The use of digital texture image analysis in determining the masticatory efficiency outcome.

The mixture level of gum samples consisting of two colours can be assessed visually, using the electronic colorimetric method, employing digital image processing techniques and specially designed software. The study investigates the possibility of an alternative method called "digital texture image analysis" (DTIA) to assess improvement of masticatory efficiency in denture wearers. The objectives were i) to evaluate whether DTIA discriminates changes in the colour mixing ability within a group over time; ii) to determine whether DTIA can be used to detect improvement in chewing ability; iii) to select the most appropriate DTIA feature that sufficiently describes masticatory efficiency in CDs wearers. The study was designed as an intra-individual evaluation of masticatory efficiency, which was assessed in participants with new dentures in three follow-up times. A set of four texture features was used in the current study. Uniformity, Contrast, Homogeneity and Entropy of the obtained chewing-gum samples were correlated to the degree of gum comminution. A statistically significant difference in masticatory efficiency was observed based on the values of the analysed DTIA variables of gum samples-Uniformity, Contrast, Homogeneity, and Entropy-have changed in the participants during the observation period. The improvement of the masticatory function in relation to the mixing ability of two-coloured chewing gum could be traced by monitoring changes in the values of DTIA variables. The most increasement of masticatory efficiency was observed by monitoring DTIA parameters such as contrast, and homogeneity.

Introduction

It is indisputable that tooth loss and edentulism have an impact on masticatory function and a consequent influence on the nutritional status [1-3]. Although the prevalence of edentulism has fallen markedly in industrialised countries [4, 5], tooth loss is among the main conditions of interest when considering oral diseases among the elderly [6], who are already affected by other systemic diseases. Deterioration of masticatory function affects the efficiency of food absorption and nutritional status [7], limiting their food selection and dietary intake of fibres, magnesium or calcium [8, 9], with potential effect on systemic health.

Edentulous people are likely to be treated with complete dentures (CDs) in order to rehabilitate their masticatory function to some extent. The ability to successfully wear a pair of CDs is, to an extent, a learned, skilled performance. Thus, it is expected that aged subjects who are first-time CDs wearers may need time to cope with a new pair of dentures.

After insertion of complete dentures, the chewing ability improves as patients get accustomed to their new dentures, and the improved fit optimises retention and stability within the limits allowed by the anatomical and tissue conditions [10]. Chewing ability expressed as masticatory efficiency is a dynamic chewing function and it is objectively evaluated by observing, measuring or quantifying food changes during mastication [11]. Various methods characterized as objective have been used in masticatory efficiency assessment so far, where researchers evaluated changes in chewed natural foods [12], or synthetic food [13], as in sieving methods. The sieving method, which measures the comminution of test materials, was considered the gold standard for assessing the masticatory efficiency, especially the multiple sieve method, which yields better results than the single one [14]. A substitution of comminution tests is the ability to mix and knead a food bolus, usually simulated by two-coloured chewing gum [15, 16] or paraffin wax [17, 18], which was also widely used in masticatory efficiency evaluation [19]. The degree of mixing of two colours was determined by optical methods [17, 19], by visual inspection [20] or by both [21]. Later, software was introduced for image processing of two-colour chewing gum mixing [22]. Colorimetric detectability of chewing-gum colour changes has proven to be as reliable method in clinical and academic masticatory efficiency assessment. These methods allow for the evaluation of gum colour changes but lack deeper insight into the changes of gum sample texture appearance. We hypothesized that using the “digital texture image analysis” (DTIA) might provide a better insight into the improvement of masticatory efficiency in denture wearers. DTIA is based on the grey level co-occurrence matrix (GLCM) method, where the texture of an image corresponds to the spatial organisation of pixels in the image, and the co-occurrence matrix describes the occurrence of grey level between two pixels separated in the image by a given distance [23]. There are up to 14 textural features of DTIA which may be selected to represent the textural characteristics of the image under investigation [23].

The DTIA method was used to evaluate the structure of osteoporotic bone based on a series of images [24], to follow the periapical bone healing [25] or differentiate soft tissue lesions [26]. Also, texture image analysis was used to characterise the impact of chewing on pasta particle size reduction [27] or food bolus characterisation [28]. Although used sporadically in dental research, there are no publications about the application of DTIA in assessment of the digital images of chewed two-colour gums during the mixing ability test in masticatory efficiency evaluation.

Therefore, we investigated the applicability of DTIA in evaluating and measuring the bolus-kneading capacity of two-coloured chewing gum samples in complete denture wearers. The objectives were i) to evaluate whether DTIA discriminates changes in the colour mixing ability within the group in follow-up time; ii) to determine whether DTIA can be used to detect improvement in chewing ability iii) to select the most appropriate DTIA feature that sufficiently describes masticatory efficiency in CDs wearers.

Materials and method

The study was designed as a prospective intra-individual cohort investigation of masticatory efficiency in edentulous subjects with new CDs lasting over 6 months. Following the requirements of the Declaration of Helsinki on ethical principles for medical research involving human subjects and according to the STROBE guidelines, prior to the very beginning of the investigation the study protocol was approved by the Ethics Committee of the School of Dental Medicine, University in Belgrade (No 35/14). All participants were given a detailed explanation of the purpose and process of the study and gave their written informed consent. The investigation was conducted from October 2017 to July 2018 (including recruitment, clinical procedures, follow-up after three and six months, and data collection).

Inclusion and exclusion criteria

Participants were recruited from a pool of patients who visited the Clinic of Prosthodontics, asking for prosthodontics rehabilitation.

The basic inclusion criterion was that the subjects in the study be of both sexes, over 65 years and edentulous. The specific inclusion criterion was related to the quality of the prosthesis supporting tissues. After clinical suitability examination, the subjects with the Kapur index higher than 14 for the mentioned parameters were included in the study [29, 30]. After the complete dentures were made, their clinical quality was analysed. In order for a subject to be included in the study, it was necessary that at the clinical examination a pair of complete dentures meet the criterion for good clinical quality of dentures and have the Kapur index higher than 6 [29, 31].

The exclusion criteria were the presence of signs of TMJ dysfunctions or neuromuscular disorders. Participants who were unable to use the dentures continuously or who were unwilling to attend the follow-up appointments were excluded from the study, as well.

Based on the defined inclusion / exclusion criteria, after a clinical suitability examination and fabrication of upper and lower CDs, the study group consisted of 20 subjects. However, for private reasons, two subjects withdrew and the definitive number of subjects in the study group was 18.

After the insertion of a new pair of CDs, verbal and written instructions about CDs insertion, removal, cleaning, and care were given to all participants.

Test sample and chewing protocol

Masticatory efficiency was assessed according to the described study protocol, by measuring the bolus-kneading capacity through the ability to mix two-coloured chewing gum [32]. The study group chewing gum samples were obtained at three different time periods, and three subgroups of samples were formed: at the baseline (T0), after 3 months (T1) and after 6 months (T2).

The samples for testing were prepared from the Five Tape sugarless gum (The Wrigley Company Ltd, Plymouth, Devon, PL6 7PR, England) in the following flavours: peppermint (green colour) and strawberry (pink colour). Dimensions of the chewing gum samples were adopted from the literature [15, 21].

Thirty mm long strips were cut out of the green chewing gum and pink chewing gum. The strips of different colours were then joined lengthwise to form a sample with two different colour layers, of the overall dimensions of 30mmx18mmx3mm.

Clinical protocol and image assessment

Upper and lower CDs were inserted and a sample of gum was placed onto the patient’s tongue with the pink side oriented towards the palate. Each subject was instructed to sit upright and to chew the sample of gum on the preferred chewing side for 20 cycles [15]. The chewing cycles were evaluated by the operator, who counted the rhythmic open-close movement of the lower jaw in the anterior plane as one chewing stroke [33, 34].

Every participant chewed two chewing gum samples, according to the described procedure; therefore, there was a total of two samples per subject for analysis. Between two consecutive chewing cycles an interval of 1 min was imposed in order to minimize the effect of fatigue. Immediately after the final chewing cycle, the samples were placed directly into a transparent plastic bag labelled with an identification code. Each specimen was compressed to a 1mm-thick piece using a custom-made polyvinyl chloride plate with a milled depression according to the described protocol [15]. Both sides of the piece were photographed with a Nikon D3200 digital camera using 18–55 mm lens and Macro filter 8x, (Nikon Corp. Japan) and saved in JPEG format as described in the literature [32].

Digital texture image analysis (DTIA)

The DTIA procedure was adopted from the literature [23-26] and applied to the gum samples in this study. Each chewed specimen was analysed from both sides, so that the images of both sides of the gum were converted from red, green, and blue (RGB) images (Fig 1A) to grayscale (GS) images (Fig 1B) by applying Image J software v 1.34s (National Institutes Health, Bethesda, MD, USA).

Fig 1

The schematic presentation of the image processing procedure.

The red, green, and blue images (RGB) and the grayscale (GS) images.

Later, the Grey Level Co-Occurrence Matrix (GLCM) algorithms were applied to obtain texture features of the gum images under investigation [23]. GLCM describes the second-order statistics in the images, enables a calculation of the textural characteristics, compares two neighbouring pixels at a time and compiles the frequency at which different grey-levels can be found within a restricted area [35, 36]. Based on the described operations, the software automatically generates values for the selected parameters. In this study, homogeneity, contrast, uniformity and entropy were selected as independent variables that describe the level of masticatory performance.

Uniformity represents the texture uniformity of the image and it is the opposite feature of entropy. It measures the number of repeated pairs, which is expected to be high if the occurrence of repeated pixel pairs is high [37]. Contrast is a measure of local variations of grey level values of pixels in the images [36]. Homogeneity measures the local homogeneity of a pixel pair. Homogeneity is expected to be large if the grey levels of each pixel are similar [37]. Entropy measures the disorder, or randomness of images, and can be used to characterize the image texture. It is an indicator of the complexity within an image, so, the more complex the images, the higher the entropy values [36, 38, 39]. A small difference in the levels of the grey scale of the pixels in the image corresponds to lower values of contrast and entropy, which means better colour mixing and consequently better masticatory performance. Also, small differences in the levels of the grey scale of the pixels in the image are in relation to higher values of uniformity and homogeneity, which means better colour mixing and consequently efficient masticatory performance. The digital texture features used were also described by Tournier et al. [23].

Colorimetric analysis

Colorimetry (analysis of variance of the hue) was performed using the freeware ViewGum© software (dHAL Software, Greece, www.dhal.com), and a step-by-step procedure for delimitation of the gum images, and VOH calculation was adopted from the literature [22]. The obtained images of specimens were imported into the custom ViewGum software, after which the software automatically executed the segmentation process. As seen in Fig 1A, after segmentation, the images were transformed from RGB values (red, green, blue) into the HSI colour space (hue, saturation, intensity) using an image processing application to analyse separately the hue, intensity and saturation [32]. A more representative measure of mixing two colours is achieved with only the hue component [22]. When the two colours of the chewing gum are not well mixed, then the neighbouring pixels belong to groups of different hues, with higher values. Therefore, the variance of hue (VOH) was set as the measure of level of colour mixture [21], which represents fusing the two colours into a single colour, where the lower VOH value corresponds to better colour mixing and consequently better masticatory performance.

Statistical analysis

Statistical analyses were performed using SPSS software (IBM SPSS Statistics 2019 Version 26, USA). All obtained values were numerical. Descriptive data were expressed as mean, standard deviation, median and the interquartile range. Numeric data were tested for normal distribution using the Koglomorov-Smirnov test. All obtained data were non-parametric; therefore, the Fridman test and Wilcoxon test were used to analyse differences between the data at different follow-up times and baseline. This analysis tested the difference in the values of the obtained parameters measured on the upper and lower side of the chewing gum sample immediately after receiving complete dentures, after three months, and after six months. The analysis of the parameters measured on the upper side of the gum evaluated the adequacy of dental restorations in the upper and lower jaw. The Mann Whitney test was used for intra-individual comparison, between the upper and lower side of the chewing gum sample. Differences were considered significant when p-value was <0.05.

Results

Participants

After all, the study included 18 participants with the mean age of 68 ± 4,6 (ranging from 65 to 80 years), and all of them completed the follow-up visits. All obtained chewing samples (N = 108) were suitable for further evaluation. The representative samples of chewing gum mixing ability images for T0, T1, and T2 are shown in Fig 2A–2C.

Fig 2

Representative samples of chewing gum mixing ability images for T0, T1, and T2.

Each patient chewed two samples of chewing gum, so there were 36 chewed gum samples per subgroup with a total of 108 chewed samples. A flow diagram of participants and total gum samples is presented in Fig 3.

Fig 3

Flow diagram of participants and total gum samples implemented in the study.

The main variables of investigation are presented in Table 1, expressed as mean, standard deviation, median and the interquartile range. Based on the intra-group comparison between the chewed samples of both cycles, significance was observed in the Uniformity group at the baseline and after three months, for Entropy at the baseline, and after three months for VOH. Furthermore, from the values presented in Table 1, one may say that the values of the analysed variables of gum samples—Uniformity, Contrast, Homogeneity, and Entropy—have changed in the participants during the observation period, from the moment of CDs delivery (T0), after three months (T1), and after six months (T2). Also, a statistically significant change of VOH in the tested gum samples was noticed for all three follow-up times, which can also be seen in Table 1.

Table 1

Parameter values in the study group at the baseline, after three and after six months.

Variables	Baseline (T0) N = 18	After three months (T1) N = 18	After six month (T2) N = 18	#Significance
Uniformity (upper side)	0.0033 (0.000575) 0.0030 (0.000)	0.0028 (0.0008) 0.0030 (0.001)	0.0031 (0.00024) 0.0030 (0.0000)	ap = 0.010
Uniformity (lower side)	0.0034 (0.000575) 0.0033 (0.000)	0.0027 (0.0003) 0.0028 (0.00022)	0.0030 (0.0008) 0.0030 (0.001)	ap = 0.001
& Significance	bp = 0.029	bp = 0.001	bp = 0.318
Contrast (upper side)	2.870 (0.641) 2.645 (0.719)	2.365 (0.414) 2.273 (0.262)	2.098 (0.356) 2.043 (0.511)	ap = 0.001
Contrast (lower side)	2.676 (0.471) 2.773 (0.196)	2.337 (0.116) 2.351 (0.108)	2.168 (0.288) 2.133 (0.272)	ap = 0.001
& Significance	bp = 0.974	bp = 0.142	bp = 0.508
Homogeneity (upper side)	0.539 (0.024) 0,542 (0,030)	0.560 (0.019) 0.566 (0.022)	0.576 (0.025) 0.581 (0.038)	ap = 0.001
Homogeneity (lower side)	0.547 (0.010) 0.543 (0.043)	0.564 (0.008) 0.562 (0.006)	0.569 (0.020) 0.572 (0.018)	ap = 0.002
& Significance	bp = 0.420	bp = 0.936	bp = 0.497
Entropy (upper side)	6.24 (0.13) 6.26 (0.173)	6.326 (0.138) 6.31 (0.124)	6.208 (0.086) 6.205 (0.134)	ap = 0.001
Entropy (lower side)	6.12 (0.096) 6.07 (0.067)	6.346 (0.067) 6.33 (0.089)	6.222 (0.086) 6.215 (0. 047)	ap = 0.001
& Significance	bp = 0.006	bp = 0.323	bp = 0.386
VOH (upper side)	0.062 (0.062) 0.050 (0.095)	0.129 (0.026) 0.127 (0.037)	0.068 (0.024) 0.066 (0.034)	ap = 0.001
VOH (lower side)	0.076 (0.059) 0.068 (0.090)	0.104 (0.026) 0.116 (0.052)	0.058 (0.006) 0.062 (0.011)	ap = 0.005
&Significance	bp = 0.235	bp = 0.020	bp = 0.299

Mean (SD) and Median (IQR) values of the parameter at the baseline, after three and after six months.

#comparison between follow-up periods;

&intergroup comparison;

a Fridman test;

bMann Whitney test.

Similarity between the upper and lower side of the gum samples is observed in the Contrast and Homogeneity values for all three follow-up times, as seen in Table 1, while differences between the chewing gum samples are found for Entropy in T1. The values for Uniformity measurements of one subject were significantly different at the beginning and three months after, and for VOH after three months.

Further analysis of the results obtained included the Wilcoxon test, a comparison between different measurement times, as shown in Table 2. Furthermore, statistically significant changes in the values are seen, especially when comparing the parameters Uniformity, Contrast and Homogeneity at the baseline (T0), with regards to the values after three months (T1) and after six months (T2). More graphical representations of the variable values are seen in Figs 1–10 in S1 Appendix. When observing the VOH values of the examined samples in all three time periods, statistically significant differences between the baseline (T0), after three months (T1) and after six months (T2) were noticed, as well as between the values of T1 and T2 (Figs 9 and 10 in S1 Appendix).

Table 2

Values for intergroup comparison.

Variables	Follow-up time	T0	T1
Uniformity (upper)	T1	p = 0.052	/
	T2	p = 0.248	p = 0.102
Uniformity (lower)	T1	p = 0.002	/
	T2	p = 0.296	p = 0.032
Contrast (upper)	T1	p = 0.001	/
	T2	p = 0.001	p = 0.001
Contrast (lower)	T1	p = 0.016	/
	T2	p = 0.000	p = 0.030
Homogeneity (upper)	T1	p = 0.002	/
	T2	p = 0.010	p = 0.004
Homogeneity (lower)	T1	p = 0.001	/
	T2	p = 0.001	p = 0.443
Entropy (upper)	T1	p = 0.048	/
	T2	p = 0.459	p = 0.002
Entropy (lower)	T1	p = 0.001	/
	T2	p = 0.001	p = 0.001
VOH (upper)	T1	p = 0.001	/
	T2	p = 0.679	p = 0.001
VOH (lower)	T1	p = 0.048	/
	T2	p = 0.500	p = 0.001

*statistical significance; Wilcoxon test.

Discussion

Summary of the results

Implementing DTIA enabled a thorough masticatory efficiency analysis, with discrimination of masticatory efficiency progression within the edentulous subjects during adaptation time. The obtained results give insight into how fast the process of adaptation is going in edentulous patients who are first-time CDs wearers in relation to masticatory efficiency. The implemented method of DTIA is new and it has been supplemented with the already published and accepted colour mixture VOH analysis [15, 21, 22, 34]. Although they analyse different aspects of chewed gum samples, both analyses proved masticatory efficiency, discriminative to the analysed study group during the observed time, and DTIA was found to be consistent with the colorimetric method, which was considered the gold standard. Also, DTIA possesses good reliability for two texture features, since there was similarity between the samples from the same subject for Contrast and Homogeneity. Therefore, Contrast and Homogeneity might be identified as a textural feature marker that could quantify chewing ability.

Strength and weakness of the study

Although some authors consider the sieving test to be the most reliable method of mastication analysis [32, 40], the chewing gum test was reported to be as reliable for quantifying masticatory performance [30], especially in CD wearers [16, 19, 32] with compromised mastication, as was the case in the conducted study. This type of test has many advantages: reduced possibility of swallowing food parts and, consequently, loss of analytical material, as well as the fact that the elasticity of the material allows for the use of maximum available mastication capacity. Also, the mixing-ability tests for masticatory efficiency are less dependent on the flow rate of saliva, which is of particular importance in older people who often suffer from dry mouth syndrome [40]. The duration of 20-cycle chewing was implemented in the study according to the recommendations in the literature, as this seems to sufficiently discriminate between the ‘physiological’ chewing efficiency of a young, healthy and fully dentate person and an edentulous elderly individual with a substantial masticatory handicap [15]. The analysis of the inter and intra-individual reproducibility suggested that, for the application of the test, a fixed number of 20 chewing cycles should be applied. Also, it is reported that, when groups of participants are to be correlated, it is better to analyse 20 strokes, since it is not known after how many strokes the comparison curves keep diverging [41]. This number of chewing cycles was found useful, whereas high repeatability allows for follow-up evaluations, for example during a dental treatment or neuromuscular rehabilitation programmes [21].

It is reported that a significant increase in masticatory efficiency can be obtained about 6 weeks after the insertion of new complete dentures, while some authors stated that biting abilities improved 2 months after the insertion of new prostheses [42, 43]. Nevertheless, we performed the first follow-up after three months. The idea was to give more time to the new wearers to get used to dentures and to perform a more realistic mixing ability test. The masticatory function test was performed 6 months after the fabrication of good-quality complete dentures, as it was reported that during that period new muscle memory patterns for mastication would have been established [44].

Edentulous patients with the adequate size of the residual alveolar ridge have been included in this study, in order to avoid the impact of residual ridge resorption on masticatory function. However, it was recently indicated that mandibular bone atrophy does not interfere directly with the masticatory function of edentulous patients [45]. Confounding factors have been minimized since the impact of ill-fitting dentures, improper denture bearing tissues and other factors were eliminated, allowing the cause (edentulism) and effect (masticatory efficiency) to be distinguished.

The limitations of this study may be the small sample size, as well as the shortcomings of chewing tests. However, the evaluation of the literature dealing with similar topics revealed similar sample sizes [41-43]. One drawback of the present study is the missing control group in the form of dentate subject. However, findings for the dentate are well documented elsewhere but data about edentulous subjects are lacking. The main shortcoming of the study may be the use of commercial chewing gum in the analysis of masticatory efficiency and not of gum specially designed for this purpose. However, commercial chewing gum was used elsewhere in the literature as specimen for the chewing gum mixing ability test [21, 46]. Also, when testing CDs wearers using this method, one must take into account the possibility of the onset of irritation and pain caused by wearing dentures during the adaptation period, which may affect the masticatory efficiency of these patients. Hence, transitory problems such as discomfort, sore spots, and injured mucosa were taken care of at the very beginning, providing minimum impact on the conducted investigation.

Interpretation of the results in the context of applied methodology

The analysis of the literature showed that, for the analysis of the mixture level between the two colours of the gum, samples might be assessed both visually and by electronic colorimetric assessment [32], or by using digital image processing techniques [15] and specially designed software [22]. The evaluation of the obtained results in the mentioned procedures is more or less based on the measurement of fusing of the original colours into a new colour, with different intermediate intensities.

As opposed to the mentioned studies, we implemented DTIA, which gave insight not only into the colour change, but into the texture of the digital gum image. The texture refers to a visual feature which is homogenous, but the homogeneity does not come from one colour. The texture features used in the study are variables such as uniformity, contrast, homogeneity and entropy of the obtained chewing gum sample images, which are in direct relation to the comminution of the gum, and their values for each participant are presented in Fig 4. Uniformity refers to the existence of irregularities within the surface of the specimen colour image, and there will be fewer of them when the sample is better blended / chewed. Contrast measures differences in the colour of the specimen, which means that it is reduced in well chewed samples, where the colours are well mixed. Homogeneity, in the context of measuring chewing efficiency, shows that a mixture of two colours was created, meaning the sample has been mixed and the colours merged into one. Entropy is a measure of the image system regulation of the specimen. The smaller it is, the sample is more arranged, or rather—better chewed.

Fig 4

DTIA variable values for each patient during the observation period (at the baseline, after three months, after six months).

Uniformity (A); Contrast (B) Homogeneity (C); Entropy (D).

The Uniformity values are the lowest at the first follow-up, as seen in Fig 4A, showing the chewiness of the sample after the third month. This suggested that the participants at the baseline faced with the chewing gum samples between CDs masticated vigorously, thus confirming the initial uniformity inside the sample. Later, after a certain period of use (T2), when analysed again, their mastication was more coordinated, not requiring vigorous mastication. These findings should be confirmed in future research with parallel evaluation of masticatory force values.

The evaluation of the obtained results shows that the easiest method of observing the masticatory efficiency in edentulous subjects is by analysing Contrast and Homogeneity. Contrast, as a measure of chewed samples, shows a decrease during the observation period, as seen in Fig 4B. This means that the contrast in the tested samples is reduced, due to higher masticatory efficiency and more complete chewing. The values for Contrast were highest at the baseline, confirming that mixing ability is scarce, with distinguished colours in the samples and great contrast between the colours. Contrast among all tested texture features showed compliance between the test-retest samples in all three follow-ups. The same is found for the Homogeneity feature, which possesses good reliability, since there is also similarity between the test-retest samples of the same subject. Tournier et al. [23] arrived at the same conclusion, which showed that contrast could be used successfully to investigate food bolus formation during chewing different breads.

The Homogeneity parameter values are increasing from T0 toward T2 with statistical significance (Table 1), showing a gradual improvement of masticatory efficiency. The graph in Fig 4C shows an increase of masticatory efficiency in terms of homogeneity after three months and after six months, which indicates that there is an increase of masticatory efficiency at the beginning with slight during the course of observing the edentulous subjects.

The Entropy values are shown on the graph in Fig 4D. Entropy is in correlation with Uniformity and confirms the claims that participants improve their masticatory efficiency after three months and that, by the sixth month, there are only improvements in the fineness of chewing.

The VHO values were lowest for the edentulous participants after three months, which is in correlation with the finding that adequate chewing leads to well-mixed colours and a low VOH [21]. In addition, as seen in Fig 5, the highest VOH results were found in poorly mixed colours through deficient chewing, whereas in this study they were found at the beginning of the period of adaptation.

Fig 5

VOH values of colorimetric analysis for each patient during the observation period (at the baseline, after three months, after six months).

Clinical implications

Complete denture wearers are the worst case scenario for chewing gum application. This means that only small changes can be expected—but were detected using DTIA. The applied method enabled observation of differences in the chewed samples within the group, but also the comparison of the obtained samples at defined time intervals. The analysed DTIA features confirmed a gradual improvement of masticatory efficiency in the study group over the evaluation period. Therefore, the improvement of the masticatory function in relation to the mixing ability of two-coloured chewing gum could be traced by monitoring changes in the values of DTIA variables. The method has proven to be reliable in complete denture wearers and should be tested with a different number of chewing cycles, as well as in dentate subjects and / or different dental restoration wearers.

Conclusion

Accepting the discussed weakness of the study, it may be presumed that DTIA can be considered a reliable tool for assessing changes in the masticatory efficiency through mixing behavior of two-coloured chewing gum samples. A hypothesized increase of masticatory efficiency was observed by DTIA, which thereby seems to be sensitive enough to detect improvement in chewing ability. This is most predictable by monitoring DTIA parameters such as contrast, and homogeneity, but not uniformity. Further studies shall clarify the reproducibility and threshold of values to better interpret the data from DTIA alone, as well as in hand with VHO.

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18 Jun 2020

PONE-D-20-18199

Within diabetic subject evaluation of masticatory efficiency using digital image texture analysis

PLOS ONE

Dear Dr. Milić Lemić,

Thank you for submitting your manuscript to PLOS ONE. After a first assessment I have to ask you for some adaptations before I am able to send it to potential reviewers.

My major points are the following:

a) you state in the introduction that edentulism is a prevalent problem

- please give references. Actually edentulism is decreasing across Europe - but

of course not world wide.

b) your number of patient (N=19) with DM is rather low for sophisticated statistical

methods and p-value fishing. Thus, I invite you to better depict the graphical data

with representation of the deviations by single dots of each measurement to allow a

c) more qualitative approach to your results and findings. This could include to track

the performance (or measurements) of each individual from T0->T2 graphically.

d) I am missing the STROBE checklist as well as data about the drop outs, exclusions

and reasons for exclusion.

e) You assessed the Kapur score - please include the data to your findings and as

highlighted in c) contrast the distributions of this score to the findings in your

variables under evaluation.

f) These measures may enable a better understanding of the results especially the

divergence between VOH and contrast from T0->T2

g) please only enclose high resolution images in line art (all Figures in TIFF are

depicted awfully - please check conversions or extraction paths)

h) please help the reader to understand the variables more clearly.

For me, the differences in variables under observation are quite small. How does

dentate patients perform in this test? This is crucial because statistical significance has

nothing to do with relevance.

i) To understand deviations please give the data of both cycles (I assume but you didnt mentioned that you

used the mean of both chewings) and perform an intra-individual analysis

about the reproducibility at each T. Therefore, I recommend to work rather with differences and deviations from

the common mean than with extraorinary statistical methods.

Finally I am looking forward to reassess and handle your manuscript as far as you are

willing to adjust the points raised by my side,

Please submit your revised manuscript by Aug 02 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Fabian Huettig, DMD, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

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18 Aug 2020

a) you state in the introduction that edentulism is a prevalent problem - please give references. Actually edentulism is decreasing across Europe – but of course not world wide.

As you have pointed the referencse are added in the text, and the paragraph was rewritten.

b) your number of patient (N=19) with DM is rather low for sophisticated statistical methods and p-value fishing. Thus, I invite you to better depict the graphical data with representation of the deviations by single dots of each measurement to allow a

Our statistician has made changes as you requested.

Also we were aware of the small sample size at the beginning and addressed it in the Discussion section: “The limitations of this study may be in the small sample size, as well as in the shortcomings of chewing tests. However, evaluation of the literature dealing with similar topics revealed the optimal sample sizes. (34.Van der Bilt A, Speksnijder CM, De Liz Pocztaruk R, Abbink JH. Digital image processing versus visual assessment of chewed two-colour wax in mixing ability tests. Journal of Oral Rehabilitation.2012; 39: 11–17.

35.Miyaura K, Morita M, Matsuka Y, Yamaashita A, Watanabe T. Rehabilitation of biting abilities in patients with different types of dental prostheses. Journal of Oral Rehabilitation. 2000;27: 1073–1076.

36.Gunne H S, Wall A K. The effect of new complete dentures on mastication and dietary intake. Acta Odontologica Scandinavica. 1985;43: 257-268)

c) more qualitative approach to your results and findings. This could include to track the performance (or measurements) of each individual from T0->T2 graphically.

Our statistician has made changes as you requested.

d) I am missing the STROBE checklist as well as data about the drop outs, exclusions and reasons for exclusion.

Beginning of the Materials and method section was rewritten and modified accordingly in order to present inclusion and exclusion criteria more clearly.

e) You assessed the Kapur score - please include the data to your findings and as highlighted in c) contrast the distributions of this score to the findings in your variables under evaluation.

The paragraph was rewritten since you revealed the lack of clearness. The Kapur scoring was done during the selection of the participants in order to eliminate potential confounders that may influence the results like improper anatomy of the bearing tissues and inadequate clinical quality of the dentures.

f) These measures may enable a better understanding of the results especially the divergence between VOH and contrast from T0->T2

The values of VOH and contrast are not supposed to be correlated but both show similar trend of diminution proposing the fact that there is improvement of masticatory efficiency from T0-T2. Smaller values mean better color mixing and thorough mastication respectively.

g) please only enclose high resolution images in line art (all Figures in TIFF are depicted awfully - please check conversions or extraction paths)

The Figures were corrected as you requested.

h) please help the reader to understand the variables more clearly. For me, the differences in variables under observation are quite small. How does dentate patients perform in this test? This is crucial because statistical significance has nothing to do with relevance.

In the Methods section we included explanation of the variables in order to have deeper insight into the study results.

i) To understand deviations please give the data of both cycles (I assume but you didnt mentioned that you used the mean of both chewings) and perform an intra-individual analysis about the reproducibility at each T. Therefore, I recommend to work rather with differences and deviations from the common mean than with extraorinary statistical methods.

Part of the manuscript involving statistics analysis was rearranged. As a result of that new Tables 1 & 2 are introduced, and the results are better graphically presented in Figures 4 & 5.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

20 Oct 2020

PONE-D-20-18199R1

Within diabetic subject evaluation of masticatory efficiency using digital image texture analysis

PLOS ONE

Dear Dr. Milić Lemić,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

In order to provide you an atleast timely answer after having a hard time to

find reviewers for your manuscript, I'd like to invite you to adapt your manuscript

towards the points raised by reviewer #1. I am following these points and stay with

him to rather adjust your "story" and to reperform ex ante calculations and to

provide assumptions for your calculations in order to validate and present your method

rather than the data itself (story lost).

If you do not feel comfortable with this recommendation, I suggest that you retract the

paper and hand it to a dental journal for publication.

Please submit your revised manuscript by Dec 04 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Fabian Huettig, DMD, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Dear Dr. Lemic,

in order to provide you an atleast timely answer after having a hard time to

find reviewers for your manuscript, I'd like to invite you to adapt your manuscript

towards the points raised by reviewer #1. I am following these points and stay with

him to rather adjust your "story" and to reperform ex ante calculations and to

provide assumptions for your calculations in order to validate and present your method

rather than the data itself (story lost).

If you do not feel comfortable with this recommendation, I suggest that you retract the

paper and hand it to a dental journal for publication.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: 1) The overall intention of this paper remains unclear due to the mix of all the paramenters: diabetic patients, full dentures and a new method to evaluate chewing efficiency.

1a) the used technology was used first time according to the authors - is the method valid and robust? After having measured all the different data - uniformity, contrast, homogeneity, entropy - contrast turned out to be the best marker - but this is post hoc and was not predefined as hypothesis. The authors´conclusion is focused only on that point, diabetes and full dentures are completly ignored in the conclusion. The goal of the study was to see how their exposure to new CDs affects their masticatory performance as treatment outcome (line 72-73). It seems, that this focus was lost somehow.

1b) it remains unclear, why diabetic patients have been selected - is there a difference to non-diabetic patients in chewing performance and/or adaptave capacity to new CDs? So, it is difficult to understand the clinical implications. A specific diabetic related altered learning period in diabetic patients cannot be identified in the conclusion. So, the lake of a control group is critical, especially in combination with the low number of participants.

2) Statistics: there are no information about a repeated measurement design.

3) Age: the range 55 to 80 is wide, and in accordance to the literature (e.g. Peyron MA, Blanc O, Lund JP, et al. Influence of Age on adaptability of human mastication. JNeurophys 2004; 92;773-9 https://doi.org/10.1152/jn.01122.2003), age is an influencing factor. A subgroup analysis for different age groups (at least two) should be included. It is an important influencing factor, that the adaptive capacity is completely different in elderly compared to younger patients groups.

4) Why the chewing test was performed on the preferred chewing side? Why not on both sides? Was preferred chewing side checked again at T1 and T2 - changes can be assumed in adequate and symmetric CD.

Overall:

The parameters diabetic patients, full dentures and a new method are not clearly separated and specified - so the interested clinician might expect an information regarding diabetes or full denture masticatory performance, and not a methodologic study on a new method.

Recommandation:

Clear focus on the new mehtod or on DM in combination CD. Maybe two split into two separated papers?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

30 Nov 2020

Response to the Reviewer

Reviewer #1: 1) The overall intention of this paper remains unclear due to the mix of all the paramenters: diabetic patients, full dentures and a new method to evaluate chewing efficiency.

1a) the used technology was used first time according to the authors - is the method valid and robust? After having measured all the different data - uniformity, contrast, homogeneity, entropy - contrast turned out to be the best marker - but this is post hoc and was not predefined as hypothesis. The authors´conclusion is focused only on that point, diabetes and full dentures are completly ignored in the conclusion. The goal of the study was to see how their exposure to new CDs affects their masticatory performance as treatment outcome (line 72-73). It seems, that this focus was lost somehow.

Thank you very much for your comment, beaacuse it forced us to thoroughly go through the text and make substantial corrections. Firstly as you suggested we focused on the novel method and greatly rewrote the introduction with specific ephasize on the existing methods for evaluating masticatory efficiency. Further we added some desctirption regarding DTIA and its contemporary use in dental research so far. As you pointed, we introduced new aims with special focus on evaluating texture features in masticatory efficiency follow up, measuring chewing ability improvement and justifying the use of DTIA in masticatory efficiency evaluation.

1b) it remains unclear, why diabetic patients have been selected - is there a difference to non-diabetic patients in chewing performance and/or adaptave capacity to new CDs? So, it is difficult to understand the clinical implications. A specific diabetic related altered learning period in diabetic patients cannot be identified in the conclusion. So, the lake of a control group is critical, especially in combination with the low number of participants.

As pointed in the Recommendations: Clear focus on the new mehtod or on DM in combination CD. Maybe two split into two separated papers?

We rewrote our manuscript with completely different focus, abandoning diabetic patients and focusing only on thourough research about the texture analysis and methodical approach with the chewing gum testing. However, we felt that new wearers are representative froup for our methodological approach since they as a new wearers certainly need more time to learn to cope with new dentures. Therefore, we were able to follow them in adequate period of time, providing our method to express itself in full.

2) Statistics: there are no information about a repeated measurement design.

Every participant chewed two chewing gum samples, according to the described procedure (line 159) in methodology section. And In Table 1 each parameter was presented with test-retest values.

3) Age: the range 55 to 80 is wide, and in accordance to the literature (e.g. Peyron MA, Blanc O, Lund JP, et al. Influence of Age on adaptability of human mastication. JNeurophys 2004; 92;773-9 https://doi.org/10.1152/jn.01122.2003), age is an influencing factor. A subgroup analysis for different age groups (at least two) should be included. It is an important influencing factor, that the adaptive capacity is completely different in elderly compared to younger patients groups.

We are completely aware of the fact you were pointing but age 55 was completely writing mistake, since we at the first place focused on older subjects our inclusion criterion was age over 65, which was written in the methodology section.

4) Why the chewing test was performed on the preferred chewing side? Why not on both sides? Was preferred chewing side checked again at T1 and T2 - changes can be assumed in adequate and symmetric CD.

The used methodology about mixing ability test with chewing double coloured chewing gum was adopted from the literature. 15. Schimmel M, Christou P, Herrmann F, Mȕller F. A two-colour chewing gum test for masticatory efficiency: development of different assessment methods. Journal of Oral Rehabilitation. 2007; 34: 671–678. In order to completely create the real life situation we accepted the proposed the methodology.

Overall:

The parameters diabetic patients, full dentures and a new method are not clearly separated and specified - so the interested clinician might expect an information regarding diabetes or full denture masticatory performance, and not a methodologic study on a new method.

Thank you very much for your comments, because you opened my eyes and I have written the text in completely different manner. Sincerely hope that this new version of the mansucript will satisfy your expectations, because we really put a great effort on it. Of course the merit is on you, but hope we deserved it.

Response to the academic editor

I'd like to invite you to adapt your manuscript towards the points raised by reviewer #1. I am following these points and stay with him to rather adjust your "story" and to reperform ex ante calculations and to

provide assumptions for your calculations in order to validate and present your method rather than the data itself (story lost).

We accepted with great pleasure each point raised by yourself and the reviewer, and are enclosing the new version of the manuscript with changed „ story" and detailed focus on the method implemented. The introduction section is now completely rewritten, with different aims and issues raised. Further text is changed accordingly to meet the new approach in the manuscript. Also we have made changes to the title to better match the text itself.

Thanks to you we hope that our new version of the manuscript possess merit for the PLOS ONE journal.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

22 Jan 2021

PONE-D-20-18199R2

The use of Digital texture image analysis in determining the masticatory efficiency outcome

PLOS ONE

Dear Dr. Milić Lemić,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

The manuscript improved, but some major aspects are still lacking to allow a

publication. In case of a sufficiently revised resubmission covering all points/comments of the reviewers, I am willing to accept the manuscript in an academically sound structure and in line

with the publication criteria (please adhere to raw data submission as well) upon Editorial decision.

==============================

Please submit your revised manuscript by Mar 08 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Fabian Huettig, DMD, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The manuscript improved, but some major aspects are still lacking to allow a

publication. In case of a sufficiently revised resubmission covering all points

I am willing to accept the manuscript as academically sound and in line

with the publication criteria (please adhere to raw data submission as well) upon

Editorial decision.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Now, the revised manuscript has a clear red line and is therefore adequately understandable.

Recommended adaptations:

a) Table 1: several Spaces missing in column Variables

b) Table 2: Spelling mistake: Entropy

c) Table 1 and Table. 2: the * to highlight significance can be ommitted (to relieve the content of the tables). The p-value is mentioned in the text (line 220 and 221)

d) Table 1 and Table 2: p<0,001 instead of p=0,000 (I gues, that the probability of error is not really zero)

e) Table 1 and Table 2: the meaning of (1) and (2) remains unclear, allthough it seems to be essential for the understanding of the statistical analysis. (1) = DTIA?; (2) = VOH?. Recommendation: no coding, but insertion of the abbreviations.

f) Table 1 and Table 2: the number should be explained in the text - or I havn´t found it.

g) The sentence page 6, lines 127 to 129 should be eliminated or rewritten, otherwise this passage is confusing and falling out of context

The data used to perform the statistical analysis are not explained. So the meaning of those data remains unclear for a reader without a specific background knowledge for DTIA and/or VOH technology. I recommend to add a short paragraph in Material&Method section (or to add a explaining subtext to the tables.

Reviewer #2: Dear authors,

your revision pushed the manuscript into a more readable and understandable form/ structure.

However it is lacking a sufficient scientific English, especially the punctation and construction of sentences should be revised by a native speaker. Basically, there should be one sentence for one thought/statement. Some recommendations were done by me within the word file.

Since your data evaluation follows one possible approach and the raw data are not submitted within the supplement, I exspect that you resubmit a SPSS or CSV or at least XLS file with the data set from the 228 images and all variables.

I suggest a major revision, because the manuscript should be orded as it is the standard. I commented within the word file which passages should be shifted. Finally, the discussion should be revisited and more balanced between your novel findings and the established standard (VHO). This encompasses the reflection of absolute and relative changes despite of the p-value hype.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Gregor Slavicek

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: Manuscript_R2-revision3.docx

Click here for additional data file.

2 Mar 2021

Dear Reviewers:

Thank you for the opportunity to revise our manuscript, "The use of Digital texture image analysis in determining the masticatory efficiency outcome" (PONE-D-20-18199R2)

We appreciate the time and effort that the reviewers dedicated to providing valuable feedback on our manuscript. We also appreciate the careful review and constructive suggestions. We have been able to incorporate changes to reflect most of the suggestions made by the reviewers. It is our belief that the manuscript is substantially improved after making the suggested edits.

Following this letter are our point-by-point responses to the reviewers’ concerns in italics, including how the text was modified. The changes made in the manuscript are highlighted in yellow.

Reviewer #1:

Now the revised manuscript has a clear red line and is therefore adequately understandable.

Thank you very much. Hope that the manuscript is even better after these modifications.

Recommended adaptations:

a) Table 1: several Spaces missing in column Variables

Changed

b) Table 2: Spelling mistake: Entropy

Changed

c) Table 1 and Table 2: the * to highlight significance can be omitted (to relieve the content of the tables). The p-value is mentioned in the text (line 220 and 221)

Thank you for this, as you recommended we omitted * in the Tables.

d) Table 1 and Table 2: p<0,001 instead of p=0,000 (I guess that the probability of error is not really zero)

Changed

e) Table 1 and Table 2: the meaning of (1) and (2) remains unclear, although it seems to be essential for the understanding of the statistical analysis. (1) = DTIA?; (2) = VOH?. Recommendation: no coding, but insertion of the abbreviations.

Thank you for your suggestion. We have inserted the meaning of (1) and (2) into the Tables and changed them accordingly. Didn`t quite understand what you meant by "no coding, but insertion of the abbreviations"

f) Table 1 and Table 2: the number should be explained in the text - or I haven´t found it.

We mentioned the table number in the text.

g) The sentence on page 6, lines 127 to 129 should be eliminated or rewritten; otherwise this passage is confusing and falling out of context. The data used to perform the statistical analysis are not explained. So, the meaning of those data remains unclear for a reader without specific background knowledge of DTIA and/or VOH technology. I recommend you to add a short paragraph in Material & Method section (or to add an explaining subtext to the tables).

Thank you for your kind notice. The sentence was omitted, and changes were made as you suggested. In the MM section we added the meaning of all DTIA and VOH variables, and our statistician author modified the paragraph explaining the statistical analysis.

Reviewer #2:

- Dear authors, your revision pushed the manuscript into a more readable and understandable form/ structure. However, it is lacking sufficient scientific English, especially the punctuation and construction of sentences should be revised by a native speaker. Basically, there should be one sentence for one thought/statement. Some recommendations were given by me within the word file.

Thank you very much for this comment. The manuscript was revised by a professional agency for academic editing. We are adding the proof as a supplementary file.

-Since your data evaluation follows one possible approach and the raw data are not submitted within the supplement, I expect that you resubmit a SPSS or CSV or at least XLS file with the data set from the 228 images and all variables.

During the first submission we submitted the XLS file and SPSS data in the supplementary files as directed by the journal policy. We will do it again in case they are missing.

-I suggest a major revision, because the manuscript should be ordered as it is the standard. I commented within the word file which passages should be shifted.

Thank you very much for your kindness. We followed the recommendations you inserted within the word file and made changes accordingly.

We also implemented the corrections and suggestions you made within the word file

-Finally, the discussion should be revisited and more balanced between your novel findings and the established standard (VHO). This encompasses the reflection of absolute and relative changes despite of the p-value hype.

As you pointed out, analyzing masticatory efficiency in complete denture wearers is the worst case scenario, and there are not many references using the colorimetric method that investigated ME in CDs wearers. We correlated the studies we analyzed to our findings and broadened the discussion in that way.

-Please restructure your abstract according to the paper and your mentioned aims (i-iii), give the variables first and thereafter the results and conclusion.

Thank you for this suggestion. We have structured the abstract in more proper way.

- Please describe how this was done – it is important for the reproducibility of your study.

The M&M section was rewritten, thanks to your comment. However, the methodology and procedure for the preparation of gum samples were adopted from the literature and we cited it properly for possible reproducibility.

-Please give the software and operation, as well as if adjustments were set for this conversion.

Thank you for this comment. It was a mistake not providing proper software details. After consulting the author KR who performed the DTIA and VOH, we made some modifications to the text as you suggested. There were no extra manual adjustments of the images, and she relied only on the software to automatically perform the analyses. That way we excluded any manual intrusion in the process.

-Please add for each variable what a clinical relevant absolute or relative change would be.

Since this was our first DTIA using and following the references regarding the method (there were not many), we decided to follow the changes of the uniformity, contract, entropy and homogeneity. Like other authors, we were not able to give exact values for each of them, but rather to discriminate them during the follow-ups. Of course, implementing the DTIA in dentate subjects might provide more insight into the absolute and relative change. That is our plan for future studies. As far as our edentulous subjects are concerned, for now DTIA has proven to be reliable, providing more detailed analyses than VOH.

-Please describe how you handled the data from both sides of the specimen from both specimens per patient and point in time. This should be described in the subheadings below as well. If you decided for one side and one specimen only – describe why and how.

Thank you for pointing to us this unclearness. Our statistician, author BM reorganized the section and wrote it in a more understandable way.

-According to STROBE, you should give a flowchart on how many patients were screened and how many dropped out/ were closed out to which reasons. This is necessary to understand a potential selection bias.

Thank you for this comment. We provided another flowchart.

-The flowchart should start above your 19 patients.

Yes, we created it accordingly. After a detailed review of our initial XLS data, and SPSS data, we, however, noticed that 18 participants were involved in the study and changed that throughout the text. Our colleague also made changes to Table 1, as requested.

-Please contrast your overall findings from the DTIA with the published standard VOH. You can detect weaknesses and strength in both approaches. This should be described

Thank you very much for this suggestion and raising that issue. We discussed the strength and weakness of DTIA at the beginning of the discussion. However, the colorimetric analysis is well established in the reference and we didn`t find ourselves competent to discuss it more. We added more sentences concerning the findings of DTIA and VOH.

The Clinical implications section was rewritten as you suggested. Thank you for pointing out.

-No: you hypothesized it - you can draw the conclusion with the reasoning. This is…

Thank you for your great help in formulating the conclusions. The whole section has been changed and we hope it has quality now and that the conclusions are drawn based on the presented data.

Submitted filename: Response to Reviewers .doc

Click here for additional data file.

9 Mar 2021

PONE-D-20-18199R3

The use of digital texture image analysis in determining the masticatory efficiency outcome

PLOS ONE

Dear Dr. Milić Lemić,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

Dear Dr. Lemic,

thank you for your revision. Your manuscript improved; but could you please fix the shortcomings mentioned

below that I can proceed the decision.

a) Table 1: There are two values in each cell (one of it in italics)

please use the headline oder the Table legend to clarify which values there are.

e.g.: "Mean(SD) and Median (IQR) values of the parameter at the baseline, after three and after six months."

--> the last row should be "&Significance" instead of "#Significance" - therefore the last column (right) should be #Significance in headline.

b) Figure 3: Please give age and gender of the participants within the step "assessment" and "exclusion"

and name the failed inclusion criteria of N=7 as well. Please add the time intervals of recall next to the three boxes.

c) S2Appendix: Please adjust the Data headline (ln 2) or provide a legend which is in hand with the wording in your manuscript and add the subjects number, gender, and age.

d) ln 332: please change edentulousness to "edentulism" and crosscheck the wording again.

Best regards, Fabian Huettig

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23 Mar 2021

Dear Editor Dr Fabian Huettig,

Thank you for the opportunity to revise our manuscript, "The use of Digital texture image analysis in determining the masticatory efficiency outcome" (PONE-D-20-18199R3)

We appreciate the time and effort that you dedicated to strengthening our manuscript pushing us further to improve it.

We also appreciate the careful review and constructive suggestions of the reviewers. It is our belief that the manuscript is substantially improved after making the suggested edits.

Following this letter are our point-by-point responses to your pointed shortcomings in italics, including how the text was modified. The changes made in the manuscript are highlighted in yellow.

a) Table 1: There are two values in each cell (one of it in italics) please use the headline oder the Table legend to clarify which values there are.

e.g.: "Mean(SD) and Median (IQR) values of the parameter at the baseline, after three and after six months."

-> the last row should be "&Significance" instead of "#Significance" - therefore the last column (right) should be #Significance in headline.

Authors: Thank you for this suggestion. We used the headline as you suggested and added it below the Table legend.

b) Figure 3: Please give age and gender of the participants within the step "assessment" and "exclusion"

and name the failed inclusion criteria of N=7 as well. Please add the time intervals of recall next to the three boxes.

Authors: We named the failed inclusion criteria for the seven subjects as you requested. Unfortunately, we are not able to give proper data concerning age and gender of the participants within the assessment and exclusion step. This information was gathered at the very beginning and since the main outcome of the study was not related to differences in age and gender, we do not have those papers with us anymore.

-Please add the time intervals of recall next to the three boxes.

Authors: We are afraid we do not understand exactly what you meant. However, we inserted that recalls were after three months. As it was noted in the text, the study lasted for ten months, and not all subject were analysed at the same time.

c) S2Appendix: Please adjust the Data headline (ln 2) or provide a legend which is in hand with the wording in your manuscript and add the subjects number, gender, and age.

Authors: Thank you for this suggestion, we corrected as you requested.

d) ln 332: please change edentulousness to "edentulism" and crosscheck the wording again.

Authors: It was changed as you requested.

Authors: We are aware that we were not able to respond to your request and are sorry about that. The information regarding age and gender of the participants within the assessment and the exclusion step are no longer with us. However, we hope that the lack of data concerning the age and gender in the supplementary files might not significantly affect the quality of the manuscript we reached after your guidance and that it will not affect your decision.

19 Apr 2021

The use of digital texture image analysis in determining the masticatory efficiency outcome

PONE-D-20-18199R4

Dear Dr. Milić Lemić,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Fabian Huettig, DMD, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for your prompt and sufficient adaptations of your manuscript.

I am looking forward to seeing it published within PLOS ONE!

Reviewers' comments:

21 Apr 2021

PONE-D-20-18199R4

The use of digital texture image analysis in determining the masticatory efficiency outcome

Dear Dr. Milić Lemić:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Fabian Huettig

Academic Editor

PLOS ONE