Evaluation of the effects of retro-cavity preconditioning with or without ethylenediaminetetraacetic acid on root surface pH and dislodgement resistance of NeoMTA2 and MTA Flow retro-fills; an ex vivo investigation

doi:10.21203/rs.3.rs-4290785/v1

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Evaluation of the effects of retro-cavity preconditioning with or without ethylenediaminetetraacetic acid on root surface pH and dislodgement resistance of NeoMTA2 and MTA Flow retro-fills; an ex vivo investigation

https://doi.org/10.21203/rs.3.rs-4290785/v1

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Background

The aim of this study was to investigate the effects of retro-cavity preconditioning with or without 17% ethylenediaminetetraacetic acid (EDTA) solution on root surface pH as well as dislodgement resistance of NeoMTA2 and MTA Flow retro-fills.

Methods

Forty-eight single-rooted human incisors were selected. After completion of endodontic treatment, root-end resections were done and retro-cavities prepared. The samples were randomly divided into two groups of A and B (n = 24 each). In group A, retro-cavities were pre-conditioned with 2.5% NaOCl, followed by 17% EDTA solution, whereas in group B, pre-conditioning was done using 2.5% NaOCl before final irrigation with normal saline. Samples in each group were randomly subdivided into two subgroups of 1 and 2. Retro-fillings in A1 and B1 subgroups were done with MTA Flow and in A2 and B2 subgroups with NeoMTA2. Root surface pH was measured in each sample at three different stages; before preparation of retro-cavities (pH0), after retro-cavity pre-conditioning (pH1), and three days after retro-filling (pH2). Subsequently, pushout bond strength (PBS) of the retro-filling materials was measured by a universal testing machine and their failure modes visualized under 64x magnification.

Results

Preconditioning with EDTA caused a significant increase in PBS for both materials (p < 0.001). There was no significant difference between average bond strength of MTA Flow and Neo MTA2 (p = 0.271). There was an increase in average pH2 compared to pH1 and pH0 in all groups (p < 0.001). Use of EDTA resulted in a significant increase in average pH2 in MTA Flow compared to Neo MTA2 (p = 0.027). Groups preconditioned with EDTA more frequently indicated a cohesive failure mode.

Conclusion

Use of EDTA significantly increased pushout bond strength of retro-fill materials to dentin. However, it did not prevent ultimate alkalinity of retro-filled cavities.

Bond strength

Calcium silicate cement

Dentin

EDTA

Root canal

Periradicular surgery is indicated to remove causative irritants when nonsurgical endodontic (re-)treatments result in persistent disease(1). It involves several steps including retro-filling the resected and prepared root end cavity with an appropriate material(2). In order to have long-term success, mineral trioxide aggregate (MTA) has been widely accepted to be used as the retro-filling material due to its sealing ability, biocompatibility, hard-tissue stimulation, and antibacterial properties(3–6). To overcome some disadvantages of MTA such as long setting time, poor handling properties, low resistance to compression and bending, and tooth discoloration potential(2, 6, 7), a few changes have been made to its original formulation. For example, Neo MTA 2 (NuSmile Avalon Biomed, Bradenton, FL, USA) is recently introduced with fine powder particles and tantalum oxide as the radiopacifier. It is mixed with a gel in a desired consistency to provide users with improved handling properties(8).

Another example is MTA Flow (Ultra dent Products Inc., South Jordan, UT), which consists of a water-soluble silicone-based gel and a gray powder containing bismuth oxide, tricalcium, and dicalcium silicate. It can be prepared in different consistencies by changing the powder-to-gel ratio, thereby providing clinicians with ease of material application and placement for different endodontic treatments(8). In addition to similar physico-chemical properties such as low solubility, acceptable radiopacity, as well as comparable ability to deposit calcium phosphate, similar alkalinizing capability of MTA Flow has also been reported in comparison with MTA Angelus after being soaked in simulated body fluid(9).

It is preferable for a retro-filling material to have a strong bond at the material-dentin interface to resist against dislodging forces during function(10). It is believed that bond strength of materials to root dentin is in line with the material sealing ability(11). Push-out bond strength (PBS) test is considered as one of the most well-recognized laboratory assessments in evaluating the bond strength between root filling material and dentin(12). Although the main advantage of PBS test over other bond testing approaches is to evaluate the material bond strength while being surrounded by dentin, there is no consensus whether the results of this assessment is clinically applicable(2).

Dentin conditioning with ethylenediaminetetraacetic acid (EDTA) has been widely advocated in endodontics. It has been demonstrated that EDTA as a chelating agent is capable of removing inorganic components in dentin structure as well as the smear layer created following dentin preparation. Promoting cyto-differentiation and tissue formation as well as provoking growth factor release throughout the root canal dentin has been pointed out as other advantages of EDTA application in endodontics(13).

On the other hand, an alkaline pH following use of calcium silicate cements (CSCs) is required to eliminate endodontic pathogens. It also can favor apatite nucleation, release of alkaline phosphatase and bone morphogenetic protein 2 which are involved in periapical healing(14, 15).

Since limited information was available concerning the physico-chemical properties of NeoMTA2 and MTA Flow, the present study aimed to evaluate the effects of retro-cavity preconditioning with or without EDTA on root surface pH changes as well as dislodgement resistance of these two recently introduced CSCs used as retro-fills in an ex vivo model.

This ex vivo investigation was approved by Research Ethics Committee of the School of Dentistry, Tehran University of Medical Sciences (IR.TUMS.Dentistry.Res.1401070) and was performed on human extracted maxillary incisor teeth.

4.2.1 Sample selection and preparation

In this study, a total of 48 caries- and resorption-free teeth which were extracted for periodontal reasons were involved. Teeth with straight roots and type I root canal anatomy based on Vertucci’s classification were included. Teeth with cracks, fractures or developmental anomalies were excluded. Radiographs were taken from all teeth in both buccolingual and mesiodistal directions to confirm Vertucci’s type I root canal configuration, apical maturity, and absence of any previous endodontic treatments. The soft tissues on the root surfaces were removed with a scaler (Universal Sickle Scaler, Nordent, Solingen, Germany) and the teeth were kept in 0.5% Chloramine-T solution (Merck, Darmstadt, Germany) for disinfection for 24 hours.

Initially, an access cavity was prepared on each tooth using a high-speed fissure bur (Livingstone, Korea) under water and air spray. Then, a #10 K-File (Micro-Mega, Besançon, France) was inserted into the root canal and extruded from the apical foramen. As soon as the file tip was seen from the root end, a one-millimeter short measurement was considered as the working length for the entire root canal preparation and obturation procedures. Root canal preparation was performed using Gold Denco rotary system (Shenzhen Denco Medical Co, China) up to #F3 rotary file according to the manufacturer’s recommendations. Root canals were irrigated with 2.5% NaOCl solution (Morvabon Co, Iran) between each file use. The teeth were obturated using cold lateral compaction with 0.02 taper gutta-percha points (Data Co, China) and AH 26 sealer (Dentsply DeTrey GmbH, Konstanz, Germany). Afterwards, the teeth were incubated at 37°C for 24h under 100% humidity. Subsequently, root-end resection was performed on each tooth root at a 3-mm distance from apex using a carbide fissure bur (Jota AG, Switzerland) under water spray and a 3-millimeter deep retro-cavity was prepared using diamond-coated retro-tips powered by an ultrasonic device (Ultra Mint Pro, Eighteenth Co, China) using E (endodontic) mode at the power of #3 under water spray.

4.2.2 Preparation and pre-conditioning of the retro cavities

The samples were randomly divided into two groups of A and B, each containing 24 samples using simple randomization aided by www.randomization.com. In group A, retro-cavities were pre-conditioned with 5mL of 2.5% NaOCl, followed by 5mL of 17% EDTA solution (Morvabon Co, Iran) and rinsed with 5mL of normal saline (Daroo-Pakhsh Co, Iran) each for 1 min. In group B, the retro-cavities were pre-conditioned using 2.5% NaOCl and irrigated by normal saline solutions using the same volumes and times.

The samples in each group were randomly divided by the same method mentioned above into two subgroups of 1 and 2. Retro-fillings in A1 and B1 subgroups were done with MTA Flow (Ultradent Products Inc, South Jordan, UT, USA) and in A2 and B2 subgroups with NeoMTA2 (NuSmile Avalon Biomed, Bradenton, FL, USA). (See Table 1.) In all subgroups, the retro-filling materials inside the cavities were 3 mm thick. The retro-cavities were completely dried prior to retro-fillings and each material was prepared and placed according to the manufacturer’s recommendation. Eventually, the surface of each retro-fill material was covered with a wet cotton pellet and placed in a specified falcon each.

The teeth were subsequently incubated at 37°C in100% humidity for 7 days to ensure completion of the retro-fill setting reactions.

Table 1

Experimental groups categorized based on their pre-conditionings and retro-fillings
Experimental group		Pre-conditioning	Retro-filling
A (n = 24)	A1 (n = 12)	2.5% NaOCl + 17% EDTA + Normal saline	MTA Flow
A (n = 24)	A2 (n = 12)	2.5% NaOCl + 17% EDTA + Normal saline	Neo MTA2
B (n = 24)	B1 (n = 12)	2.5% NaOCl + Normal saline	MTA Flow
B (n = 24)	B2 (n = 12)	2.5% NaOCl + Normal saline	Neo MTA2

4.2.3 Measurement of pH

Measuring root surface pH was carried out using a digital pH meter device pen (Sentek Co, UK) within the specified falcon for each sample and the number displayed by the device monitor was recorded as the sample’s pH. Prior to each measurement, a neutral substance with a known pH of 7 was tested to calibrate the device according to the manufacturer’s recommendation. Measurement of pH in each sample was done at three different stages:

A. At the beginning of the study, before the preparation of retro-cavities (recorded as the baseline or pH0),
B. Immediately after retro cavity preconditioning (recorded as pH1), and
C. Three days after placing the retro-fill materials (recorded as pH2).

4.2.4 Pushout bond strength (PBS) test

After one week, the setting of the retro-filling materials was confirmed by a size #B endodontic spreader (Dentsply-Maillefer, USA). The samples were placed in a mold filled with self-curing acrylic resin (Beta Dent, Iran). The samples were buried from the coronal region so that the apical end of the cavity was tangential to the distal edge of the acrylic resin. After complete setting of the acrylic molds, the samples were removed from the mold to prepare one-millimeter disks, at a one-millimeter distance coronal to the resected apex. Initially, the samples, which were held in cylinders, were fixed horizontally on the stand of the cutting machine using a glue (Super glue. Razi, Iran). Then, the samples were horizontally fixed from the apical area and were cut with a diamond disk under water and air spray at a slow speed by a cutting machine (Pars Mechatronic, Iran). The samples were visualized under 3.5X magnification to rule out any fractures or defects. Then, the surface roughness of each sample was smoothed out using a soft sandpaper. After preparing all the discs, push-out bond strength was measured using a universal testing machine (UTM) (Santam Co. Iran).

In order to perform the pushout bond strength (PBS) test, the prepared disks were fixed with a piece of wax in the universal testing machine and a plunger, 0.7mm in diameter, was fit to the retro-filled portion at the center of the disc to apply pushout force at the speed of 0.5mm per minute until dislodgement occurred. Meanwhile, a diagram related to the application of force was drawn by SCM-3000 software (Microtest; Madrid, Spain). Therefore, within the force diagram drawn by the software, which had an upward trend, an immediate drop was observed which indicated failure. The maximum force required to dislodge the retro-filling material was recorded in Mega Pascals (Mpa).

Eventually, the sections were examined under 64x magnification using a scanning stereomicroscope (Olympus, Japan) in order to investigate the state of bond failure. Then a photomicrograph of each disk sample was taken to visualize the type of failure.

Types of bond failure situations were classified as:

a)Adhesive failure which occurred between the retro-filling material and the dentin. (Fig. 3.A)

b) Cohesive failure which occurred within the retro-filling material (Fig. 3.B)

and

c) Mixed failure which encompassed both above mentioned failure types. (Fig. 3.C)

4.2.5 Statistical analysis

Kolmogorov–Smirnov test was used to assess normal distribution of data and two-way ANOVA test was used to determine the effect of pre-conditioning on PBS of the retro-filling material as well as the root surface pH values. The p value of less than 0.05 was considered statistically significant.

This investigation evaluated the effects of the use of 17%EDTA as a pre-conditioner on dislocation resistance of Neo MTA2 and MTA Flow retro-fills, as well as root surface pH changes after 3 days.

4.3.1 Push-out bond strength (PBS):

As shown in Fig. 1, pre-conditioning the retro-cavities with 17%EDTA caused a significant increase in PBS of the samples (p < 0.001). The results showed that there was not a statistically significant difference between A1 and A2 subgroups (p = 0.271).

4.3.2 pH changes:

4.3.2.1 pH1: (after retro cavity preconditioning)

The results showed that there was a significant decrease in average root surface pH1 in group A compared with group B (p < 0.001). There was not any significant difference between average pH1 in A1 and A2 subgroups (p = 0.212).

3.3.2.2 pH2: (3days after placing the retro-filling materials)

The average pH2 showed significantly lower values in A2 subgroup compared to B2 (p = 0.011).

Additionally, while comparing different retro-fill materials, higher pH2 values were observed in A1 subgroup compared to A2 (p = 0.027). However, there was no significant difference between B1 and B2 subgroups (p = 0.141). There was not any significant difference between average pH2 values in A1 and B1 subgroups (p = 0.5 11).

4.3.2.3 Comparison of pH1 and pH2:

As shown in Fig. 2, regardless of the type of retro-filling material used or whether or not preconditioning with17%EDTA was done, there was a significant increase in the average pH2 in comparison with pH1 (p < 0.001).

4.3.2.4 Comparison of pH1 and pH2 with pH0:

The average values for pH1 were significantly higher than those for pH0 in B1 and B2 subgroups (p < 0.001). However, no significant difference between pH0 and pH1 values was observed between A1 and A2 subgroups. As demonstrated in Table 2, there was a significant increase in pH2 values compared with pH0 in all subgroups (p < 0.001).

Table 2

Comparison of pH1 and pH2 with the baseline pH (= 7.5) in experimental groups with or without preconditioning with EDTA
Material	Preconditioning with EDTA	pH2	p	pH1	p
MTA Flow	No	11.16 ± 0.37	p < 0.001	8.59 ± 0.23	p < 0.001
MTA Flow	Yes	11.24 ± 0.23	p < 0.001	7.64 ± 0.24	p = 0.073
Neo MTA2	No	11.45 ± 0.55	p < 0.001	8.45 ± 0.21	p < 0.001
Neo MTA2	Yes	10.87 ± 0.47	p < 0.001	7.55 ± 0.46	p = 0.692

4.3.3 Modes of failure:

The prevalent form of bond failure observed was cohesive within the A1 and A2 subgroups, while demonstrating a mixed nature within B1 and B2 subgroups. (See Fig. 4 for details.)

Providing adequate apical seal against trafficking of bacteria and their byproducts is among the most important factors influencing the long-term success of periradicular surgery(16). Adequate frictional or micromechanical bond strength between calcium silicate cements(CSCs) used as retro-fillings and radicular dentin is advantageous in providing a sustainably impervious interface(17). In this realm, dislodgement resistance of a retro-filling material can be typically evaluated using a PBS test(18). It has been documented that PBS can correspond with clinical situations in which forces are applied parallel to the long axis of the tooth, creating a shear force at the material-dentin interface(19, 20). PBS test has been considered by a number of investigators as an efficient, reliable and practical method in evaluating bond strength of a (retro-)filling material to root canal dentin(21–29). Several factors are influential in the results of PBS of an endodontic (retro-)filling material such as the irrigants used prior to the material application, containment of the retro-fill material within radicular dentin (increased C-factor), presence or absence of the smear layer, and the tubular size and density in surrounding dentin(10, 30–32). In this study, Neo MTA2 and MTA Flow were the CSCs used as retro-fillings due to their recent introduction as well as improved handling and structural properties. To the best of the authors’ knowledge, limited studies were found in the available literature to evaluate their physico-chemical properties. Therefore, the purpose of this study was to investigate the effects of retro-cavity preconditioning with or without EDTA on dislodgement resistance and failure modes of NeoMTA2 and MTA Flow retro-fills as well as its effect on root surface alkalinity in an ex vivo model.

Use of 17% EDTA solution has been advocated by some authors in pre-conditioning retro-cavities(33–35). EDTA is a weak acidic solution with a pH ranging from 4 to 6. It is used in endodontics to help remove the organic components of the smear layer. Some authors have supported the use of EDTA in endodontic and periodontal surgeries(36–38). Acting as an anti-oxidant, EDTA is capable of removing the so-called “oxygen-rich layer” which can, in turn, improve the interaction between CSCs used as retro-fillings and radicular dentin(39). EDTA performs as a chelating agent that has an affinity to divalent cations e.g. calcium ions and can make them into soluble chelates(40). Its chelating effect on bacterial cell wall which results in more susceptibility to antibiotics and antibacterial agents can be responsible for its bactericidal effect(40). It is applied to remove inorganic components of smear layer and is able to release growth factors from dentin and stimulate cell differentiation(13). However, compromising dentin structure due to microscopic erosions as well as decreased bond strength and microhardness of CSCs has been reported following use of EDTA due to its interference with CSCs hydration(13, 24, 41).Nevertheless, in the current investigation, pre-conditioning the retro-cavities with EDTA resulted in an increase on bond strength of NeoMTA2 and MTA Flow with dentin wall. This finding is in line with those of Gokturk et al who similarly stated that a significant increase in dislocation resistance of MTA Flow and MTA Angelus was found following application of 17% EDTA compared to chitosan-based silver nanoparticles (AgNPs-chitosan), and maleic acid(17). Consistently, Valencia et al stated that 17% EDTA was able to improve PBS of calcium silicate cement to dentin when used as a retro-filling material(42). This was also supported by Ballal et al who reported that use of 17% EDTA as an irrigation solution could increase PBS of Biodentine(20). Likewise, Anju et al also showed that, maximal increase in PBS of NeoMTA Plus was achieved, when 17% EDTA was used as the pre-conditioner(43). This can be due to the capability of EDTA application following NaOCl irrigation in removing the smear layer that could aid in forming retro-fill material tags into conditioned dentin, increasing the micromechanical surface area at the bond interface(44, 45). These results are contradictory to finding of Paulson et al., who concluded that in root canals irrigated with 2.5% sodium hypochlorite and 17% EDTA, lower PBS values were recorded in EDTA-treated groups following use of Biodentine as the rertro-filling(46). Authors claimed that acidic nature of EDTA could have detrimental effects on the hydration of the CSCs that could, in turn, result in impaired bonding capabilities. Additionally, this contradictory result can be related to the difference in dentin structure where the samples had been taken from. Contrary to our investigation, the dentin samples in Paulson et al’s study were taken from the middle third of the root which have different tubular and structural pattern compared with apical dentin used in the current investigation(46) In addition, Gade et al. demonstrated that use of 17% EDTA could compromise PBS of CSCs to dentin. This might be related to immersion of root sections in sodium hypochlorite for 5 minutes and in 17% EDTA for 30 minutes which could deteriorate dentin structure for further assessment(47).

It has been stated that, higher frequency of cohesive failure while performing PBS test can indicate a stronger resistance against dislocation in comparison with that of adhesive type material disintegration. This can be supported by the findings of some authors who pointed out that a strong bond is formed between CSCs and dentin via formation of interfacial tag-like structures during biomineralization through a diffusion-controlled reaction(48). Confocal laser as well as scanning electron microscopic images revealed that the flowable consistency of CSCs could permit intra-tubular penetration of the cements. In addition, a structurally altered inter-tubular dentin subjacent to the investigated CSCs was visualized at the cement-dentin interface referred to as “mineral infiltration zone” which conceptually resembled dentin-adhesive interface(49). The type of failure for each retro-filling material under dislodgement forces can be closely related to the amount of PBS(46). In the present study, subgroups preconditioned with EDTA showed higher PBS records, meanwhile indicating cohesive failure mode as the most common feature encountered. This finding can endorse stronger bonds between retro-fill materials and EDTA-treated dentin surfaces. However, mixed type of failure mode was observed in non-EDTA treated subgroups as the most frequent finding. An alkaline pH following release of hydroxyl and calcium ions from the CSCs during endodontic surgical interventions favors elimination of the remaining pathogenic microorganisms(14, 15) via reacting carbon dioxide(15, 50) as well as fostering apatite nucleation and increased hard-tissue formation via upregulating alkaline phosphatase and bone morphogenetic protein 2 (BMP2) which, in turn promotes sealing ability of CSCs(15, 51–53). In this study NeoMTA2 and MTA Flow were able to alkalinize the environment similar to other CSCs. (9, 54–56)

The present study showed that 17% EDTA used to precondition the retro-cavities could initially decrease environmental pH, but did not interfere with the secondary alkalinity induced by the CSCs after 3 days. The present study showed that MTA Flow was more effective than NeoMTA2 in creating a higher pH on the root surface after three days. This finding may probably be due to a more pronounced capability of MTA Flow to release hydroxyl ions to a pre-conditioned environment in comparison with NeoMTA2 which needs further evaluation(15). Since pH differences was not considered statistically significant in non-preconditioned groups, this might be attributable to a chemical interaction between MTA Flow and EDTA at the presence of dentin(15).Although this should be corroborated by more detailed investigations. Guimarães et al demonstrated that more alkalinity of MTA Flow compared with MTA Angelus in a similar time interval(9).

According to the point that the results obtained by laboratory studies cannot be extrapolated to the clinical situations, more detailed investigations are recommended to evaluate the effect of preconditioning retro-cavities on the long term stability of the retro-filling materials as well as their effect on the healing process of the apical periodontitis.

Use of EDTA significantly increased pushout bond strength of the NeoMTA2 and MTA Flow to dentin. However, it did not prevent ultimate alkalinity of retro-filled cavities in 3 days.

ethylenediaminetetraacetic acid (EDTA)
mineral trioxide aggregate (MTA)
Push-out bond strength (PBS)
calcium silicate cement (CSC)
Mega Pascals(Mpa)

Ethical approval

This ex vivo investigation received approval from the Research Ethics Committee of the School of Dentistry, Tehran University of Medical Sciences (IR.TUMS.Dentistry.Res.1401070) and was conducted using human extracted maxillary incisor teeth.

Consent for publication

This section is not applicable pertaining to this investigation.

Consent to Participate declaration

Given the absence of human participants in this study, the inclusion of this section is deemed “NOT APPLICABLE” for this manuscript.

Availability of data and materials

The data supporting the findings of this investigation will be available upon request from the corresponding author [SAA and HA].

Competing interests

The authors of the manuscript affirm that they have not received any financial support by any producing company pertaining to the study and therefore they have no conflicts of interest to declare pertaining to this work.

Funding

The research leading to these results has received funding from a research project titled " Evaluation of the effects of retro-cavity preconditioning with or without EDTA on root surface pH and dislodgement resistance of NeoMTA2 and MTA Flow retro-fills; an ex vivo investigation " in the frame of a postgraduate dissertation under the Grant Agreement number 133-62905 dated February 2023.

Authors contribution

Sedigheh Khedmat DDS, MSc contributed to conceptualization and methodology, validated the raw data and performed the formal analysis, while also managing resources and data curation.

Hadi Assadian DDS, MSc, played a pivotal role in conceptualization and methodology, data validation, and also took charge of writing the manuscript draft and subsequent review and editing.

Seyyed Ali Abaee DDS, MSc performed sample preparation and original data extraction under the supervision of [SK] and [HA].

Project administration was overseen by [SK] and [HA], with funding acquisition managed by [SK and [SAA].

Acknowledgements

The authors extend their sincere gratitude to Dr. Mohammad Javad Kharrazifard for his exceptional support and expertise in conducting the statistical analyses.

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Evaluation of the effects of retro-cavity preconditioning with or without ethylenediaminetetraacetic acid on root surface pH and dislodgement resistance of NeoMTA2 and MTA Flow retro-fills; an ex vivo investigation

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Abstract

Background

Methods

Results

Conclusion

Figures

Background

Methods

4.2.1 Sample selection and preparation

4.2.2 Preparation and pre-conditioning of the retro cavities

4.2.3 Measurement of pH

4.2.4 Pushout bond strength (PBS) test

4.2.5 Statistical analysis

Results

4.3.1 Push-out bond strength (PBS):

4.3.2 pH changes:

4.3.2.1 pH1: (after retro cavity preconditioning)

3.3.2.2 pH2: (3days after placing the retro-filling materials)

4.3.2.3 Comparison of pH1 and pH2:

4.3.2.4 Comparison of pH1 and pH2 with pH0:

4.3.3 Modes of failure:

Discussion

Conclusions

Abbreviations

Declarations

Ethical approval

Consent for publication

Consent to Participate declaration

Availability of data and materials

Competing interests

Funding

Authors contribution

References

Additional Declarations

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