SDH activity. Knoop hardness of the materials also was measured at 0 and 8 weeks to determine whether surface breakdown of the materials in artificial saliva contributed to cytotoxic responses. RESULTS: Materials with traditional methacrylate chemistries (Herculite, Premise, Filtek Supreme) were severely (>50%) cytotoxic throughout the 8-week interval, but materials with newer chemistries or filling strategies (Hermes, CeramXDuo, and Quixfil) improved over time of aging in artificial saliva. Hermes showed the least cytotoxicity at 8 weeks, and was statistically equivalent to Teflon negative controls. Hardness of the materials was unaffected by exposure to artificial saliva. CONCLUSIONS: Newer polymerization and filling strategies for dental composites show promise for reducing the release of unpolymerized components and cytotoxicity.

Dynamics of composite polymerization mediates the development of cuspal strain.

OBJECTIVES: In the current study, we used electronic speckle pattern interferometry (ESPI) to measure tooth deformation in response to polymerization of five resin composites with a range of polymerization shrinkage. Our hypothesis was that composites with higher polymerization shrinkage should cause more cuspal strain as measured by ESPI. METHODS: Standardized MOD cavities were prepared and placed into the ESPI apparatus before the cavities were filled with composites (n=10). The ESPI apparatus was constructed to measure the out-of-plane displacement of the lingual cusps of the teeth during the polymerization of the restorative material. A thermocouple was attached to the specimen to monitor thermal changes throughout the polymerization process. RESULTS: Experiments with empty preparations demonstrated that the ESPI technique was temporally responsive and sensitive to dimensional changes. However, the correlation between polymerization shrinkage of composite resins and ESPI-measured tooth deformation was not straightforward. In particular, a flowable material did not deform the tooth significantly more that a conventional hybrid. Further, an experimental silorane material (with the lowest axial shrinkage) induced the least tooth deformation. SIGNIFICANCE: We concluded that ESPI is a viable method for assessing cuspal strain induced by shrinkage of bonded composite restorations, but that polymerization shrinkage data may overestimate shrinkage-induced tooth deformation. The rate of polymerization shrinkage appeared to mediate the development of cuspal strain.

One-year cyclophosphamide treatment combined with methylprednisolone improves cognitive dysfunction in progressive forms of multiple sclerosis.

We conducted an evaluation of changes in cognition in progressive multiple sclerosis (MS) patients receiving monthly intravenously pulse of cyclophosphamide (700 mg/m2) with methylprednisolone (1g). Twenty-eight consecutive progressive MS patients (10 primary progressive, 18 secondary progressive MS) were evaluated before and after six and 12 months of treatment. The WAIS-R score, memory and executive functions were evaluated. Under treatment we found a significant improvement in global cognitive efficiency, encoding abilities, planning abilities and inhibition after six and 12 months. However, mechanisms of action of the positive effect of these anti-inflammatory and immunosuppressive treatments on cognition remain unclear.

Single-bottle adhesives behave as permeable membranes after polymerization. I. In vivo evidence.

OBJECTIVES: This study tested the hypothesis that single-bottle total-etch adhesives are effective in reducing dentine permeability under in vivo conditions. METHODS: Crown preparations on vital human teeth were performed under local analgesia as part of the treatment plan for prosthetic rehabilitation. Four single-bottle adhesives (Single Bond, 3M ESPE; Excite DSC, Ivoclar Vivadent; Prime and Bond NT Dual-Cure, Dentsply DeTrey and One-Step, Bisco Inc.) were applied to the cut dentine after acid-etching. Polyvinyl siloxane impressions were taken, using an ultra-low viscosity impression material, of the smear layer-covered dentine before applying the adhesives to deep vital dentine, and after adhesive placement. Additional impressions were taken of the adhesive-sealed dentine following the removal of the provisional prostheses after a 7-10 day period. Epoxy resin replicas of the crown preparations were examined with scanning electron microscopy to evaluate the extent of dentinal fluid transudation during pre-bonded, immediately bonded and post-bonded periods. RESULTS: Dentinal fluid transudation from localised areas that were close to the dental pulp was universally observed from all epoxy resin replicas, irrespective of the adhesive employed. The transudation of dentinal fluid from the control smear layer-covered dentine of each crown preparation was comparatively mild when compared to the extent that was observed after total-etching and application of the single-bottle adhesive. Dentinal fluid droplets were specifically located over the surface of the adhesive layer. Continuous transudation of dentinal fluid occurred even after the removal of the provisional prostheses, and was considerably more profuse in subjects who opted for the fitting of the permanent prostheses without the use of a local anaesthetic solution. CONCLUSION: Single-bottle adhesives, because of their lack of a comparatively more hydrophobic bonding resin layer, behave as permeable membranes after polymerisation. They permit the continuous transudation of dentinal fluid and do not provide a hermetic seal in vital deep dentine. Although the relatively slow rate of diffusion of dentinal fluid is unlikely to result in post-operative cold sensitivity, it may interfere with the optimal polymerisation of dual-cured or auto-cured composites or resin cements in both direct and indirect restorations.

Future directions in bonding resins to the dentine-pulp complex.

Resin-based materials are rapidly becoming the primary restorative material to replace tooth structure and the low percentage of biological problems reported for resin-based restorations is testimony to their relative biocompatibility. Despite considerable advances in the understanding of the mechanisms, which govern the integration of resin composite materials with the dentine-pulp complex, considerable potential for improvement remains. Most of these improvements centre around a better understanding of the biological and microbiological risks of resin materials and will only be possible with improved in vitro and in vivo models. There are also a number of possible future developments in materials, clinical techniques and diagnostic methods that may improve the long-term success and reliability of resin-based restorations. The remainder of this paper describes the most likely avenues for future improvements.

Blue light differentially modulates cell survival and growth.

Previous studies have reported that blue light (400-500 nm) inhibits cell mitochondrial activity. We investigated the hypothesis that cells with high energy consumption are most susceptible to blue-light-induced mitochondrial inhibition. We estimated cell energy consumption by population doubling time, and cell survival and growth by succinate dehydrogenase (SDH) activity. Six cell types were exposed to 5 or 60 J/cm(2) of blue light from quartz-tungsten-halogen (QTH), plasma-arc (PAC), or argon laser sources in monolayer culture. Post-light SDH activity correlated positively with population doubling time (R(2) = 0.91 for PAC, 0.76 for QTH, 0.68 for laser); SDH activity increased for cell types with the longest doubling times and was suppressed for cell types with shorter doubling times. Thus, light-induced exposure differentially affects SDH activity, cell survival, and growth, depending on cell energy consumption. Blue light may be useful as a therapeutic modulator of cell growth and survival.

Ag, Cu, Hg and Ni ions alter the metabolism of human monocytes during extended low-dose exposures.

The monocyte and macrophage play an important role in the biological response to dental biomaterials. However, the effects of low-level, extended exposures of monocytes to metal ions which are known to be released from dental alloys is not known. Thus, in the current study we characterized the metabolic activity of monocytes in the presence of low doses of Ag, Cu, Hg and Ni ions for up to 4 weeks. THP-1 human monocytes were exposed in vitro to concentrations of metal ions at 1-10% of those known to be lethal during 24 h exposures. Mitochondrial function [succinic dehydrogenase (SDH) activity] and total cellular protein [bicinchoninic acid (BCA) assay] were assessed at weekly intervals during metal exposure. Each metal ion caused a unique pattern of effects from the monocytes. These effects were sometimes delayed until several weeks into the exposure (Cu, Ni). Large increases in total protein or SDH activity per cell were observed (Cu 150%, Hg 40-60%, Ni 50%), but these increases were always transient. The differences between concentrations with minimal effects and those which were lethal (8 versus 12 micromol L(-1) for Ag, 1.0 versus 1.5 micromol L(-1) for Hg) were small. Finally, concentrations which caused total suppression of cellular activity were sometimes preceded by an increased activity (Hg, Ni). We concluded that metal ions alter monocyte metabolic activity during extended exposures in vitro, but that the concentrations required are often near long-term lethal levels. Clinically, these results imply that the levels of metals released from dental alloys may be significant to monocytic function.

Long-term cytotoxicity of resin-based dental restorative materials.

Highly filled composites, Ormocers (organically modified ceramics) and 'smart' materials have been developed to overcome the polymerization shrinkage problems of conventional composite materials. The purpose of the current study was to investigate the effect of longer-term (up to 8 weeks) ageing of these resin-based dental restorative materials and determine the effect of post-curing on cytotoxicity. Twelve discs of each material (Colombus/IDR, Definite/Degussa, Ariston pHc/Vivadent) were either light-cured (Lc) or light-cured and post-cured (Pc). For cytotoxicity testing, the discs were placed in contact with cell culture medium (DMEM) and incubated at 37 degrees C. Extracts from composite materials were collected after 24 h and weekly over a time period of 8 weeks. Cytotoxicity of the eluates to cultured fibroblasts (Balb/c3T3) were measured by the succinic dehydrogenase (SDH) activity (MTT assay) and the results expressed in percentage of negative controls (Teflon discs). The results showed that ageing significantly influenced the cytotoxicity of the materials. Except for Ariston pHc, materials were less cytotoxic after 8 weeks of ageing than they were in early intervals and post-curing was not generally useful in reducing cytotoxicity. The Ariston pHc was initially moderately toxic, but then become highly cytotoxic for 5 weeks before returning to initial levels. The current study demonstrated the importance of assessing the cytotoxicity of resin composite materials at multiple times.

Bonding characteristics to dentin walls of class II cavities, in vitro.

OBJECTIVES: The objective of this study was to test the hypothesis that bond strengths to intact class II cavity wall surfaces are lower than those measured on corresponding flat surfaces isolated by cutting away the rest of the cavity walls. METHODS: Class II (MOD) cavities were prepared in extracted human third molars and then divided into two groups: Intact cavity bonding group or flat surface group. All prepared surfaces were acid-etched and bonded with Scotchbond Multi-Purpose Plus adhesive system and restored with Z100 resin composite. After storage in water for 2 days, the teeth were divided mesio-distally into two equal halves. One half was vertically serially sectioned, while the other half was horizontally serially sectioned to yield a series of 0.5mm thick slabs. Each slab was trimmed into an hour-glass outline with the narrowest cross-sectional area at the region of interest (i.e., axial resin-dentin interface). RESULTS: The mean bond strengths obtained in the cavity bonding group were significantly lower (p<0.05) than those of the flat bonding group. However, within either group, there were no significant consistent differences among the various regions. SIGNIFICANCE: The large flat surfaces used in most laboratory bonding studies may overestimate the bond strengths that can be achieved in complex cavities prepared and restored under clinically relevant conditions.

Bond strength of composite to dentin using conventional, one-step, and self-etching adhesive systems.

OBJECTIVES: This in vitro study compared the dentin bonding performance of eight adhesive systems using a microtensile bond strength test. METHODS: Thirty bovine teeth were ground to 600-grit to obtain flat root-dentin surfaces. Two conventional adhesive systems (Scotchbond Multipurpose Plus, OptiBond FL), four one-step adhesive systems (Scotchbond 1, Asba S.A.C., Prime and Bond NT, Excite) and two self-etching adhesive materials (Clearfil Liner Bond 2 V and Prompt L-Pop) were evaluated. Each bonding system was applied according to manufacturer's instructions and followed by composite (Z100) application. Immediately after bonding, the teeth were prepared for microtensile testing. Bond strength to dentin was measured using a Vitrodyne V-1000 universal tester. There were 14 replicates for each material. Fractured specimens were further observed by SEM. RESULTS: Scotchbond Multipurpose Plus exhibited significantly (p<0.05) higher bond strength values (30.3+/-9.4 MPa) than all other materials. The bond strengths of the other materials were (from highest to lowest): Opitbond FL (22.4+/-4.3 MPa); Scotchbond 1(18.9+/-3.2); Clearfil Liner Bond 2 V (18.9+/-3.0); Prime and Bond NT (18.3+/-6.9); Asba S.A.C. (14.4+/-2.9); Excite (13.8+/-3.7); and Prompt L-Pop (9.1+/-3.3). Statistical comparisons frequently overlapped, but Optibond was significantly (p<0.05) greater than Asba, Excite, and Prompt L-Pop; whereas, Scotchbond 1 was only significantly (p<0.05) greater than Prompt L-Pop. Asba, Excite and Prompt L-Pop were not significantly different. The fracture modes were mostly adhesive. CONCLUSIONS: The conventional adhesive systems produced higher bond strengths to root dentin than most one-step adhesives and one self-etching adhesive; with the exception of one material in each respective system.

Effects of sodium hypochlorite and RC-prep on bond strengths of resin cement to endodontic surfaces.

There is concern that the use of sodium hypochlorite (NaOCl) and RC-Prep may lower the bond strength of resin cements. The objective of this study was to evaluate the effect of 5% NaOCl and RC-Prep treatment on the bond strength of a resin cement, C&B Metabond. Control roots (group 1) were biomechanically prepared using 0.9% NaCl as an irrigant; group 2, roots with 5% NaOCl; group 3, roots with RC-Prep; group 4, roots with 0.9% NaCl followed by 10% ascorbic acid; group 5, roots with 5% NaOCl followed by 10% ascorbic acid (pH 4); group 6, roots with 5% NaOCl followed by 10% neutral sodium ascorbate; and group 7, roots with RC-Prep followed by 10% ascorbic acid. All roots were then filled with C&B Metabond, incubated in water for 24 h, and then cross-sectioned into six 1-mm thick slabs representing cervical and middle root dentin. The slabs were trimmed and tested for tensile bond strength. The results demonstrated that both 5% NaOCl and RC-Prep produced significantly (p < 0.05) large reductions in resin-dentin bond strengths, and the reductions could be completely reversed by the application of either 10% ascorbic acid or 10% sodium ascorbate.

Effect of sub-lethal concentrations of HEMA (2-hydroxyethyl methacrylate) on THP-1 human monocyte-macrophages, in vitro.

OBJECTIVE: Resin monomers such as HEMA (2-hydroxyethyl methacrylate) can be released from restorative materials and diffuse into the tooth pulp over long periods of time. Although the short-term toxicity of resin monomers has been well documented, little is known about the risk for chronic toxicity resulting from low concentrations of resins. Thus, the hypothesis tested in this study was that sub-lethal concentrations of HEMA alter the functions of macrophages after long-term exposure. METHODS: Human THP-1 monocyte-macrophages were exposed to concentrations of HEMA between 0 and 1.5 mmol/l for up to 6 weeks. Cellular proliferation was measured by a hemocytometer with trypan-blue dye exclusion. Mitochondrial activity was measured by the MTT assay, and total cellular protein was measured using the bicinchoninic acid assay. RESULTS: Macrophage proliferation was inhibited 40-50% (significant, p < 0.05) by as little as 0.75 mmol/l after 1 week of exposure. Inhibition of proliferation remained constant after 1 week. The total protein per cell increased by as much as 80% (significant, p < 0.05) after 2 weeks and remained elevated through 6 weeks. Mitochondrial activity per cell increased 60-80% (significant, p < 0.05) after 2 weeks, then decreased. However, mitochondrial activity remained significantly elevated above controls through 6 weeks. SIGNIFICANCE: Findings from the current study indicate that 6-week exposures of monocytes to HEMA alter their proliferation and other activities at concentrations substantially lower than previously reported. This is particularly relevant in light of evidence that such concentrations have been previously shown to come through dentin by diffusion.

Mechanical disruption of dentin collagen fibrils during resin-dentin bond testing.

PURPOSE: To determine if collagen fibrils on the dentin side of failed resin-dentin interfaces undergo mechanical disruption during microtensile bond testing. MATERIALS AND METHODS: Extracted, caries-free human third molars were divided into four groups. The occlusal enamel was removed, leaving a flat dentin surface for bonding. Resin composite buildups were made after the acid-conditioned dentin was bonded with either Single Bond (S) or One-Step (O), and using either moist bonding (M) or air drying for 5 s (D). After storage in water for 24 h, the teeth were vertically sectioned into an array of 0.9 x 0.9 mm resin composite-dentin beams. They were stressed to failure using the nontrimming version of the microtensile bond test. Fractured dentin and resin composite sides of representative beams from each group that exhibited adhesive failures under light microscopy examination were prepared for scanning (SEM) and transmission electron microscopy (TEM). RESULTS: A two-way ANOVA showed that moist bond strengths were significantly higher than those made to dry dentin (M > D; p < 0.001), but that there was no difference between the adhesives (S vs O; p = 0.547). SEM analysis showed the presence of loose collagen fibrils within fractured hybrid layers in the dry groups, but not in the moist groups. TEM examination of the dry-bonded groups revealed collagen fibrils that were thinner and exhibited abnormally wide interfibrillar spaces when hybrid layers were intact. Within dry-bonded fractured hybrid layers, broad mechanical disruption zones could be seen, consisting of fibrils that were devoid of cross banding, defibrillation of the subfibrillar architecture, and gross disaggregation into microfibrils. In the moist-bonded groups, only short mechanical disruption zones were found along the torn edges of the collagen fibrils. The rest of the fibrils beyond the fracture site were intact and retained their periodicity. Mechanical testing of demineralized matrices yielded a maximum modulus of elasticity of 43.9 +/- 6.1 MPa. CONCLUSION: We speculate that adhesive resin has a protective function for demineralized collagen in well-infiltrated hybrid layers. We propose that both the collagen and resin contribute to load sharing during stress application until the final moment of rupture. On the other hand, collagen fibrils in poorly infiltrated hybrid layers, being unsupported by resin, undergo various degrees of irreversible mechanical disruption depending on how the stress is dissipated. The collagen fibril network has a much lower modulus of elasticity compared to those of resin-infiltrated fibrils or the demineralized dentin.