Balancing Demineralisation and Collagen Integrity: A Nanoscale Analysis of Etching Time in Human Dentin

Abstract

Objectives

To investigate the effect of varying phosphoric acid etching times on dentin demineralisation and collagen fibril morphology and to identify a time-dependent balance between sufficient mineral removal and preservation of collagen nanostructure.

Methods

Dentin specimens from human third molars were etched with 37% phosphoric acid for 6 different durations: 0, 5, 10, 15, 30, and 60 seconds. Demineralisation was assessed using attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy by calculating the phosphate-to-amide I absorbance ratio. Collagen integrity was evaluated using scanning electron microscopy (SEM) and atomic force microscopy (AFM) imaging, with the percentage of visible D-banding serving as a morphometric indicator.

Results

FTIR analysis revealed a progressive reduction in the phosphate signal with increasing etching time, following an exponential decay pattern and approaching a plateau at approximately 10 seconds. AFM imaging revealed that the D-banding periodicity reached its peak at 10 seconds (mean: 71.7%) and subsequently decreased, with notable degradation observed at 30 and 60 seconds. Statistical analysis indicated significant differences in D-banding between the 10-second group and longer etching durations (P < .05).

Clinical significance

This study provides nanoscale evidence that phosphoric acid etching of dentin for approximately 10 seconds achieves effective demineralisation while preserving collagen integrity. Longer etching times compromise fibril structure, underscoring the need for precise, time-controlled protocols to optimise resin–dentin bonding performance.

Introduction

Resin-based restorations have become the primary choice for the conservative management of carious lesions; however, their clinical success depends highly on proper technique and operator skill.^1,2 Achieving a durable bond to dentin requires strict adherence to adhesive protocols—whether etch-and-rinse, self-etch or universal systems—along with optimal field isolation and meticulous control of surface conditioning and resin application.³ Even minor deviations from the protocol, such as inadequate moisture control, improper etching or incomplete adhesive infiltration, can compromise outcomes and lead to premature restoration failure.³ Unlike amalgam restorations, which rely primarily on macromechanical retention and are less technique sensitive, resin restorations depend on the formation of a complex hybrid layer between the biomaterial and tooth structure to obtain micromechanical adhesion.^4,5 Therefore, optimising each step of the adhesive process is crucial to enhance clinical longevity.¹ Among adhesive strategies, the etch-and-rinse technique remains the gold standard for effectively removing the smear layer and conditioning dentin.⁶ It typically involves applying 32%-38% phosphoric acid for 15 seconds; this method aims to demineralise the superficial dentin layer and expose the collagen fibrils for adhesive infiltration.^5,6 However, prolonged etching may collapse the collagen matrix and impair resin monomer penetration, while insufficient etching can leave a residual smear layer and reduce micromechanical retention.^7,8 Therefore, the final quality of demineralisation strongly depends on the duration of acid application. For example, studies have examined how different etching times affect the micromorphology of dentin using scanning electron microscopy (SEM) and light microscopy to assess the demineralisation depth.9, 10, 11 Hashimoto et al. demonstrated that extended etching with 35% phosphoric acid increases hybrid layer thickness but leaves a demineralised zone not thoroughly infiltrated by resin, thereby weakening bond strength due to unsupported collagen fibrils.¹² In addition, a study by Marshall et al. highlighted that both the chemical formulation and delivery mode of phosphoric acid can influence etching patterns, particularly in the intertubular and peritubular regions.¹³ Several studies have evaluated the effect of varying phosphoric acid etching times on resin–dentin bond strength.14, 15, 16 Although these studies examine the morphological effects of phosphoric acid on dentin, the optimal etching time that balances mineral removal with preservation of collagen structure remains unclear. The biodegradation of the hybrid layer is a major contributor to resin–dentin bond failure.17, 18, 19 A key structural component of this layer—collagen fibrils—is particularly vulnerable to deterioration caused by host-mediated matrix metalloproteinase (MMP) activity and oral biofilm–related degradation, such as Streptococcus mutans–derived collagenase.^17,19 Consequently, the decline in bond strength observed in aged resin restorations is largely attributed to progressive collagen breakdown within the hybrid layer.^19,20 This study aims to identify a critical etching threshold that supports effective demineralisation while maintaining collagen structure with minimal damage to better withstand factors affecting resin-based dental restoration longevity.

Materials and methods

The University of Toronto Research Ethics Board (REB 44300) approved the collection of extracted, sound human permanent third molars. The teeth were scaled to remove soft tissue, stored in a 0.1% sodium azide solution, and refrigerated at 4 °C until use. All mineralised dentin sections were prepared using a water-cooled low-speed diamond blade mounted on an Isomet low-speed saw. Figure 1 provides an overview of the experimental workflow.

Fig. 1.

Workflow chart for the study. AFM, atomic force microscopy; ATR-FTIR, attenuated total reflectance-Fourier-transform infrared; DI, deionised; H₃PO₄, phosphoric acid; SEM, scanning electron microscopy.

Demineralisation protocol of dentin

A total of 108 coronal dentin specimens (1 ± 0.2 mm thick) were sectioned and randomly assigned to 1 of 6 etching time groups: control (no etch), 5, 10, 15, 30, and 60 seconds. Thirty-seven-percent phosphoric acid was applied in either gel form or solution form (prepared from 85% phosphoric acid stock). The acid–dentin reaction was neutralised in a Milli-Q water stream for 10 seconds. The samples were placed in a sonic bath for 10 minutes in Milli-Q water to remove any phosphoric acid gel deposits or solutions and any smear layer remaining on the sample surface. For infrared analyses, 2 types of acid etching forms were used: gel and solution (n = 6). After demineralisation, the samples were left to air-dry for 30 minutes before characterisation. For SEM imaging, after demineralisation with phosphoric acid solution (n = 3), the samples were fixed with 4% glutaraldehyde for 15 minutes at 4 °C and then sonicated in Milli-Q water for 5 minutes. Subsequently, they were dehydrated using an ethanol series comprising 50%, 70%, 95% and 100% concentrations. The specimens were then immersed in a series of hexamethyldisilazane (HMDS) solutions at 30%, 70% and 100% concentrations for 5 minutes each. Finally, they were air-dried in a fume hood for 10 minutes before characterisation. For atomic force microscopy imaging (AFM), the samples were polished with silicone-carbide grit papers (grades 500, 1200 and 2400), then washed with Milli-Q water for 15 seconds. To prevent residual debris on the surface, a 37% phosphoric acid solution (n = 3) was used instead of gel. After demineralisation and rinsing, the samples were fixed with 4% glutaraldehyde for 15 minutes at 4 °C and sonicated in Milli-Q water for 5 minutes. Finally, the samples were air-dried for 30 minutes before characterisation.

Rate of dentin demineralisation

Attenuated total reflectance – Fourier transform infrared spectroscopy (ATR-FTIR) was used to monitor the rate of demineralisation. The demineralised side of each disc was oriented towards the diamond window of a GladiATR accessory mounted in an iS20 FTIR spectrometer, operating at a resolution of 4 cm^-1 with 32 scans per spectrum. Spectra were baseline corrected and normalised before processing. The areas under the curve of the bands of interest (phosphate and amide I) were integrated, and the resulting phosphate/amide ratios were plotted using Origin software.

The time required to achieve 90% decay in the phosphate/amide ratio (t₉₀) was calculated using the exponential decay equation:

$$90\%decay=-t\times \text{ln}\frac{\left(0.1\times A0\right)-Y0)}{A0}$$ (1)

An empirical 90% threshold marks the beginning of the plateau phase, where further change becomes minimal. The demineralisation time constant (t) was obtained by fitting the exponential decay curve of the phosphate/amide ratio over time. A0 represents the initial phosphate-to-amide ratio at t = 0, and Y0 is the plateau value reached after the decay has stabilised (the asymptote).

Gross morphology of demineralised dentin

Scanning electron microscope imaging was performed using a FlexSEM 1000 II VP-SEM with an accelerating voltage of 10 kV. Seven random locations were chosen for each section and imaged at 10x and 20x magnification. The objective was to observe the effects of acid demineralisation on smear layer removal, exposure of dentinal tubules and any alterations in bulk demineralised dentin morphology, explicitly focusing on the localisation of 'amorphous/degraded collagen' in both peri- and intertubular demineralised dentin.

Nanoscale morphometry of demineralised dentinal collagen

Topographical changes on acid-etched dentin discs were visualised using an AFM operated with a Nanoscope IIIa controller. Samples were imaged in contact mode using MSLN-10 cantilevers. All imaging locations were standardised using the instrument’s motorised X–Y translator stage. After acquiring the first scan, the stage was translated laterally so that each subsequent imaging site was positioned at least 100 µm away from the previous location. This spacing ensured that no 2 scans overlapped and that each frame represented an independent region of the demineralised dentin surface. The same procedure was applied consistently for all specimens and experimental groups. Six 3 × 3 μm² images per sample were captured and processed using WSxM software.²¹ After thoroughly examining all images acquired from each sample and verifying that no intra-sample variation was present, 3 representative images per sample were randomly selected for quantitative analysis. Each image was divided into 36 (500 × 500 nm²) focus areas (n = 324 Areas of Interest per group). Three blinded, calibrated examiners assessed each mini-image for collagen D-banding periodicity (Figure 2). Given that the assessment involved more than 2 raters, inter-rater reliability was quantified using Fleiss’ Kappa analysis. Further details on this procedure are provided in Khattignavong et al.²² Kappa values were interpreted according to the Landis and Koch scale,²³ in which 0.01-0.20 indicates slight agreement, 0.21-0.40 fair, 0.41-=0.60 moderate, 0.61-0.80 substantial and >0.80 almost perfect agreement. The mean D-banding periodicity percentages were calculated for various etching times (5, 10, 15, 30, 60 seconds). The control group was excluded because it lacked an exposed collagen matrix.

Fig. 2.

Examples of different areas of interest (AOI). The scoring criteria are illustrated based on the presence or absence of D-banding periodicity. A score of 0 is assigned when no visible D-banding is observed in the AOI, whereas a score of 1 is assigned if any D-banding is visible in the image.

Statistical analysis

Statistical analyses were performed to evaluate the effects of etching time and phosphoric acid formulation on dentin demineralisation. Assumptions of normality and homogeneity of variance were assessed before analysis. For the phosphate-to-amide I ratio, a 2-way ANOVA was conducted, followed by Tukey's post hoc test for pairwise comparisons. In contrast, analysis of D-banding periodicity revealed a significant violation of homogeneity of variances (Levene’s test, P < .001); therefore, Welch’s ANOVA with Games-Howell post hoc tests was applied. All statistical analyses were conducted using OriginPro, with a significance threshold set at P < .05.

Results and discussions

Rate of demineralisation

The ideal etching procedure in dentistry aims to effectively remove the smear layer and open dentinal tubules while exposing collagen without causing any damage to generate a structurally stable substrate for resin infiltration.²⁴ However, acid exposure can lead to the breakdown of collagen into molecular fragments by selectively breaking intermolecular crosslinks, which are typically present to stabilise the fibrillar structure.²⁵ Therefore, the etching technique must achieve a balance between adequately exposing collagen fibrils within the dentin matrix and preserving their structural integrity. Figure 3A shows the ATR-FTIR spectra of the control dentin (black line) after exposure to 37% phosphoric acid at 5 different etching times, ranging from 5 to 60 seconds (red line). There was no notable difference in the FTIR spectra of dentin samples exposed to 37% phosphoric acid solution compared to those exposed to gel. In the control group, the mineral content, primarily represented by the phosphate band at 1,000 cm^-1, dominates the spectral fingerprint compared to the collagen matrix, which is represented by the amide groups. The amide groups include amide I at 1,650 cm^-1 (resulting from the stretching and bending of the carbonyl group, C=O), amide II at 1,550 cm^-1 (characterised by the N–H and C–N bonds), and amide III at 1,230 cm^-1. In the control sample, amides I and II appear very weak compared to the phosphate band, as the collagen fibrils are embedded within the mineralised matrix. Therefore, the absorbance of the collagen can be measured as negligible. After 5 seconds of etching, the phosphate₄ band decreases, and the amides I and II increase as the collagen becomes less mineralised. After 10 seconds, the phosphate band almost wholly vanishes, and amides I and II have become more notable. A 2019 study achieved similar findings.²⁶ By calculating the absorbance intensity ratios of the phosphate band to the amide I band (Figure 3B) we assessed demineralisation on the dentin surface within a 2-µm depth. As expected, the phosphate-to-amide I ratio decreased significantly with increasing etching time, reflecting progressive demineralisation of the dentin surface. The unetched control group (0 s) exhibited the highest ratio in both the gel (10.99 ± 2.99) and solution (10.69 ± 3.19) groups, consistent with fully mineralised dentin. All etched groups (5–30 s) showed statistically significant reductions in phosphate/amide I ratio compared to the control (P < .05), confirming the rapid onset of mineral loss following phosphoric acid application. However, there were no statistically significant differences between the gel and solution forms of phosphoric acid (P = .5893), indicating comparable demineralisation efficacy. Plotting the phosphate/amide I ratio (Figure 3C) reveals an exponential decay in both the gel and the solution (Adjusted R² = 0.85 and 0.89, respectively). Demineralisation reached a plateau at 90% decay after approximately 10.2 ± 1.6 seconds in the gel and 7.6 ± 1.1 seconds in the solution. The ATR crystal's evanescent infrared waves penetrate 2 µm, corresponding to the minimum demineralisation depth after 15 seconds of etching with 37% phosphoric acid.²⁷ To ensure complete resin monomer infiltration throughout the demineralised layer, it is advisable to minimise the etching depth, as this promotes the formation of a homogeneous hybrid layer without gaps. Previous studies indicated that bond strength at the resin–dentin interface correlates more positively with the quality of the hybrid layer—characterised by homogeneity and complete resin infiltration into the collagen matrix—than with its thickness.27, 28, 29, 30 Based on these considerations and our findings, we propose that an etching time of 10 seconds is sufficient to achieve 90% demineralisation while preserving the integrity of the collagen matrix.

Fig. 3.

Monitoring the rate of demineralisation as a function of time. A, FTIR spectra of control teeth pre-demineralisation (black line) and post-demineralisation (red line) with various etching times (0-60 s). B, The phosphate/amide I ratio as a function of demineralisation is presented in mean and standard deviation (P < .05). C, Exponential decay plot of the phosphate/amide I intensity band ratio for the gel and solution groups (Gel t1 = 4.06 ± 0.6; Adj.R² = 0.85, Solution t1 = 3.11 ± 0.4; Adj.R² = 0.89).

Gross morphology of demineralised dentin

Although the results from ATR-FTIR provided insights into the time required for a 37% phosphoric acid etchant to demineralise the dentin surface, they cannot provide the necessary information on the quality of the organic collagenous matrix post-demineralisation. Figure 4 shows the qualitative changes in the surface of dentin as a function of acid demineralisation. In the control samples (Figure 4A), the smear layer covers the whole surface, as it is difficult to observe any dentinal tubules. After 5 seconds of acid etching, some dentinal tubules were exposed; however, the smear layer is still evident on parts of the surface (Figure 4B). The groups of 10-60 seconds showed complete removal of the smear layer and exposure of dentinal tubules (Figure 4C-F). The smear layer is the debris of crushed hydroxyapatite and fragmented collagen created after tooth preparation.³¹ When phosphoric acid contacts the smear layer, its hydrogen ions react with the hydroxyl ions of hydroxyapatite, forming water and calcium phosphate ions in solution, thereby demineralising the mineral content within the smear layer.³² Furthermore, it hydrolyses the intermolecular crosslinks of the fragmented collagen,^25,33 resulting in the dissolution of the smear layer, followed by further demineralisation of peritubular and intertubular dentin. It is worth noting that the enlarged dentinal tubule in the 60-s group is a result of excessive demineralisation of the peritubular dentin. The 15-s group exhibits scattered bundles of collagen fibrils across the surface (Figure 4D). However, visualising and evaluating the quality of the collagen matrix and determining whether 37% phosphoric acid harmed its fibrillar structure was challenging using SEM. Therefore, AFM was employed to assess topographical changes at the nanoscale.

Fig. 4.

Representative SEM images of dentin as a function of phosphoric acid etching: 0 s (fully mineralised), 5 s, 10 s, 15 s, 30 s and 60s (10x magnification).

Nanoscale morphometry of demineralised dentinal collagen

One characteristic feature of healthy collagen is the presence of a 67-nm D-banding periodicity, which reflects the highly ordered, staggered arrangement of collagen molecules within the fibril.^34,35 Any reduction or loss of this periodicity indicates a disruption of this supramolecular organisation and reflects underlying biochemical and mechanical alterations in the fibrillar matrix. Acid exposure can induce breakdown of the intermolecular crosslinking networks that stabilise molecular alignment, resulting in diminished fibrillar organisation, reduced stiffness and increased susceptibility to enzymatic degradation.^34,36 These nanoscale structural changes are highly relevant to the performance of the resin–dentin interface, as compromised collagen architecture undermines hybrid layer stability and contributes to reduced long-term bond durability.^9,19,20 Figure 5A presents AFM topographical imaging of dentin surfaces after various etching times (0-60 s). In the control group with mineralised dentin, collagen is absent (Figure 5A-i); after 5 seconds, discrete fibrils become visible on the dentin surface, as shown in Figure 5A-ii. The complete exposure of the collagen matrix occurs after 10 seconds, at which point the D-banding of the fibrils is visible (Figure 5A-iii). This is also evident at the 15-s mark (Figure 5A-iv). Although collagen fibrils remain visible in the 30-s group, portions of the surface exhibit loss of fibrillar structure, characterised by absent or indistinct D-banding and areas of amorphous, disorganised topography (Figure 5A-v). By 60 seconds, the fibrillar structure of the collagen matrix is no longer distinguishable because of the extensive breakdown of fibrillar crosslinks, leading to the collapse of the collagen matrix (Figure 5A-vi).

Fig. 5.

A, Representative topographical AFM images of dentin as a function of acid etching: 0 s (ctrl), 5 s, 10 s, 15 s, 30 s and 60 s. Arrows in A-ii point to some of the collagen fibril D-banding. B, Box-plot of the D-banding periodicity percentage among demineralised groups presented as mean ± SD (n = 324). Statistical significance of P < .05 was defined using Welch’s ANOVA and Games-Howell pairwise comparisons.

To quantify the impact of acid on the collagen structure, 3 calibrated examiners measured the percentage presence of D-banding on the surface of the dentin as a function of the demineralisation time based on the scoring system illustrated in Figure 2. The inter-rater kappa value was 0.81, indicating substantial agreement between examiners. Figure 5B presents the box plot of the mean for D-banding periodicity percentage among the acid-etched groups. Upon 5 seconds of etching, only a negligible amount of D-banding was detected (mean = 17.6%). Based on the FTIR phosphate/amide I ratio for this group (Figure 3B), this limited visibility is attributed to residual mineral masking the collagen surface rather than fibrillar disruption. After 10 seconds, collagen fibrils and D-banding periodicity were evident in most areas, with a mean coverage of 71.7% over the sample surface. In the 15-second group, the mean percentage dropped to 57.3%. However, the difference in mean percentage of D-banding periodicity between 10 and 15 seconds was not statistically significant (P > .05). Increasing the etching time to 30 seconds resulted in a statistically significant decrease in D-banding, with a mean of 28.2% compared to the 10-second group (P < .05). Importantly, the FTIR phosphate/amide I ratio for this group demonstrated ∼97% mineral loss relative to the fully mineralised control, confirming that the reduced D-banding cannot be attributed to residual hydroxyapatite. Instead, the combined chemical and morphological evidence indicates true collagen denaturation, consistent with acid-induced hydrolysis of intermolecular crosslinks and collapse of the fibrillar architecture. This is particularly important when using selective enamel etching (SEE), as a 2023 study found that the SEE technique resulted in inadvertent dentin conditioning near the DEJ.³⁷ Marshall et al. first used AFM to examine the topography of dentin after demineralisation with dilute phosphoric acid. They focused on depth changes in the peritubular matrix and intertubular region rather than the quality of collagen fibrils and the D-banding.¹⁴ To our knowledge, no systematic research has been done to characterise the submicron topographical changes of dentin with a clinically relevant duration of 37% phosphoric acid etching.

Clinical relevance of etching time in resin–dentin bond durability

Biodegradation at the resin–dentin interface remains a primary cause of restoration failure, often initiated by marginal discrepancies and microgaps that facilitate bacterial infiltration and enzymatic degradation of both the restorative material and the underlying dentinal collagen.^17,38 While phosphoric acid etching is essential for effective smear layer removal and resin monomer infiltration, particularly in etch-and-rinse systems, its acidic nature simultaneously activates endogenous matrix metalloproteinases (MMPs), which can degrade unprotected collagen fibrils and potentially compromise the structural integrity of the bonded interface.^17,39 Furthermore, excessive etching may disrupt the fibrillar structure of the matrix by breaking down intermolecular crosslinks,³³ leading to collapse of the collagen network, hindering resin monomer penetration and creating gaps that can activate MMPs and initiate collagen degradation.⁴⁰

Although manufacturers commonly recommend a 15-second phosphoric acid etching time for dentin,⁴¹ several investigations have suggested that substantially shorter application periods may still achieve adequate smear layer modification and collagen exposure for effective adhesive infiltration.^15,42 A systematic review reported improved long-term bond strength in etch-and-rinse systems when reduced etching durations were used in primary dentin.⁴³ Clinical findings appear to support this trend, with a randomised trial showing better, though statistically comparable, outcomes when a 7-second etch was used rather than the traditional 15-second protocol in primary teeth.⁴⁴ Micromorphological studies show that increasing etching time from 5 to 15 seconds was associated with a progressively thicker uninfiltrated collagen zone, indicating deeper demineralisation and producing fibrillar regions more vulnerable to collapse and degradation.⁴⁵ Scheffel et al. extended this understanding to permanent dentin, showing that etching durations shorter than 10 seconds significantly reduced μTBS, whereas 10- and 15-second etching produced comparable bond strengths with a 2-step etch-and-rinse adhesive. The authors therefore identified approximately 10 seconds as the minimum effective duration for adequate demineralisation in permanent dentin, noting that further etching did not improve bonding performance.¹⁶ Recent evidence further reinforces this time-controlled rationale.

A study by Santos et al. demonstrated that reducing etching time to 3 seconds resulted in superior bond strength and lower long-term nanoleakage compared with the conventional 15-second protocol when used with a universal adhesive system.¹⁵ These external findings closely parallel our FTIR and AFM observations. Our kinetic analysis demonstrated that the transition into the demineralisation plateau occurs at approximately 10 seconds, beyond which additional mineral loss is minimal (Figure 3C). AFM imaging further showed that fibrils etched for 10 seconds exhibited the highest degree of structural organisation (Figure 5). Although differences between the 10- and 15-second groups did not reach statistical significance, the subtle reduction in D-banding periodicity and early signs of fibrillar disorganisation observed at 15 seconds may reflect the initial stages of collagen structural alteration associated with extended acid exposure. The present study demonstrates that etching dentin with 37% phosphoric acid for approximately 10-15 seconds achieves a balance between effective smear layer removal and preservation of collagen matrix architecture. As adhesive failure and secondary caries continue to be the leading causes of restoration replacement,¹⁷ approaches that preserve collagen integrity, as one component of interface stability, are worth exploring. Therefore, we proposed limiting etching to 10 seconds, given that it constitutes the most conservative choice: it achieves necessary smear layer removal and collagen exposure while minimising overetching, reducing unnecessary demineralisation and preserving collagen architecture that may otherwise become more susceptible to enzymatic degradation. Future research should validate these in vitro results through large-scale clinical trials and examine their effectiveness across various substrates, including caries-affected and sclerotic dentin. Moreover, although the 10-second etching protocol is expected to be suitable for both 2-step and 3-step etch-and-rinse systems, which follow similar procedures of smear layer removal, collagen exposure and resin infiltration, the actual bonding performance may vary according to the adhesive’s chemical formulation.^3,46 A related consideration is the advent of universal adhesives, formulated to be compatible with total-etch, self-etch and selective-etch approaches, allowing clinicians greater flexibility and simplifying operative protocols.47, 48, 49 Many of these systems contain functional monomers such as 10-MDP that chemically interact with residual hydroxyapatite. In such formulations, shorter etching times may be particularly advantageous because they preserve more mineral for MDP–Ca salt formation, thereby promoting a more stable chemical interaction within the hybrid layer.50, 51, 52

Conclusion

This study employed ATR-FTIR, AFM and SEM to assess the nanoscale effects of phosphoric acid etching on dentin, revealing a critical threshold around 10 seconds beyond which collagen integrity may be compromised. Consistent trends highlight the value of combining chemical and morphological analyses to optimise adhesive protocols and improve the quality of the dentin–biomaterial interface during resin restoration. Further investigation through clinical trials is warranted to validate these findings and support evidence-based recommendations.

Author contributions

Conceptualisation: Alzubaidi, Bozec; Visualisation: Alzubaidi, Somogyi-Ganss, Bozec; Funding acquisition: Bozec; Methodology: Alzubaidi; Investigation: Alzubaidi, Ganss; Formal analysis: Alzubaidi, Vaez, Neshatian, Aguayo, Somogyi-Ganss, Bozec; Writing—original draft: Alzubaidi, Somogyi-Ganss, Bozec; Writing—review and editing: Alzubaidi, Vaez, Neshatian, Aguayo, Somogyi-Ganss, Bozec.

Conflict of interests

None declared