Uterine sutures at prior caesarean section and placenta accreta in subsequent pregnancy: a case-control study.

OBJECTIVE: To clarify the effects of uterine myometrial suture techniques at prior caesarean section on the incidence of pathologically diagnosed placenta accreta in placenta praevia with prior caesarean section (PPPC). DESIGN: Case-control study. SETTING: Eleven tertiary referral hospitals in central Japan. POPULATION: A total of 98 cases of placenta praevia, a history of one or more prior caesarean sections, and a history of uterine transverse incision and usage of only absorbable thread for myometrial sutures at the prior caesarean section. Exclusions were a history of myomectomy or Strassmann's operation. METHODS: Cases were grouped into a pathologically diagnosed placenta accreta group (38 cases) and a no accreta group (60 cases). Clinical characteristics including uterine suture methods at prior caesarean section were compared (single-layer versus double-layer closure; continuous versus interrupted sutures in the inner myometrial layer). MAIN OUTCOME MEASURE: The incidence of placenta accreta. RESULTS: No difference was found comparing single-layer with double-layer closure in the incidence of placenta accreta (37.1 versus 39.7%, P = 0.805); however, a significant difference was found comparing continuous with interrupted sutures (58.1 versus 29.9%, P = 0.008). Multivariable logistic regression analysis with stepwise selection for the eight factors meeting the criterion of P < 0.10 in univariate analysis was used, and four independent factors were selected, as follows: gravidity ≥ 3 (adjusted odds ratio, aOR, 3.4, 95% confidence interval, 95% CI, 0.99-11.6, P = 0.050); total praevia (versus non-total, aOR 18.4, 95% CI 3.2-107.0, P = 0.001); anterior/centre placenta (versus posterior, aOR 16.4, 95% CI 3.7-72.2, P < 0.001); and continuous sutures (versus interrupted, aOR 6.0, 95% CI 1.4-25.2, P = 0.015). CONCLUSIONS: In this limited study, a history of continuous sutures on the inner side of the uterine wall showed potential to influence the development of placenta accreta in PPPC patients.

An infrared imaging method for high-throughput combinatorial investigation of hydrogenation-dehydrogenation and new phase formation of thin films.

We have developed an infrared imaging setup enabling in situ infrared images to be acquired, and expanded on capabilities of an infrared imaging as a high-throughput screening technique, determination of a critical thickness of a Pd capping layer which significantly blocks infrared emission from below, enhancement of sensitivity to hydrogenation and dehydrogenation by normalizing raw infrared intensity of a Mg thin film to an inert reference, rapid and systematic screening of hydrogenation and dehydrogenation properties of a Mg-Ni composition spread covered by a thickness gradient Pd capping layer, and detection of formation of a Mg2Si phase in a Mg thin film on a thermally oxidized Si substrate during annealing.

Model, prediction, and experimental verification of composition and thickness in continuous spread thin film combinatorial libraries grown by pulsed laser deposition.

Pulsed laser deposition was used to grow continuous spread thin film libraries of continuously varying composition as a function of position on a substrate. The thickness of each component that contributes to a library can be empirically modeled to a bimodal cosine power distribution. We deposited ternary continuous spread thin film libraries from Al(2)O(3), HfO(2), and Y(2)O(3) targets, at two different background pressures of O(2): 1.3 and 13.3 Pa. Prior to library deposition, we deposited single component calibration films at both pressures in order to measure and fit the thickness distribution. Following the deposition and fitting of the single component films, we predict both the compositional coverage and the thickness of the libraries. Then, we map the thickness of the continuous spread libraries using spectroscopic reflectometry and measure the composition of the libraries as a function of position using mapping wavelength-dispersive spectrometry (WDS). We then compare the compositional coverage of the libraries and observe that compositional coverage is enhanced in the case of 13.3 Pa library. Our models demonstrate linear correlation coefficients of 0.98 for 1.3 Pa and 0.98 for 13.3 Pa with the WDS.

In vitro effect of nanoleakage expression on resin-dentin bond strengths analyzed by microtensile bond test, SEM/EDX and TEM.

This study evaluated the effect of multiple consecutive adhesive resin coatings of adhesive bonded to human dentin on nanoleakage and resin-dentin bond strength. Resin bonded dentin specimens were prepared using a total-etch adhesive (One-Step Plus) applied as multiple consecutive coating, or using two self-etch adhesive systems (iBond or Fluoro Bond). For the total-etch adhesive, resin application and air evaporation were performed 1, 2, 3, or 4 times. The self-etch adhesives were applied according to manufacturers' instructions. Resin-dentin bonded beams were prepared and immersed in water (control) or ammoniacal silver nitrate. After storage, microtensile bond strengths were measured. The fractured surfaces were examined by scanning and transmission electron microscopy (SEM and TEM), and energy-dispersive X-ray spectrometry (EDX). No significant differences in bond strength were found between water and silver nitrate storage groups. Several types of silver depositions (spotted, reticular, or water trees) were found in adhesive joints. The bond strengths of the single coated specimens of the total-etch adhesive were significantly lower than those receiving 2-4 coatings. Single coats produced more nanoleakage than multiple coats. However, no correlation was found between the bond strengths and nanoleakage between the different adhesives (total-etch adhesive with different conditions or self-etch adhesives).

Degradation of resin-dentin bonds using NaOCl storage.

OBJECTIVE: Several recent studies have reported collagen hydrolysis within bonds over the long-term. This may be one reason for the degradation of the bonds. This study therefore aimed to determine the effects of NaOCl on adhesive system bonds (total-etch bonding system vs. self-etching primer system) to dentin in order to accelerate the durability testing. METHODS: Resin-dentin bonded specimens were produced using Liner Bond 2V (Kuraray), a self-etching primer system, and OptiBond SOLO (Kerr), a total-etch bonding system, according to the manufacturers' instructions. The bonded specimens were serially sectioned in both x and y directions across the adhesive interface to obtain beams (adhesive area: 0.9 mm(2)). The specimens were immersed in 10% NaOCl solution for 1-5h after being stored in water at 37 degrees C for 24h. Control specimens were tested without exposure to NaOCl. After storage, micro-tensile bond tests were performed. Results were analyzed by two-way ANOVA and Fisher's PLSD tests (p<0.05). All fractured surfaces were observed by SEM, and examined using an image analyzer. RESULTS: The bond strengths decreased with increasing storage time in NaOCl. Fractography showed that NaOCl had a greater effect on the bond structure of OptiBond SOLO than on that of Liner Bond 2V, although both adhesives were susceptible. SIGNIFICANCE: Deterioration of the bonds was responsible for the effect of NaOCl on the hybrid layer. This deterioration may occur in humans in cases of deproteinization within the bonds.

Over-etching effects on micro-tensile bond strength and failure patterns for two dentin bonding systems.

OBJECTIVES: The purpose of this study was to determine (1) the weakest zone of resin-dentin bonds and (2) the relation between bond strength and failure mode to clarify the effect of demineralized dentin. METHODS: Human premolars were sectioned to expose the dentin surfaces, and the dentin surfaces were conditioned with phosphoric acid for 15, 60, 120, or 180s. Resin-dentin bonded specimens were produced using two adhesives: One-Step (Bisco) and OptiBond Solo (Kerr). Each sample was sectioned to produce a beam (adhesive area: 0.9mm(2)). Microtensile bond tests were then conducted, and the mean bond strengths (n=12 for each group) were statistically compared using two-way ANOVA and Duncan's multiple-range test (p<0.05). The fractured surfaces of all specimens were examined using SEM, and the areas of failure were measured using an image analyzer. RESULTS: For One-Step, the bond strength decreased with increase in acid-conditioning time (15s: 50.7+/-9.7, 60s: 40.8+/-11.0, 120s: 23.6+/-4.9 and 180s: 12.1+/-4.6MPa) (p<0.05). For OptiBond Solo, the bond strength in the case of 15s acid-conditioning time (42.6+/-7.9MPa) was significantly greater than that for the other times (60s: 31.9+/-10.3, 120s: 31.8+/-14.4 and 180s: 31.8+/-7.4MPa) (p<0.05). Fractography showed that the area percentage of the hybrid layer increased with increase in etching time for both systems. CONCLUSIONS: The integrity of the hybrid layer, especially the top part, has an effect on bond strength.

Autosomal-dominant hypoplastic form of amelogenesis imperfecta caused by an enamelin gene mutation at the exon-intron boundary.

Amelogenesis imperfecta (AI) is currently classified into 14 distinct subtypes based on various phenotypic criteria; however, the gene responsible for each phenotype has not been defined. We performed molecular genetic studies on a Japanese family with a possible autosomal-dominant form of AI. Previous studies have mapped an autosomal-dominant human AI locus to chromosome 4q11-q21, where two candidate genes, ameloblastin and enamelin, are located. We studied AI patients in this family, focusing on these genes, and found a mutation in the enamelin gene. The mutation detected was a heterozygous, single-G deletion within a series of 7 G residues at the exon 9-intron 9 boundary of the enamelin gene. The mutation was detected only in AI patients in the family and was not detected in other unaffected family members or control individuals. The male proband and his brother showed hypoplastic enamel in both their deciduous and permanent teeth, and their father showed local hypoplastic defects in the enamel of his permanent teeth. The clinical phenotype of these patients is similar to that of the first report of AI caused by an enamelin gene mutation. Thus, heterogeneous mutations in the enamelin gene are responsible for an autosomal-dominant hypoplastic form of AI.

Human periodontal ligament cells derived from deciduous teeth induce osteoclastogenesis in vitro.

The receptor activator of NF-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), are the important proteins involved in osteoclastogenesis. In this study, we investigated the expressions of RANKL and OPG in cultured human periodontal ligament cells derived from deciduous teeth (DPDL cells) and their roles in osteoclastogenesis. Northern blotting revealed that the OPG mRNA was down-regulated by application of 10(-8) M 1 alpha, 25(OH)2 vitamin D3 [1,25-(OH)2D3] and 10(-7) M dexamethasone (Dex). In contrast, RANKL mRNA was up-regulated by the same treatment. Western blotting demonstrated a decrease in OPG following application of 1, 25-(OH)2D3 and Dex. Tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNCs) were induced when DPDL cells were co-cultured with mouse bone marrow cells in the presence of 1,25-(OH)2D3 and Dex. TRAP-positive MNCs increased significantly when the DPDL cells were co-cultured with bone marrow cells in the presence of anti-human OPG antibody together with 1, 25-(OH)2D3 and Dex. These results indicate that PDL cells derived from deciduous teeth synthesize both RANKL and OPG and could regulate the differentiation of osteoclasts.

The extent to which resin can infiltrate dentin by acetone-based adhesives.

The combined methodologies of fractography and laser-Raman spectroscopic analysis were used for evaluation of the resin-dentin bonds made with wet and dry bonding. Resin-dentin-bonded beams were produced by means of 2 acetone-based adhesives (One-Step and Prime & Bond NT). The micro-tensile bond test was conducted, and the fractured surfaces of all specimens were examined by SEM and an image analyzer. The amount of resin infiltration within the hybrid layer was quantified by means of a laser-Raman spectroscope. In Raman analysis, the amount of resin impregnation within the hybrid layer of the dry bonding was found to be significantly lower (approximately 50%) than that in the wet one. Under fractographic analysis, a correlation was found between the bond strength and the failure mode. Based on those findings, it was suggested that the integrity between the bonding resin and the top of the hybrid layer played a major role in bond strength.

Delayed eruption of premolars with periodontitis of primary predecessors and a cystic lesion: a case report.

Apical periodontitis after pulp therapy in a primary tooth can cause delayed eruption of the permanent successor. A case of bilateral delayed eruption of mandibular premolars is presented. The patient. a 13-year-old girl, was referred by her dentist. Oral findings showed that the right first and left second primary molars were retained. Other premolars had erupted. An orthopantomogram revealed apical periodontitis, affecting both retained primary molars. The right first mandibular premolar was impacted against the alveolar bone and root of the second premolar, and there was a large cystic lesion in close association with the left second mandibular premolar. Both primary molars were extracted, and the cystic lesion was treated by marsupialization. Fenestration and traction were performed on the right first premolar. Correct tooth alignment was achieved with orthodontic appliances. If the problem had been detected earlier, treatment of the premolars might have been easier. Clinical and radiological follow-up, therefore, of primary teeth that have undergone pulp therapy procedures should be performed until eruption of succedaneous teeth.

Fractured surface characterization: wet versus dry bonding.

OBJECTIVE: Fractographic analysis was conducted to evaluate the resin-dentin bond structures made under wet and dry conditions. METHODS: Resin-dentin bonded specimens were prepared using two adhesive resin systems (Single Bond/SB; 3M and All Bond 2/AB2; Bisco Inc) under wet and dry conditions. The specimens were sectioned perpendicular to the adhesive interface to produce a square bar-shaped specimen (adhesive area: 0.9 mm(2)) by means of a diamond saw. The mean bond tensile test was then conducted at a crosshead speed of 1.0 mm/min. The mean bond strengths were statistically compared with two-way ANOVA and Fisher's PLSD test (p<0.05). Subsequently, the fractured surfaces of all specimens were examined using SEM and the area fractions of failure modes (%) were measured using an image analyzer on SEM microphotographs. RESULTS: No significant differences in tensile-bond strength were observed between SB (60.1+/-16.4MPa) and AB2 (69.8+/-17.4MPa) (p>0.05) under wet conditions. However, the bond strength either of SB or AB2 made under wet conditions was significantly greater than those made under dry conditions (SB: 26.2+/-12.5MPa and AB2: 6.8+/-3.3MPa) (p<0.05). Under fractographic analysis, the major portion at the fractured surface was occupied by the cohesive failure of bonding resin and the resin composite for the wet conditions, and the top of the hybrid layer for the dry conditions in both systems. SIGNIFICANCE: The interaction between the top of the hybrid layer and the bonding resin influenced the bond integrity.

Resin-tooth adhesive interfaces after long-term function.

PURPOSE: To characterize with scanning electron microscopy (SEM), the morphology of adhesive interfaces that had been functioning in an oral cavity for several years. MATERIALS AND METHODS: Cavities were prepared in vivo in caries-affected primary molars under local anesthesia. After removal of the entire carious lesion, the cavity was restored with a dentin adhesive system (Scotchbond Multi-Purpose). After several yrs, as the successor permanent teeth erupted, the resin-filled teeth were extracted. Then, the extracted resin-restored primary molars were cross-sectioned perpendicular to the adhesive interface using a low speed diamond saw and then polished for SEM examination. RESULTS: Morphological changes, such as digestion of the collagen fibrils and deterioration of the bonding resin and hybrid layer, were observed at the interface of the oral environment specimens. The results of this study demonstrated that degradation of resin-dentin bond structures appeared to occur in the human oral environment over time.

The in vitro cytotoxicity of eluates from dentin bonding resins and their effect on tyrosine phosphorylation of L929 cells.

OBJECTIVES: The aim of this study was to examine the relationship between the monomers eluted from dentin-bonding systems and their cytotoxicities, and to investigate the biochemical effect of the monomers on tyrosine phosphorylation, especially relating to the cell growth activity, of L929 cells in vitro. METHODS: The primers, uncured or cured adhesives (3M and Kuraray) were tested to determine the cytotoxicity of confluent L929 cells cultured by Eagle's MEM medium supplemented with 10% FCS. The area of cells affected by the eluted monomers were evaluated with an image analyzer and the concentrations of monomers eluted into the medium were measured with high performance liquid chromatography (HPLC) after 24h incubation. The protein composition of the stimulated cells was compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tyrosine phosphorylation was detected by Western blot. RESULTS: The primer and uncured adhesives revealed variable cytotoxicities. 2-hydroxyethyl-methacrylate (HEMA) was the major component eluted from uncured primers and adhesives. Small amounts of triethylene glycol dimethacrylate (TEGDMA) were also detected from the uncured adhesives. The cytotoxicities of the adhesives decreased as photo activation time increased. The amount of monomers eluted from the cured adhesives was almost undetectable and did not reach a sufficient concentration to suppress cell viability or cell growth. The cytotoxicities of the primers and adhesives correlated well with the amounts of either HEMA or TEGDMA eluted. Moreover, a high concentration of HEMA (4 mg/ml medium) affected intracellular tyrosine phosphorylation, which is related to cellular activities. SIGNIFICANCE: Although the monomers present in dentin bonding resins are cytotoxic to L929 cells, the amount from cured bonding resin is very small and does not provide a cytotoxic dose. This data does however suggest that clinical exposure to the uncured primers and adhesives of dentin bonding resins should be minimized.

Circadian rhythm of nitric oxide production in the dorsal region of the suprachiasmatic nucleus in rats.

Extracellular concentration of nitrite (NO2-), an oxidized product of nitric oxide (NO), was measured consecutively in the dorsal region of the rat suprachiasmatic nucleus (SCN) by means of in vivo microdialysis. The NO2- concentrations in the dialysates showed robust circadian rhythm under a 12:12 h light/dark cycle and were higher during the dark phase than during the light phase. When the rats were transferred to constant darkness, the 24 h rhythm of NO2- persisted without damping the amplitude. The NO2- level was significantly lowered by an injection of NO synthase inhibitor (NG-monomethyl-L-arginine, 10 mg/kg i.p.). These findings indicate that the daily fluctuation of NO2- in the dorsal region of the SCN, which represents endogenous rhythm of NO, is regulated independently of photic inputs into the SCN and may be related to the circadian clock functions.

[A case of hookworm infestation with dissociation values between FDP-E and FDP-D dimer].

We previously reported a five-year-old girl showing bleeding tendency and transient morphological and functional platelet abnormalities probably due to a hookworm, Necator Americanus, infestation. In this report, we describe the rarely accelerated fibrinogenolysis and/or fibrinolysis in this patient whose value of fibrinogen and/or fibrin degradation products(FDP) determined with an FDP-E assay was much higher than that determined with a D-dimer assay. Namely, on day-1 and day-13 of hospitalization, her D-dimer values were only 10 to 20% of the prospected values from FDP-E values. We speculated this phenomenon was induced by circulating protease(-like) agent(s) produced by hookworm, because the only slightly participation of plasmin and/or granulocyte elastase was evaluated by the determination of enzyme-inhibitor complexes. And the other possibility of fibrinogen degradation by blast- or tumor-associated protease was excluded by the clinical manifestations and primary disorders. In conclusion, we report a very rare case with the accelerated fibrinogenolysis and/or fibrinolysis in a patient with the hookworm infestation. We are interested in the mechanism that manifested the patient's bleeding tendency accompanied with morphological and functional platelet abnormalities.

Fractographical analysis of resin-dentin bonds.

PURPOSE: To characterize resin-dentin bond structures of three adhesive resin systems using fractographic analysis to measure the area of failure at the fractured surface. MATERIALS AND METHODS: Flat dentin surfaces were ground perpendicular to the long axis of the tooth. The prepared dentin surfaces were treated by one of three adhesive resin systems (Mac Bond II, One-Step, and Single Bond). The samples were sectioned perpendicular to the adhesive interface to produce a square bar-shaped specimen (adhesive area: 0.9 mm2) using a diamond saw. A micro-tensile test was then conducted at a crosshead speed of 1.0 mm/min. The mean tensile bond strengths were statistically compared using one-way ANOVA and Fisher's PLSD test (P< 0.05). The fractured surfaces of all specimens were examined using SEM and the area of failure was measured using an image analyzer on SEM microphotographs. RESULTS: No significant differences in tensile bond strength were observed between Single Bond (62.1+/-18.2 MPa) and One-Step (53.8+/-13.1 MPa) (P > 0.05). However, the bond strength of Mac Bond II (36.5+/-13.7 MPa) was significantly lower than that of One-Step or Single Bond (P< 0.05). At the fractured surface, except for the cohesive failure of the bonding resin and resin-based composite, different failure patterns were observed for each resin system as follows: the failure of the hybrid layer and demineralized dentin was observed in the two wet bonding systems (One-Step and Single Bond) and of the hybrid layer but not the demineralized dentin in the self-etching primer system (Mac Bond II). The results demonstrated that the integrity of the hybrid layer depends on the adhesive system.

Effect of serial extraction alone on crowding: spontaneous changes in dentition after serial extraction.

We examined changes in dentition after serial extraction in subjects who wore no appliances to determine the relationships between changes in dentition and improvement in dental crowding. Mandibular dental casts and lateral cephalograms from 31 subjects who had undergone serial extraction without orthodontic treatment were analyzed at 3 stages: before extraction of the deciduous canines (T1), after extraction of first premolars (T2), and at the end of the observation period (T3). Although movements of the first molar cusp and apex from T2 to T3 were significantly greater than from T1 to T2, movements of the incisor cusp and apex from T1 to T2 were significantly greater than from T2 to T3. The first molar tipped mesially from T1 to T2 but tipped distally from T2 to T3. The distal tipping of the incisor from T1 to T2 was significantly greater than from T2 to T3. These results suggest that the main changes in dentition from T1 to T2 are different from those from T2 to T3. The correlations between the annual change in the canine movement or tipping from T2 to T3 and the annual change in the irregularity index at the same time were significant. These results suggest that the canine movement or tipping contributed to the correction of the anterior crowding from T2 to T3.

Feeding-induced c-fos expression in the nucleus of the solitary tract and dorsal medullary reticular formation in neonatal rats.

We investigated feeding-associated activation of neurons in the medulla oblongata during the suckling period in rats, using the c-fos gene-encoded protein (Fos) immunohistochemistry. After an isolation from mothers for 12 h, neonates were either breast-fed intensively or further isolated for another 3 h, and sacrificed on postnatal day 1, 3, 5, 7 and 14 (P1-14). In the former pups, Fos-immunoreactive (FI) neurons were predominantly localized in the nucleus of the solitary tract (NST) and in the dorsal medullary reticular formation (RF). The number of FI cells peaked on P5-7 and decreased on P14 in the NST, and increased remarkably on P3 and was consistently high until P14 in the dorsal RF. In contrast, much fewer FI cells were found in the NST and RF in the latter pups. The results indicated that not only the NST but also the dorsal RF were implicated in feeding behavior in the suckled pups.

Synchronization of circadian firing rhythms in cultured rat suprachiasmatic neurons.

The circadian clock in mammals is located in the suprachiasmatic nucleus (SCN) which consists of multiple oscillating neurons. Integration of the cellular oscillations is essential for the generation of a single circadian period in the SCN. By using a multielectrode dish (MED), we measured circadian firing rhythms in individual SCN neurons for more than 2 weeks continuously, and examined the involvement of synaptic communication in the synchronization of circadian rhythms. Cross-correlation analysis of spontaneous action potentials revealed that a neuron pair was functionally connected by synapses when their circadian rhythms were synchronized. No correlation was found between the paired neurons whose circadian rhythms were not synchronized. Calcium (Ca2+)-dependent synaptic transmission in the cellular communication was indicated by dose-dependent lengthening of an intercellular spike interval and loss of spike correlation with a Ca2+ channel blocker. Approximately 60% of the SCN neurons in culture were immunoreactive to antibodies against gamma-aminobutyric acid (GABA) or glutamic acid decarboxylase (GAD). Spontaneous firing of all the neurons tested was either increased or decreased by bicuculline, the GABAA receptor antagonist. These findings indicate that synaptic communication plays a critical role in the synchronization of circadian rhythms in individual SCN neurons and the GABAergic transmission is involved in the synchronization mechanism.

The effect of hybrid layer thickness on bond strength: demineralized dentin zone of the hybrid layer.

OBJECTIVES: The purpose of this study was to evaluate the correlation between hybrid layer thickness and bond strength using specimens acid-conditioned for varying lengths of time. METHODS: The dentin surfaces of human premolars, sectioned to remove the enamel from the labial surface, were conditioned with 35.0% phosphoric acid of an adhesive resin system (Scotchbond Multi-Purpose; 3M) for 15 (as directed by the manufacturer), 60, 120, or 180 s (experimental acid-conditioning times). The bonded specimens were then sectioned perpendicular to the adhesive interface to measure the hybrid layer thickness by SEM. The specimens for the micro-tensile test were sectioned perpendicular to the adhesive interface and trimmed to an hourglass-shape. Then, the micro-tensile test was performed at a crosshead speed of 1.0 mm/min. The bond strengths and hybrid layer thickness were statistically compared with Student's t-test (p < 0.05). All fractured surfaces were also observed by SEM. RESULTS: Significant differences between the groups exposed to acid for 15 and 60 s, and those exposed for 120 and 180 s were observed in hybrid layer thickness and bond strength (p < 0.05). SEM observation of the fractured surfaces revealed that a demineralized dentin zone without resin impregnation remained within the hybrid layer. SIGNIFICANCE: A demineralized dentin zone was formed in the bond structures after prolonged acid-conditioning, resulting in low bond strength. The shrinkage of the hybrid layer due to desiccation during the SEM examination process provided evidence of the presence of the demineralized dentin zone within the hybrid layer.