Are CAD-CAM milled glass fiber posts better than prefabricated or custom glass fiber posts for endodontically treated teeth? A systematic review with meta-analysis.

STATEMENT OF PROBLEM: Prefabricated and custom glass fiber posts have been successfully used to reconstruct endodontically treated teeth. However, the performance of computer-aided design and computer-aided manufacture (CAD-CAM) milled glass fiber posts is unclear. PURPOSE: The purpose of this systematic review with meta-analysis was to compare the fracture and bond strength and cement layer thickness of CAD-CAM milled glass fiber posts with prefabricated or custom glass fiber posts. MATERIAL AND METHODS: The protocol was registered in the Open Science Framework (http://osf.io/65jm7). Two reviewers searched the PubMed/MEDLINE, Web of Science, Embase, Scopus, and ProQuest databases for articles up to September 2023. In addition, the reference lists were hand searched. A meta-analysis was performed by using the RevMan 5.4 program. The risk of bias was assessed using the RoBDEMAT tool. RESULTS: After screening, a total of 18 studies were included. The CAD-CAM milled glass fiber posts showed higher fracture strength (P=.02; Standardized Mean Difference [SMD]: 0.57; 95% Confidence Interval [CI]: 0.07 to 1.07), bond strength (P=.010; SMD: 1.07; 95% CI: 0.26 to 1.89), and lower cement layer thickness (P=.009; SMD: -2.94; 95% CI: -5.15 to -0.73) when compared with prefabricated glass fiber posts. However, fracture strength (P=.53; SMD: 0.38; 95% CI: -0.79 to 1.54) and bond strength (P=.90; SMD: -0.05; 95% CI: -0.81 to 0.72) were statistically similar between CAD-CAM milled and custom glass fiber posts. Significant and substantial heterogeneity was observed in all meta-analyzes (P<.01; I>60%). The studies sufficiently reported most domains related to bias, except for randomization of samples, sample size rationale and reporting and operator blinding. CONCLUSIONS: CAD-CAM milled and custom glass fiber posts provide an effective and safe option for restoring endodontically treated teeth, especially for weakened teeth or enlarged root canals. However, further well-designed clinical research is recommended to strengthen these findings.

Root canal dentin wear during final irrigation in endodontic retreatment with passive ultrasonic irrigation and easy clean instruments.

Context: Complementary procedures have been proposed to improve the endodontic retreatments. Aim: The aim of this study was to evaluate, by microcomputed tomography (micro-CT), the dentin wear caused by passive ultrasonic irrigation (PUI) and easy clean (EC) instruments, when used for the final removal of filling material during endodontic retreatment. Methods: Thirty-six mesial roots of the lower first molars were divided into four groups (n = 9), according to the final irrigation and sealer: PUI/AH Plus, EC/AH Plus, PUI/Total Fill (TF), and EC/TF. Canal volume was evaluated, both before and after the final irrigation, by micro-CT analysis. The Kruskal-Wallis test was used to compare the volumes among groups and the paired Wilcoxon test was used to compare the volume values before and after the final irrigation within each group. Results: Both complementary cleaning procedures promoted dentin wear that was observed only when the volume was analyzed for each third of the root canal. In the apical third, both instruments showed significant wear, where the use of the EC instrument resulted in greater wear than the PUI, in teeth filled with both AH Plus and TF BC Sealers (P < 0.05). Conclusion: Both complementary cleaning procedures promoted dentin wear and must be used cautiously.

Can the addition of surfactants to NaOCl irrigation impact on the percentage of voids of root canal filling?

This study evaluated the impact of canal irrigation using surfactants associated to NaOCl on the percentage of voids of root canal filling through micro-computed tomographic (micro-CT) analysis. 27 mandibular mesial roots with Vertucci type II canal configuration with isthmus were selected and scanned in a micro-CT device. The specimens were assigned to three groups (n = 9), according to the irrigation solution during instrumentation: 2.5% NaOCl without additives (control), 2.5% NaOCl with 0.1% Benzalkonium Chloride and 2.5% NaOCl with 0.1% Tween 80. After obturation, the specimens were rescanned, and the percentage of voids were calculated. Data were analysed using one-way ANOVA with a significance level of 5%. The percentage of voids were similar among the groups (p > 0.05). None of the specimens showed completely voids-free areas. The addition of surfactants to NaOCl irrigation did not improve the quality of root canal filling of mandibular mesial root canals.

Chronic alcohol and nicotine consumption as catalyst for systemic inflammatory storm and bone destruction in apical periodontitis.

AIM: To assess the periapical alveolar bone pattern and the serum levels of proinflammatory cytokines, biochemical markers and metabolites in rats subjected to chronic alcohol and nicotine consumption and induced apical periodontitis. METHODOLOGY: Twenty-eight male Wistar rats were divided into four groups: Control, Alcohol, Nicotine and Alcohol+Nicotine. The alcohol groups were exposed to self-administration of a 25% alcohol solution, while the other groups were given only filtered water. The nicotine groups received daily intraperitoneal injections of a nicotine solution (0.19 µL of nicotine/mL), whereas the other groups received saline solution. Periapical lesions were induced by exposing the pulps of the left mandibular first molars for 28 days. After euthanasia, the mandibles were removed and the percentage bone volume, bone mineral density, trabecular thickness, trabecular separation and trabecular number of the periapical bone were measured using micro-computed tomography images. Serum samples were collected for analysis of proinflammatory cytokines (IL-1ß, IL-4, IL-6 and TNF-α), biochemical and metabolomic analysis. Statistical analysis was performed with a significance level of 5%. Nonparametric data were analysed using the Kruskal-Wallis test followed by Dunn's test, while one-way anova followed by Tukey's test was performed for parametric data. RESULTS: The groups exposed to alcohol or nicotine consumption exhibited an altered bone pattern indicating lower bone density and higher levels of IL-1ß, IL-6 and TNF-α compared to the Control group (p < .05). Significant differences were observed among the groups in the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, cholesterol, triglycerides, urea, creatinine, albumin, uric acid, bilirubin and calcium. Metabolomic analysis revealed significant differences in glycine, phosphocholine, lysine, lactate, valine, pyruvate and lipids (CH2 CH2 CO), n(CH2 ) and n(CH3 ). Most of these parameters were even more altered in the simultaneous consumption of both substances compared to single consumption. CONCLUSION: Alcohol and nicotine chronic consumption altered several metabolic markers, impaired liver and kidney function, increased the production of systemic proinflammatory mediators and harmed the periapical bone microarchitecture in the presence of apical periodontitis. The simultaneous consumption of alcohol and nicotine intensified these detrimental effects.

Effect of the progressive taper enlargement of buccal root canals of three-rooted maxillary molars: A stepwise micro-CT study.

This study evaluated unprepared canal areas, volume of dentine removal, and dentine thickness after the progressive enlargement of the buccal canals of 22 maxillary molars, with and without the MB2, using instruments with the same tip size (0.25 mm) but 4 different tapers (0.03, 0.05, 0.06 and 0.08 v). Micro-CT scans were performed after each instrument. Data were compared using a general linear mixed model post hoc comparisons with Kenward-Roger for Wald F tests (α = 5%). After each treatment step, it was observed an increase in the percentage volume of removed dentine and a decrease in the unprepared areas and dentine thickness at the pericervical region of all canals (p < 0.05). The percentage reduction of dentine thickness in MB2 canal was higher than in the mesiobuccal and distobuccal canals (p < 0.05). Knowledge of pre-operative dimensions and dentine thickness before enlargement of buccal canals of maxillary molars is an important factor to avoid excessive dentine removal.

Volumetric changes in root canal sealers in ex vivo and a novel animal model approach.

AIM: This study aimed to evaluate the volumetric change of root canal sealers through micro-computed tomographic analysis using a novel in vivo model and to compare the results with those obtained using an ex vivo test. METHODOLOGY: Eighteen single-rooted teeth were cut to 5 mm length from the root apex. The root canals were uniformly enlarged and filled with EndoSequence BC Sealer or AH Plus Jet root canal sealers. Samples were stored at 37°C and 95% relative humidity for 24 h and then scanned with a micro-CT device. Twelve samples (n = 6 for each sealer) were implanted in the subcutaneous tissue of Wistar rats, while six samples (n = 3 for each sealer) were immersed in 20 mL of phosphate-buffered saline (PBS) at 37°C at neutral pH. After 7 and 30 days, teeth were removed from subcutaneous tissue or PBS and rescanned. Statistical analysis of volume changes was performed using Shapiro-Wilk's test and independent t-test (p < .05). RESULTS: AH Plus Jet had smaller volume changes (-2.2 to +0.77%) than EndoSequence BC Sealer (-2.0 to +4.0%) (p < .05), in the two tested models. The volume of the root canal sealers decreased over time (p < .05), in vivo. AH Plus Jet results varied between the in vivo and ex vivo results (p < .05), while EndoSequence BC Sealer presented similar volume losses for both experimental models (p > .05). CONCLUSION: EndoSequence BC Sealer lost more volume than AH Plus Jet. The experimental conditions influenced the volumetric change of AH Plus Jet but not the EndoSequence BC Sealer. The ex vivo model should be further explored as a methodological alternative to assess the volumetric changes of root canal sealers without causing harm to animals.

Marginal gaps and voids using two warm compaction techniques and different sealers: a micro-CT study.

OBJECTIVE: To assess the percentage of marginal gaps and voids in oval-shaped canals obturated by using two warm compaction techniques with a Bio-C sealer and AH Plus Jet. MATERIALS AND METHODS: Forty canines with oval canals were scanned by microcomputed tomography (micro-CT), and root canal preparation was performed with an XP-endo Shaper system and irrigated with 5.25% sodium hypochlorite. Then, the specimens were paired into four groups (n=10) according to the root canal filling technique and endodontic sealer: Bio-C sealer and continuous wave of condensation, Bio-C sealer and Tagger's hybrid, AH Plus Jet and continuous wave of condensation, and AH Plus Jet and Tagger's hybrid. After root canal filling, a new scan was performed. The percentage of marginal gaps and voids was calculated with the ImageJ software, and the data were analyzed statistically using two-way ANOVA and Tukey tests, with a significance level of 5%. RESULTS: The percentage of marginal gaps was significantly lower in the Bio-C sealer than in AH Plus Jet (p=0.021) regardless of the technique. However, no difference was found in the percentage of voids between root canal filling techniques and the endodontic sealer (p>0.05). CONCLUSION: Both sealers and techniques demonstrated good quality of root canal filling. However, the use of the Bio-C sealer enhanced the filling ability by reducing marginal gaps, regardless of the root canal filling technique. CLINICAL RELEVANCE: This study highlights the better performance of the Bio-C sealer in the quality of the root canal filling, reducing marginal gaps when compared to AH Plus Jet independent of the technique.

pH influences the volumetric change of calcium silicate- and epoxy resin-based root canal sealers: An ex vivo micro-CT study.

This study assessed the influence of pH variation on the volumetric change of EndoSequence BC Sealer compared to AH Plus Jet. Eighteen single-rooted teeth were uniformly prepared and filled with one of the sealers. After 24-h storing, samples were scanned in a micro-CT device. Then, roots were immersed in 20 ml phosphate-buffered saline (PBS) with pH of 5, 7 or 12 at 37°C for 7 and 30 days and rescanned. Statistical analysis was performed using Shapiro-Wilk's test, one-way ANOVA and Tukey's or Student's t-tests (p < 0.05). Differences between sealers were observed only at neutral pH and after 30 days of acidic pH exposure, with a higher loss of EndoSequence (p < 0.05). After 7 days, alkaline pH reduced and increased the volumetric loss of EndoSequence and AH Plus, respectively, compared to neutral pH (p < 0.05). It can be concluded that the pH directly influenced the volumetric change of both tested root canal sealers in different ways.

Effects of Two In-Office Bleaching Agents with Different Compositions on the Bond Strength to Enamel.

This study evaluates the effect of two in-office bleaching agents with different compositions on the bond strength to enamel surface. Fifty bovine teeth were divided into five groups (n = 10 teeth per group), according to the bleaching agent used and the time elapsed to perform the restorative procedures: restorative procedures performed without bleaching (control group); bleaching with 35% hydrogen peroxide (HP), with restorative procedures 24 hours or 7 days after bleaching (HP/24h and HP/7d groups, respectively); and bleaching with 35% HP with calcium compost, with restorative procedures 24 hours or 7 days after bleaching (HP AutoMixx/24h and HP AutoMixx/7d groups, respectively). The specimens were stored at 37°C in artificial saliva. Restored teeth were submitted to a micro-shear bond strength test. The specimens were analyzed using a stereoscope to determine the fracture pattern, classified as adhesive, cohesive, or mixed. The results of the bond strength test were evaluated by analysis of variance, with significance set at P < .05. The groups showed similar bond strength values without significant difference among them (P > .05). There was a predominance of the adhesive-type fracture pattern in all groups. The bleaching agents with different compositions showed similar bond strength values when the restoration was performed 24 hours and 7 days after bleaching, and the results were similar to the control group.

Photodynamic therapy for root canal disinfection in endodontics: an umbrella review.

To evaluate and synthesize the evidence from the individual reviews that evaluated the efficacy of PDT therapy in root canal disinfection by undertaking an umbrella review. The protocol of the review was registered in the PROSPERO database under number CRD42021214056. The literature search was conducted using the following electronic databases: PubMed, Scopus, Web of Science, BVS, Cochrane Database of Systematic Reviews, Embase, and Epistemonikos, from inception to July 2021. Systematic reviews that evaluated the efficacy of PDT for root canal disinfection were included. Two authors independently performed a literature search, data extraction, and quality assessment of the included studies. Any disagreements were resolved by a third reviewer. The quality of the reviews was assessed using the AMSTAR 2 tool and the final categorization of each systematic reviews was classified as of "high," "moderate," "low," or "critically low" quality. Six systematic reviews were included in the current umbrella review and all of them were graded as critically low quality. From the critically low-quality evidence available, this umbrella review showed that the efficacy of PDT in root canal disinfection remains yet undetermined.

Assessment of marginal gaps and image quality of crowns made of two different restorative materials: An in vitro study using CBCT images.

Background: The present study assessed the quality of images and the presence of marginal gaps on cone-beam computed tomography (CBCT) images of teeth restored with all-ceramic and metal-ceramic crowns and compared the gap sizes observed on CBCT images with those obtained on micro-CT images. Methods: Thirty teeth restored with metal-ceramic and all-ceramic crowns, properly adapted and with gaps of 0.30 and 0.50 mm, were submitted to micro-CT and CBCT scans. Linear measurements corresponding to the marginal gap (MG) and the absolute marginal discrepancy (AMD) were obtained. The objective assessment of the quality of CBCT images was performed using the contrast-to-noise ratio (CNR), and the subjective assessment was defined by the diagnoses made by five examiners regarding the presence or absence of gaps. Results: The measurements were always higher for CBCT, with a significant difference regarding AMD. No significant difference in image quality was observed using CNR between the crowns tested. Low accuracy and sensitivity values could be observed for both crowns. Conclusion: Marginal mismatch measures were overestimated in CBCT images. No difference in image quality was observed between the crowns. The correct diagnosis of gaps was considered low, irrespective of crown type and gap size.

Marginal gaps and voids of three root-end filling materials: A microcomputed tomographic study.

A root-end filling material is required to fill the root-end cavity without gaps or voids, to prevent root canal reinfection and to provide periapical healing. Thus, this study evaluated the volume of marginal gaps and voids of three root-end filling materials using microcomputed tomography (micro-CT). Thirty maxillary incisors were prepared and filled with gutta-percha and endodontic sealer. The specimens were scanned using micro-CT and distributed into three groups (n = 10): White MTA, MTA Repair HP, and Bio-C Repair. The root tips were resected at 90° to the longitudinal axis and the cavity (3 mm depth) was prepared with an ultrasonic tip. The materials were handled, and the cavities were filled. The specimens were rescanned and the percentual volume of gaps and voids were analyzed. The data were analyzed using Kruskal-Wallis and Dunn tests (p < .05). No statistical difference was found in the percentage of gaps among the tested materials (p > .05). White MTA presented less voids than Bio-C and MTA Repair HP (p < .05). The materials presented a similar percentual volume of gaps and White MTA presented less voids than other tested materials.

Shaping ability and apical debris extrusion after root canal preparation with rotary or reciprocating instruments: a micro-CT study.

OBJECTIVES: The aim of this study was to evaluate the shaping ability of the TruShape and Reciproc Blue systems and the apical extrusion of debris after root canal instrumentation. The ProTaper Universal system was used as a reference for comparison. MATERIALS AND METHODS: Thirty-three mandibular premolars with a single canal were scanned using micro-computed tomography and were matched into 3 groups (n = 11) according to the instrumentation system: TruShape, Reciproc Blue and ProTaper Universal. The teeth were accessed and mounted in an apparatus with agarose gel, which simulated apical resistance provided by the periapical tissue and enabled the collection of apically extruded debris. During root canal preparation, 2.5% sodium hypochlorite was used as an irrigant. The samples were scanned again after instrumentation. The percentage of unprepared area, removed dentin, and volume of apically extruded debris were analyzed. The data were analyzed using 1-way analysis of variance and the Tukey test for multiple comparisons at a 5% significance level. RESULTS: No significant differences in the percentage of unprepared area were observed among the systems (p > 0.05). ProTaper Universal presented a higher percentage of dentin removal than the TruShape and Reciproc Blue systems (p < 0.05). The systems produced similar volumes of apically extruded debris (p > 0.05). CONCLUSIONS: All systems caused apically extruded debris, without any significant differences among them. TruShape, Reciproc Blue, and ProTaper Universal presented similar percentages of unprepared area after root canal instrumentation; however, ProTaper Universal was associated with higher dentin removal than the other systems.

The ability of reciprocating glide path instruments to reach the full root canal working length.

This study evaluated the ability of reciprocating glide path instruments to reach the full working length (RFWL). Thirty mesial roots of mandibular molars were matched into 2 groups (n = 15 teeth, 30 root canals), according to the system used for glide path: WaveOne Gold Glider (WOGG) and R-Pilot. The samples with fractured instruments or when it was not possible to RFWL were scanned using micro-computed tomography. Fractured instruments were also visualised through a scanning electron microscopy to verify the type of fracture. The parameters were compared statistically by Fisher's and chi-square tests (P < 0.05). No instrument fractures were observed in the R-pilot, while 2 fractures occurred in the WOGG group (P > 0.05). RFLW was achieved in 29 canals (96.66%) and 28 canals (93.33%) with R-pilot and WOGG instruments, respectively (P > 0.05). The instruments are able to RFWL in mesial canals of mandibular molars. WOGG showed two fractures while no R-Pilot instruments fractured.

Shaping ability of two root canal instrumentation systems in oval-shaped canals: A microcomputed tomography study.

The objective was to evaluate the shaping ability of XP-endo Shaper and Mtwo systems in oval-shaped canals preparation by microcomputed tomography (micro-CT) along the entire canal. The volume, surface area and percentage of unprepared area were evaluated by image processing in entire canal and apical third (5 mm). Apical transportation and centring ability were evaluated at 3, 5 and 7 mm from the apex. Forty single-canal oval canines were paired in two groups (n = 20) according to the instrumentation system: XP-endo Shaper and Mtwo. The teeth were scanned by micro-CT before and after instrumentation, using a thermal vat at 37°C. The XP-endo Shaper system was more effective in the instrumentation of oval-shaped canals when compared to Mtwo system, resulting in greater volume increases and lower percentage of unprepared canals walls in apical region. Regarding apical transportation and centring ability, no statistical difference was observed.

The importance of bioceramics and computed tomography in the late clinical management of a horizontal root fracture: A case report.

Root fractures resulting from dental trauma involve dentin, cementum and pulp. The present study aimed to demonstrate the importance of cone-beam computed tomography (CBTC) and bioceramics in the correct planning and intervention of a horizontal root fracture case in tooth 11 with late treatment in an 18-year-old patient. Clinical and radiographic examinations revealed tooth displacement, pain on vertical percussion and images suggestive of a horizontal root fracture. Pulp necrosis was diagnosed and CBTC was requested for treatment planning. Subsequently, endodontic treatment was performed using a bioceramic apical plug. A 2-year follow-up indicated the absence of root resorption and normal periodontal and periapical tissues. It was concluded that endodontic treatment associated with the use of bioceramics and the aid of CBTC is an effective therapeutic option in cases of permanent horizontal root fractures. Key words:Bioceramics, Cone-beam computed tomography, dental Injuries, root fracture.

Antimicrobial effectiveness of grape seed extract against Enterococcus faecalis biofilm: A Confocal Laser Scanning Microscopy analysis.

This study evaluated the antimicrobial effectiveness of 6.5% Vitis vinifera grape seed extract (GSE) against Enterococcus faecalis biofilm using confocal laser scanning microscopy (CLSM). Saline solution (SS), 5.25% sodium hypochlorite (NaOCl) and 2% chlorhexidine (CHX) were used for comparison. Dentin discs were inoculated with E. faecalis strain establishing a 3-week-old biofilm. Discs (n = 10) were exposed to 5.25% NaOCl, 2% CHX, 6.5% GSE and SS (negative control) for 10 min. Discs were stained with the fluorescent LIVE/DEAD-BacLight™ dye and analysed using CLSM. The proportion of dead cells in biofilm was analysed using one-way anova and Tukey tests (P < 0.05). A higher proportion of dead cells was found in GSE group compared with CHX and SS (P < 0.05). NaOCl group was associated with the highest proportion of dead cells (P < 0.05). GSE presented antimicrobial activity against E. faecalis; however, NaOCl was the most effective irrigant solution. GSE was more effective than CHX and SS.

Effect of previous irrigation with chlorhexidine on the push-out bond strength of a calcium silicate-based material.

This study evaluated the effect of previous irrigation with chlorhexidine (CHX) on the bond strength of a calcium silicate-based material, Biodentine, when used for furcal repair. Furcal perforations were produced in 30 extracted mandibular molars. Teeth were divided into 3 groups according to the irrigant used: distilled water (DW), CHX followed by DW (CHX), and CHX followed by ethylenediaminetetraacetic acid (EDTA) and DW (CHX/EDTA). Biodentine was used to repair the perforations. A push-out bond strength test was performed after 7 days, and data were statistically analyzed using Kruskal-Wallis and Dunn tests (P < 0.05). The CHX/EDTA group showed significantly lower values than the DW and CHX groups (P < 0.05). The failure mode of the DW group was mainly mixed, while that of the CHX group was cohesive. The CHX/EDTA group exhibited adhesive and mixed failures. Irrigation with CHX prior to furcation repair did not result in a statistically significant difference, compared to the use of DW, in the push-out bond strength of Biodentine.

Evaluation of root canal morphology of maxillary premolars: a cone-beam computed tomography study.

The aim of this study was to evaluate the root canal configuration of maxillary premolars using cone-beam computed tomography (CBCT). CBCT images of 999 maxillary premolars from a database were analysed to determine the frequency of the number of roots, root canals and Vertucci's classification. The associations among these variables were evaluated by Chi-square test (P < 0.05). In the first premolars, 42.2% of single-rooted teeth had a type II configuration, whereas 98.7% of 2-rooted teeth showed type IV. In the second premolars, type I was the most prevalent (49.9%). The presence of two roots was more prevalent in first premolars, and the presence of one root was more prevalent in second premolars (P < 0.05). Male patients had a higher percentage of two roots compared with female patients (P < 0.05). Type IV and I was more prevalent in first and second premolars respectively (P < 0.05). There was a high frequency of 2-rooted and single-rooted teeth among maxillary first and second premolars respectively.

The internal root canal morphology of single-rooted mandibular canines revealed by micro-computed tomography.

CONTEXT: The lack of thorough knowledge regarding morphological and anatomical variations of the root canal system can result in failure to identify all root canals and lead to inadequate instrumentation and consequent failure of the endodontic treatment. AIMS: The aim of the study was to evaluate the anatomy of single-rooted mandibular canines using micro-computed tomography (micro-CT). SUBJECTS AND METHODS: Eighty mandibular canines were scanned by micro-CT to determine anatomic parameters: Vertucci's classification, number of lateral canals, roundness, and minor and major diameters. The shape of root canal orifices, volume, surface area, and convexity structure model index (SMI) were also evaluated. ANALYSIS USED: The CTAn and CTvol software were used to three-dimensional and morphometric analysis. RESULTS: All canals were classified as Vertucci Type I. Lateral canals were verified in 42.4% of the roots, in apical third. The canal diameters were decreased near the apex. The cross sections at the cementoenamel junction and 1 mm from the apex were oval in 38.3% and 79.4% of the canals, respectively. The mean canal volumes and surface areas were 11.48 ± 4.41 mm3≥ and 67.29 ± 19.18 mm2, respectively. The SMI values indicated that mandibular canine was approximately rod like. CONCLUSIONS: The morphology of the root canal of single-rooted canines varied widely at different root levels.