Influence of irradiated dentin, biofilm and different artificial saliva formulations on root dentin caries development

Influence of irradiated dentin, biofilm and different artificial saliva formulations on root dentin caries development | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Influence of irradiated dentin, biofilm and different artificial saliva formulations on root dentin caries development Beatriz Martines de Souza, Aline Silva Braga, Mariele Vertuan, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3787488/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose To evaluate the influence of radiation as well as of new formulations of artificial saliva on the development of root caries lesions. Methods Bovine root samples were divided into: irradiated (70 Gy) dentin or not; the type of biofilm (from irradiated or non-irradiated patients) and the type of artificial saliva (for the condition irradiated dentin/biofilm): Saliva A (inorganic); Saliva A + 1mg/ml hemoglobin; Saliva A + 0.1mg/ml cystatin; Saliva A + hemoglobin + cystatin; Bioextra (positive control) and water (negative control) (n = 12/group). Biofilm was produced using human biofilm and McBain saliva (0.2% of sucrose, 37 o C and 5% CO 2 ); the treatments were done 1x/day, for 5 days. Colony-forming units (CFU) counting was performed; demineralization was quantified by transversal microradiography. Two-way ANOVA/Bonferroni or Sidak test for the comparison between biofilm x dentin and ANOVA/Tukey or Kruskal-Wallis/Dunn for comparing artificial saliva were done (p < 0.05). Results The type of biofilm had no influence on CFU and demineralization. Sound dentin under control biofilm presented the lowest Lactobacillus ssp. and Streptococcus mutans CFU and the lowest mean mineral loss (R) (25.6 ± 2.2; 23.7 ± 2.9%) compared to irradiated dentin (26.1 ± 2.8; 28.1 ± 3.3, p < 0.004) for both types of biofilms (irradiated and no irradiated, respectively). Bioextra was the only one that reduced R (10.8 ± 2.5%) and LD (35 ± 15µm) compared to water (17.3 ± 3.3%, 81 ± 18µm, p < 0.0001). Conclusion Irradiation of dentin has impact on caries development; none of the experimental saliva was able to reduce its occurrence. biofilms dental caries head and neck neoplasms radiotherapy Figures Figure 1 Figure 2 Introduction Despite the technological advances related to radiotherapy (conformational and modulated) to minimize secondary radiation to tissues adjacent to the tumor, this side effect still occurs even at a lower intensity [ 1 ]. Considering that radiotherapy is the main treatment of the head and neck cancer (HNC), the side-effects may involve the oral tissues, such as salivary glands, oral mucosa and dental structures [ 2 ] with a negative impact on the health and quality of life of this population. Dentin has shown significant morphological changes after radiotherapy [ 3 ], which may compromise adhesive restorations performed after the cancer treatment period [ 4 ]. There seems to be an increase in the expression and activity of MMP-2 and MMP-9 in irradiated dentin, suggesting greater susceptibility to degradation [ 5 ]. In addition, it is expected reduced salivary flow, increased viscosity, reduced buffering capacity and changes in concentrations of electrolytes and proteins with antimicrobial function in saliva. All these alterations can increase the individual susceptible to the development of dental caries [ 6 ]. The caries lesions incidence in patients submitted to head and neck radiotherapy is about 16% higher after the first year, reaching up to 74% higher after 7 years of treatment [ 7 , 8 ]. Interventions to prevent this type of dental disease are needed and very important to provide an improved quality of life to the patients. Besides the strategies to control dental caries, such as sugar consumption control and oral hygiene with fluoride toothpaste, the use of artificial saliva has been an important strategy applied to control the symptoms of hyposalivation in these patients, especially with respect to oral dryness. There are several types of artificial saliva or saliva substitutes on the market, some containing only minerals and humectants, as well as, more complex formulas containing some proteins [ 9 , 10 ]. However, the vast majority of artificial saliva options do not have the ability to control caries development [ 11 , 12 ], especially in root dentin. Cystatin, a typical saliva protein, and hemoglobin, a typical blood protein, were identified in saliva proteomics, showing to be acid-resistant and, consequently, to have a significant anti-caries role [ 13 – 15 ]. Sugarcane cystatin, known as Cane CPI-5, has been used as a substitute for human cystatin, due to its low cost, a good affinity for hydroxyapatite, high acid resistance [ 16 ] and antibiofilm effect in vitro [ 17 ]. Hemoglobin also has a strong affinity to hydroxyapatite [ 18 ] and good results against the initial erosion in vitro as with Cane CPI-5 [ 19 ]. Therefore, the aim of this study was to test the influence of radiation on the development of root dentin caries as well as of new formulations of artificial saliva containing Cane CPI-5 and hemoglobin on the development of root caries lesions, with the objective of reducing the development of radiation caries, focusing on its possible use by patients who are undergoing radiotherapy treatment of the HNC. The null hypothesis are: (1) there is no difference between sound and irradiated dentin in relation to carious lesion development using a microcosm biofilm model, regardless of the biofilm quality (from irradiated or nonirradiated patients); (2) There is no difference between the biofilm from irradiated patients and healthy patients in relation to carious lesion development using a microcosm biofilm, regardless of the type of the dentin (sound vs. irradiated); (3) there is no difference between experimental artificial saliva formulations compared to the negative control with respect to antibiofilm and anticaries effects. Materials and methods Biofilm Collection This study was previously approved by the local Ethics Committee (CAAE: 97497318.00000.5417). Written informed consent was obtained from all individual participants included in the study. Biofilm considered control was collected from two healthy patients (1 male: 60 years old with 26 teeth and 1female: 56 years old with 26 teeth) and pooled. The inclusion criteria followed previously reported protocols [20]. The collection of irradiated biofilms was performed from two donors (1 male: 65 years old with 20 teeth and 1 female: 57 years old with 24 teeth) who received the total head and neck 3D radiotherapy (final dose: 70 Gy), 5 months previously to the study, following inclusion criteria previously determined and described by de Souza et al. [20]. The biofilms were diluted in 0.9% saline solution (proportion 2 mg: 1 ml), and then vortexed (30 s) and sonicated (5% amplitude, 3 pulses of 10 s each). Thereafter, 1 ml aliquots (70% biofilm solution and 30% glycerol) were prepared and stored at -80ºC [20, 21]. Tooth Specimen Preparation and treatment groups This study was first approved by ethics committee on animal research (CEUA, Number: 004/2018). The bovine teeth were donated by food manufacturing industry (Frigol S.A, Lençóis Paulista, São Paulo, SP, Brazil). One hundred and twenty bovine root samples (4 mm x 4 mm) were prepared and minimum polished (5 s), to allow bacterial adhesion [20]. Of these, 96 dentin samples were irradiated with a total dose of 70 Gy while 24 were not irradiated (sound dentin). Two parts of each third of the surface were covered with red nail polish (Love-Risqué ® , Guarulhos, SP, Brazil) to allow later appropriate analysis of tooth demineralization by transverse microradiography - TMR. Thereafter, the samples were sterilized by exposure to ethylene oxide [20] and randomly distributed to the groups, considering roughness values (measured by using a contact profilometer - Mahr Perthometer, Göttingen, Germany and the software MarSurf XCR-20 -Mahr Perthometer, Göttingen, Germany) as criteria (mean: 0.28 ± 0.02 µm). Forty-eight dentin samples (n=24 irradiated and n=24 non-irradiated) were submitted to two distinct microcosm biofilm formations (using irradiated biofilm or healthy biofilm), totalizing four groups (n=12/group): (1) Irradiated dentin and irradiated biofilm; (2) Irradiated dentin and healthy biofilm; (3) Sound dentin and irradiated biofilm; (4) Sound dentin and healthy biofilm. While seventy-two samples of irradiated dentin were submitted to microcosm biofilm formation using biofilm from irradiated patients and divided into 6 treatments with artificial saliva (n=12/group): (A) Artificial Saliva A: pH 5.6 – NaHCO 3 (0.219%); K 2 HPO 4 (0.127%); CaCI 2 .2H 2 O (0.0654%); MgCl 2 .6H 2 O (0.0125%); KCl (0.082%); Methylparaben (0.01%); Propylparaben (0.01%); Carboxymethylcellulose (0.8%); (B) Saliva A + 1 mg/mL hemoglobin (human hemoglobin, Sigma Aldrich, Saint Louis, MI, USA, H7379) (pH 5.4) [19]; (C) Saliva A + 0.1 mg/mL sugarcane cystatin (Cane CPI-5) (pH 5.5) [16]; (D) Saliva A + 1 mg/mL hemoglobin + 0.1 mg/mL sugarcane cystatin (Cane CPI-5) (pH 5.5); (E) BioXtra ® (positive control) [active components: lysozyme; lactoferrin, lactoperoxidase; colostrum extrac. Other ingredients: water, Propylene Glycol, Xylitol, Sodium Monofluorophosphate (1500 ppm F - )., Poloxamer 407, Hydroxyethylcellulose, Aroma, Aloe Barbadensis Leaf Juice, EDTA, Lactic Acid, Sodium Benzoate, Limonene, Linalool, CI42090. Lifestream Pharma, Seneffe, Belgium] (pH 6.3); (F) Deionized Water (negative control) (pH 7.1). All the experiments were done in biological triplicate (n=4/replicate, n final=12). The flowchart of the study is in Online Resource 1. Microcosm biofilm formation The biofilm-glycerol stock was diluted in McBain artificial saliva [22], at a ratio of 1:50 (inoculum) [20]. The microcosm biofilm was grown on the dentin samples placed into the 24-well plates, for five days. On the first day, each dentin sample was exposed to 1.5 ml of inoculum for 8 hours. After this time, the inoculum was removed, the samples were washed with PBS (1.5 ml, 5 s) and received 1.5 ml fresh medium (McBain Saliva with 0.2% of sucrose) until completing 16 hours. From the second to the fifth day, the medium with sucrose was changed once a day and the plates were incubated at 5% CO 2 and 37°C [17, 20]. Treatments with artificial saliva were performed between medium changes, once/day for 1 minute, during the following 4 days of microcosm biofilm formation (schematic diagram of biofilm microcosm in Online Resource 2). Colony-forming unit (CFU) counting After 5 days, the microbial suspension from each well plate, obtained by adding 1 ml of 0.89% NaCl solution over the formed biofilm, was sonicated (Sonifier Cell Disruptor B-30, Branson) for 30 s at 20 W. Bacterial suspensions were diluted (10 −4 or 10 -5 ) and spread on Petri dishes (25 μL/dish) and then, the dishes incubated under 5% CO 2 and 37 °C for 48 h [17, 20]. The four agar culture media used were [20]: 1) brain heart infusion agar (BHI; Difco, Detroit, USA) for total microorganisms (dilution factor 10 -5 ); (2) mitis salivarius agar (MSA; Neogen, Indaiatuba, Brazil) for total streptococci ( dilution factor 10 -4 ); (3) SB-20M for mutans streptococci ( S. mutans and S. sobrinus ) (dilution factor 10 -4 ); and (4) MRS (Kasvi, Curitiba, Brazil) for Lactobacillus sp . (dilution factor 10 -5 ). After 48h, the CFU numbers were counted and used, together with the dilution factor, to calculate the total CFU for each type of microorganism per group. The data were transformed in log 10 CFU/mL [20]. Transverse microradiography (TMR) - demineralization analysis Dentin samples were cleaned with sterile gauze, transversally sectioned and polished (100-120 µm thickness). The procedure of exposure to X-ray (20kV and 20 mA, Softex, Tokyo, Japan), development and analysis of the microradiography plates was performed as previously described [20], using the TMR from Inspektor Research System (Amsterdam, Netherlands) (schematic diagram of the sample preparation for TMR in Online Resource 3). The mineral content was calculated assuming 50 vol% of mineral content for sound dentin and that the lesion depth ends when dentin contains around of 47.5% of mineral volume [20]. The integrated mineral loss (ΔZ, %vol. µm), the average mineral loss over the lesion depth (mean mineral loss, R, %vol) and the lesion depth (LD, µm) were calculated [20]. Statistical analysis The data were submitted to statistical analysis using the Graph Pad Prism 7.04 program (San Diego, USA). (p<0.05). The normal distribution and homogeneity were checked using Kolmogorov–Smirnov and Bartlett tests, respectively. For the comparison between biofilm (irradiated or healthy) x dentin (irradiated or sound) 2-way ANOVA/Bonferroni test (CFU counting) and 2-way ANOVA/Sidak test (TMR parameters) were done. For comparison between treatments with artificial saliva, ANOVA/Tukey test was applied for CFU counting (total microorganisms and Lactobacillus spp) and for TMR parameters (R and LD) and Kruskal-Wallis/Dunn for CFU counting (total streptococci and mutans streptococci ) and for the Integrated Mineral Loss (ΔZ). Statistical sample size (n=12) was based on previous study using irradiated dentin and microcosm biofilm model [20]. Results CFU counting There was no difference between the type of dentin and biofilm on the growth of total microorganisms. Regarding total streptococci, sound dentin had a higher number of CFU compared to irradiated dentin, in the case of irradiated biofilm, but no difference was seen in the case of healthy biofilm. For Lactobacillus sp. and mutans streptococci, there was no difference between the type of dentin (irradiated or sound), while the irradiated biofilm had a higher number of CFU compared to healthy biofilm for sound dentin. For irradiated dentin, no difference was found (Table 1). With respect to the artificial saliva formulations comparisons, for total streptococci and mutans streptococci , treatments with experimental saliva, especially those containing hemoglobin, had a higher CFU counting than the negative control (water), differing significantly (Table 2). The positive control (BioXtra ® ) significantly reduced the CFU counting for all analyzed species compared to the experimental saliva (sample power of 97%, calculated from http://powerandsamplesize.com/Calculators ); however, it did not differ from negative control (water) with respect to Lactobacillus sp. , mutans streptococci and total streptococci growth. TMR analysis No differences were found between the type of biofilm and type of dentin regarding the integrated mineral loss (ΔZ). For lesion depth (LD) and mean mineral loss (R), sound dentin presented lower demineralization compared to the irradiated one (Table 3). Figure 1 shows representatives TMR images from each group. The experimental saliva formulations were not able to reduce the caries lesion development under this experimental model. Only the positive control (BioXtra ® ) was able to reduce LD and R significantly compared to the experimental saliva and the negative control (Table 4, Figure 2). Discussion Irradiated biofilm has no influence on dentin caries lesion formation while irradiation of dentin increases the susceptibility to demineralization under the tested model. Therefore, the first null hypothesis was rejected and the second one accepted. There are changes in the composition and structure of root dentin at exposure to doses greater than 60 Gy with reduction of the mineral/organic matrix ratio [ 23 ], in addition, to possible changes in the composition of the organic and collagen content [ 23 – 25 ]. The mineral content also appears to be altered due to the radiation, with a decrease in the calcium and phosphate ratio, changing dentin hardness value [ 3 ]. The cited alterations in the mineral content and mainly in the organic content can make dentin more susceptible to demineralization [ 25 ]. Previous study has shown no difference in the formation of the microcosm biofilm with respect to the inoculum source, whether it comes from saliva or from dental biofilm [ 21 ]. Considering that hyposalivation is one of the main consequences of head and neck radiotherapy, and that this condition can persist even 6 months after the end of treatment [ 26 ], we chose to collect dental biofilm in the present study. Change in the oral microbiota of patients submitted to doses greater than 30 Gy has been detected [ 26 – 28 ], with a high level of Lactobacillus (58%) and mutans streptococci (35%) even 1 year after radiotherapy [ 27 ]. In the present study, there is a small difference in the results of CFU counting, with a slight increase of cariogenic bacteria in the irradiated biofilm, although these differences were not significant to influence the demineralization parameters, which may be due to the continuous sucrose exposure during microcosm biofilm formation. Thus, the irradiated biofilm in this experimental model appears to exert no influence on the formation of the initial dentin caries lesion. A recent work [ 29 ] demonstrated that the initial bacteriological profile of the inoculum did not interfere in the formation of the initial caries lesion under microcosm biofilm model, once the conditions for biofilm growth have been standardized in vitro [ 21 , 29 ]. If a less cariogenic model would have been applied, the distinction between the sources of microorganism could have been different. More specific techniques, such as biofilm microbiome, would be relevant for understanding eventual microbiological differences between dental biofilm sources (from irradiated vs. healthy patient). The experimental artificial saliva with proteins (Cane CPI-5 and hemoglobin) did not present promising results in the reduction of demineralization or not even in the reduction of the CFU counting when compared to water (negative control); thus, the third null hypothesis was accepted. In fact, the negative control group, with deionized water washing, showed better results than the tested experimental saliva. This fact could be explained by the washing effect of water, which could have disorganized the biofilm or/and remove the acid product, since this group was subjected to washing procedure twice (PBS and water). This also helps to justify differences in dentin demineralization between negative control vs. irradiated dentin/irradiated biofilm (not compared statistically), since irradiated dentin/irradiated biofilm group was not exposed to any type of washing. Cane CPI-5 has shown promising results in preventing erosive wear (enamel and dentin) in vitro [ 30 , 31 ] and in situ at a concentration of 0.1 mg/ml [ 32 ]. Regarding the prevention of enamel caries, a recent study also under microcosm biofilm showed that different concentrations of Cane CPI-5 (0.05, 0.1 and 0.5 mg/ml) were effective in significantly reducing the integrated mineral loss when compared to the negative control (PBS) [ 17 ]. Furthermore, the lowest concentrations (0.05 and 0.1 mg/ml) were the most effective in reducing the CFU counting for cariogenic bacteria ( Lactobacillus sp , total streptococci and mutans streptococci ) [ 17 ]. When the Cane CPI-5 concentration was increased to 1 mg/ml, the protective antibacterial effect was lost [ 33 ]. The concentrations that seem to be most effective, both for preventing tooth erosion and for enamel caries control, are 0.05 and 0.1 mg/ml. However, we did not find similar results for dentin in our study, using a concentration of 0.1 mg/ml. Lower concentrations of Cane CPI-5 (0.025 and 0.05 mg/ml) have already been tested in microcosm biofilm for root dentin and they were not able to reduce the CFU counting for any bacterial group evaluated neither to reduce demineralization compared to negative control (PBS) [ 34 ]. Hemoglobin at a concentration of 1 mg/ml showed similar results to Cane CPI-5 in preventing erosive tooth wear in vitro [ 19 ]. In addition, the association of hemoglobin (1 mg/ml) and Cane CPI-5 (0.1 mg/ml) was the one that presented the best protective result against erosive tooth wear in vitro when compared to the isolated proteins [ 35 ]. Thus, the combination of both proteins could be more effective in preventing dental caries as well. No previous studies evaluated hemoglobin and the association between hemoglobin and Cane CPI-5 for preventing dental caries. However, our study did not show additive effect of the combination. Differently from enamel, dentin has a higher organic content that, once exposed to demineralization, is subjected to the activation of matrix metalloproteinase (MMP) responsible for degrading its organic content. MMPs are considered important mediators of the degradation of the dentin organic matrix, being collagenases and gelatinases the most associated with dentin caries [ 36 ]. Radiotherapy has been associated with the increased expression and activation of MMPs in some tissues [ 36 ]. In addition, there is a greater presence of cariogenic bacteria in dentinal lesions compared to enamel lesions [ 37 , 38 ] as well as the presence of proteolytic bacteria [ 39 ], which could have digested the tested proteins added in experimental saliva and their derivates. All these factors may have influenced the low efficacy of the experimental saliva, and perhaps for the specific condition (dentin + radiotherapy) the concentration of Cane CPI-5 and hemoglobin should have been higher to those tested in the present study. It is also important to consider that, despite the good results of proteins in previous studies with enamel, both Cane CPI-5 (pH 7.88) and hemoglobin were tested diluted in deionized water [ 17 , 19 , 32 ]. Only Pelá et al. [ 40 ] evaluated Cane CPI-5 incorporated into a chitosan gel (prepared with acetic acid), pH 5.5, against erosive tooth wear. When these proteins are added in a more complex vehicle in terms of the number of reagents, such as the artificial saliva used in the present work, many variables may influence their effect, such as the final pH of the product and possible interactions between components, both affecting the protein bioavailability. Accordingly, carboxymethylcellulose, which has a negative charge, could have interacted with the tested proteins, whose charge is positive [ 41 ]. Carboxymethylcellulose is added into artificial saliva since it plays an important role in improving oral lubrication [ 42 ]. More studies need to be carried out focusing on the maintenance of the protective action of proteins and their stability, minimizing possible interactions within dental products as gels, mousses, mouthwashes, and toothpastes. It was not possible to adjust the pH of the experimental saliva (pH values between 5.4 and 5.6) to be similar to the positive control – BioXtra (pH 6.3), since it could alter the stability and reactivity of the saliva. The only saliva that had a protective potential was the positive control (BioXtra®), which has proteins lysozyme, lactoferrin and lactoperoxidase as active ingredients, with broad-spectrum bacteriostatic activity [ 43 ] and it is used to relieve symptoms of dry mouth after radiotherapy [ 42 ]. No study has evaluated saliva BioXtra® with respect to antimicrobial or anticaries effect. Formulations of toothpaste and gel (with the same active ingredients of artificial saliva) have shown to reduce the number of Lactobacillus sp. and S. mutans in previous clinical studies [ 43 , 44 ]. In addition, sodium monofluorophosphate (1,500 ppm F − ) is present in its composition, described as an inactive ingredient. Although described as an inactive ingredient, in our experimental model, fluoride into the artificial saliva might have reduced demineralization and increased remineralization [ 45 ]. The influence of the presence of fluoride in BioXtra® saliva should be studied in the future. As the work had focus on artificial saliva, we did not include F solution as a control group. This shall be done in the future to better understand the results. In conclusion, irradiated biofilm has no influence, while the type of dentin has impact on dentin caries development; none of the experimental saliva was able to reduce its occurrence. Considering that no previous study had compared the impact of tooth irradiation and biofilm from irradiated patients on the dentin caries formation, our study highlighted the impact of the irradiation in increasing the susceptibility of dentin to demineralization. Other important finding is that BioXtra® saliva is able to reduce dentin caries lesion development, while the experimental solutions were not, which should be confirmed in future studies. Declarations Funding: The authors B.M.S. and A.C.M. received by São Paulo Research Foundation (FAPESP) (grant number 2019/07241-0 and 2019/21797-0, respectively). Competing Interests: All the authors have no relevant financial or non-financial interests to disclose. Ethics Approval: This study was approved by ethics committee on animal research of the Bauru School of Dentistry, University of São Paulo, Brazil (CEUA, Number: 004/2018). All procedures performed in studies involving human participants were in accordance with the ethical standards of the Institutional and/or National Research Committee (Ethics Committee of the Bauru School of Dentistry, University of São Paulo, Brazil, number 97497318.00000.5417) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Consent to participate: Informed consent was obtained from all individual participants included in the study. Data Availability Statement: All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author. References Jensen SB, Pedersen AM, Vissink A, Andersen E, Brown CG, Davies AN, Dutilh J, Fulton JS, Jankovic L, Lopes NN, Mello AL, Muniz LV, Murdoch-Kinch CA, Nair RG, Napeñas JJ, Nogueira-Rodrigues A, Saunders D, Stirling B, von Bültzingslöwen I, Weikel DS, Elting LS, Spijkervet FK, Brennan MT (2010) Salivary gland hypofunction/xerostomia section, oral care study group, multinational association of supportive care in cancer (MASCC)/international society of oral oncology (ISOO). A systematic review of salivary gland hypofunction and xerostomia induced by cancer therapies: prevalence, severity and impact on quality of life. Support Care Cancer 18(8):1039-1060. https://doi.org/10.1007/s00520-010-0827-8. Pereira IF, Firmino RT, Meira HC, Do Egito Vasconcelos BC, De Souza Noronha VRA, Santos VR (2019) Radiation-induced oral mucositis in brazilian patients: Prevalence and associated factors. In vivo 33(2):605-609. https://doi.org/10.21873/invivo.11517. Velo MMAC, Farha ALH, da Silva Santos PS, Shiota A, Sansavino SZ, Souza AT, Honório HM, Wang L (2018) Radiotherapy alters the composition, structural and mechanical properties of root dentin in vitro. Clin Oral Investig 22(8):2871-78. https://doi.org/ 10.1007/s00784-018-2373-6. Rodrigues RB, Soares CJ, Junior PCS, Lara VC, Arana-Chavez VE, Novais VR (2018) Influence of radiotherapy on the dentin properties and bond strength. Clin Oral Investig (2):875-883. https://doi.org/10.1007/s00784-017-2165-4. Queiroz AM, Bonilla CMC, Palma-Dibb RG, Oliveira HF, Nelson-Filho P, Silva LAB, Lucisano MP, Paula-Silva FWG (2019) Radiotherapy Activates and Protease Inhibitors Inactivate Matrix Metalloproteinases in the Dentinoenamel Junction of Permanent Teeth. Caries Res 53(3):253-259. https://doi.org/10.1159/000492081. Makkonen T(1988) Studies on oral complications of head and neck cancer radiotherapy. Proc Finn Dent Soc 84 Suppl 4-5:1-111. Hamilton SN, Arshad O, Kwok J, Tran E, Fuchsia Howard A, Serrano I, Goddard K (2019) Documentation and incidence of late effects and screening recommendations for adolescent and young adult head and neck cancer survivors treated with radiotherapy. Support Care Cancer 27(7):2609-2616. https://doi.org/10.1007/s00520-018-4559-5. Siala W, Mnejja W, Khabir A, Ben Mahfoudh K, Boudawara T, Ghorbel A, Frikha M, Daoud J (2019) Toxicité neurologique tardive après traitement des carcinomes nasopharyngés (Late neurotoxicity after nasopharyngeal carcinoma treatment). Cancer Radiother 13(8):709-714.French. https://doi.org/10.1016/j.canrad.2009.05.006. Meyer-Lueckel H, Umland N, Hopfenmuller W, Kielbassa AM (2004) Effect of mucin alone and in combination with various dentifrices on in vitro Remineralization. Caries Res 38(5):478-483. https://doi.org/10.1159/000079630. Kielbassa AM, Oeschger U, Schulte-Monting J, Meyer-Lueckel H (2005) Microradiographic study on the effects of salivary proteins on in vitro demineralization of bovine enamel. J Oral Rehabil 32(2):90-96. https://doi.org/10.1111/j.1365-2842.2004.01392.x. Smith G, Smith AJ, Shaw L, Shaw MJ (2001) Artificial saliva substitutes and mineral dissolution. J Oral Rehabil 28(8):728-731. https://doi.o rg/10.1046/j.1365-2842.2001.00803.x. Meyer-Lueckel H, Schulte-Mönting J, Kielbassa AM (2002) The effect of commercially available saliva substitutes on predemineralized bovine dentin in vitro. Oral Dis 8(4):192-198. https://doi.org/10.1034/j.1601-0825.2002.01762.x. Fábián TK, Hermann P, Beck A, Fejérdy P, Fábián G (2012) Salivary defense proteins: their network and role in innate and acquired oral immunity. Int J Mol Sci 13(4):4295-4320. https://doi.org/10.3390/ijms13044295. Delecrode TR, Siqueira WL, Zaidan FC, Bellini MR, Moffa EB, Mussi MC, Xiao Y, Buzalaf MA (2015) Identification of acid-resistant proteins in acquired enamel pellicle. J Dent 43(12):1470-1475. https://doi.org/10.1016/j.jdent.2015.10.009. Ventura TMDS, Cassiano LPS, Souza E Silva CM, Taira EA, Leite AL, Rios D, Buzalaf MAR (2017) The proteomic profile of the acquired enamel pellicle according to its location in the dental arches. Arch Oral Biol 79:20-29. https://doi.org/10.1016/j.archoralbio.2017.03.001. Santiago AC, Khan ZN, Miguel MC, Gironda CC, Soares-Costa A, Pelá VT, Leite AL, Edwardson JM, Buzalaf MAR, Henrique-Silva F (2017) A new sugarcane cystatin strongly binds to dental enamel and reduces erosion. J Dent Res 96(9):1051-1057. https://doi.org/10.1177/0022034517712981. Araujo TT, Camiloti GD, Valle AD, Silva NDG, Souza BM, Carvalho TS, Câmara JVF, Shibai PYT, Henrique-Silva F, Cruvinel T, Magalhães AC, Buzalaf MAR (2021) A sugarcane cystatin (CaneCPI-5) alters microcosm biofilm formation and reduces dental caries. Biofouling 37(1):109-116. https://doi.org/10.1080/08927014.2021.1881065. Yu YD, Zhu YJ, Qi C, Jiang YY, Li H, Wu J (2017) Hydroxyapatite nanorod-assembled porous hollow polyhedra as drug/protein carriers. J Colloid Interface Sci 496:416-424. https://doi.org/10.1016/j.jcis.2017.02.041. Martini T, Rios D, Dionizio A, Cassiano LPS, Taioqui Pelá V, Silva CMS, Taira EA, Ventura TM, Magalhães AC, Carvalho TS, Baumann T, Lussi A, de Oliveira RB, Palma-Dibb RG, Buzalaf MAR (2020) Salivary hemoglobin protects against erosive tooth wear in gastric reflux patients. Caries Res 54(5-6):466-474. https://doi.org/10.1159/000507110. de Souza BM, Silva MS, Braga AS, Bueno PSK, da Silva Santos PS, Buzalaf MAR, Magalhães AC (2021) Protective effect of titanium tetrafluoride and silver diamine fluoride on radiation-induced dentin caries in vitro. Sci Rep 11(1):6083. https://doi.org/10.1038/s41598-021-85748-8. Signori C, van de Sande FH, Maske TT, de Oliveira EF, Cenci MS (2016) Influence of the Inoculum Source on the Cariogenicity of in vitro Microcosm Biofilms. Caries Res 50(2):97-103. https://doi.org/10.1159/000443537. McBain AJ (2009) Chapter 4: In vitro biofilm models: an overview. Adv Appl Microbiol. 69:99-132. https://doi.org/10.1016/S0065-2164(09)69004-3. de Miranda RR, Silva ACA, Dantas NO, Soares CJ, Novais VR (2019) Chemical analysis of in vivo-irradiated dentine of head and neck cancer patients by ATR-FTIR and Raman spectroscopy. Clin Oral Investig 23(8):3351-3358. https://doi.org/10.1007/s00784-018-2758-6. Campi LB, Lopes FC, Soares LES, de Queiroz AM, de Oliveira HF, Saquy PC, de Sousa-Neto MD (2019) Effect of radiotherapy on the chemical composition of root dentin. Head Neck 41(1):162-169. https://doi.org/10.1002/hed.25493. Douchy L, Gauthier R, Abouelleil-Sayed H, Colon P, Grosgogeat B, Bosco J (2022) The effect of therapeutic radiation on dental enamel and dentin: A systematic review. Dent Mater 38(7):e181-e201. https://doi.org/10.1016/j.dental.2022.04.014. Gaetti-Jardim E Jr, Jardim ECG, Schweitzer CM, da Silva JCL, Oliveira MM, Masocatto DC, dos Santos CM (2018) Supragingival and subgingival microbiota from patients with poor oral hygiene submitted to radiotherapy for head and neck cancer treatment. Arch Oral Biol 90:45-52. https://doi.org/10.1016/j.archoralbio.2018.01.003. Al-Nawas B, Grötz KA (2006) Prospective study of the long-term change of the oral flora after radiation therapy. Support Care Cancer 14(3):291-296. https://doi.org/10.1007/s00520-005-0895-3. Eliasson L, Carlén A, Almståhl A, Wikström M, Lingström P (2006) Dental plaque pH and micro-organisms during hyposalivation. J Dent Res 85(4):334-338. https://doi.org/10.1177/154405910608500410. Viana CS, Maske TT, Signori C, Van de Sande FH, Oliveira EF, Cenci MS (2021) Influence of caries activity and number of saliva donors: mineral and microbiological responses in a microcosm biofilm model. J Appl Oral Sci 29:e20200778. https://doi.org/10.1590/1678-7757-2020-0778. Santos LA, Martini T, Câmara JVF, Reis FN, Ortiz AC, Camiloti GD, Levy FM, Shibao PyT, Honorio HM, Henrique-Silva F, Pieretti JC, Seabra AB, Cardoso CAB, Buzalaf MAR (2021) Solutions and gels containing a sugarcane-derived cystatin (CaneCPI-5) reduce enamel and dentin erosion in vitro. Caries Res 55(6):594-602. https://doi.org/10.1159/000520261. Gironda CC, Pelá VT, Henrique-Silva F, Delbem ACB, Pessan JP, Buzalaf MAR (2022) New insights into the anti-erosive property of a sugarcane-derived cystatin: different vehicle of application and potential mechanism of action. J Appl Oral Sci 30:e20210698. https://doi.org/10.1590/1678-7757-2021-0698. Pelá VT, Lunardelli JGQ, Tokuhara CK, Gironda CC, Silva NDG, Carvalho TS, Santiago AC, Souza BM, Moraes F, Henrique-Silva F, Magalhães AC, Oliveira RC, Buzalaf MAR (2021) Safety and In Situ Antierosive Effect of CaneCPI-5 on Dental Enamel. J Dent Res 100(12):1344-1350. https://doi.org/10.1177/00220345211011590. Pelá VT, Braga AS, Camiloti GD,Lunardelli JGQ, Pires JG, Toyama D, Santiago AC, Henrique-Silva F, Magalhães AC, Buzalaf MAR (2021) Antimicrobial and anti-caries effects of a novel cystatin from sugarcane on saliva-derived multi-species biofilms. Swiss Dent J 2021;131(5):410-416. Frazão Câmara JV, Araujo TT, Mendez DAC, da Silva NDG, Medeiros FF, Santos LA, de Souza Carvalho T, Reis FN, Martini T, Moraes SM, Shibao PYT, Groisman S, Magalhães AC, Henrique-Silva F, Buzalaf MAR (2021) Effect of a sugarcane cystatin on the profile and viability of microcosm biofilm and on dentin demineralization. Arch Microbiol 203(7):4133-4139. https://doi.org/10.1007/s00203-021-02403-6. Pelá VT, Buzalaf MAR, Niemeyer SH, Baumann T, Henrique-Silva F, Toyama D, Crusca E, Marchetto R, Lussi A, Carvalho TS (2021) Acquired pellicle engineering with proteins/peptides: Mechanism of action on native human enamel surface. J Dent 107:103612. https://doi.org/10.1016/j.jdent.2021.103612. Gomes-Silva W, Prado Ribeiro AC, de Castro Junior G, Salvajoli JV, Rangel Palmier N, Lopes MA, Rocha MM, de Goes MF, Brandão TB, Santos-Silva AR (2017) Head and neck radiotherapy does not increase gelatinase (metalloproteinase-2 and -9) expression or activity in teeth irradiated in vivo. Oral Surg Oral Med Oral Pathol Oral Radiol 124(2):175-182. https://doi.org/10.1016/j.oooo.2017.04.009. Richards VP, Alvarez AJ, Luce AR, et al. Microbiomes of Site-Specific Dental Plaques from Children with Different Caries Status. Infect Immun 2017;85(8):e00106-17. https://doi.org/10.1128/IAI.00106-17. Bhaumik D, Salzman E, Davis E, Bedenbaugh M, Mitchell ML, Burne RA, Nascimento MM (2022) Plaque Microbiome in Caries-Active and Caries-Free Teeth by Dentition. JDR Clin Trans Res 23800844221121260. https://doi.org/10.1177/23800844221121260. Takahashi N, Nyvad B (2016) Ecological hypothesis of dentin and root caries. Caries Res 50(4):422-431. https://doi.org/10.1159/000447309. Pelá VT, Brito L, Taira EA, Henrique-Silva F, Pieretti JC, Seabra AB, de Almeida Baldini Cardoso C, de Souza EP , Groisman S, Rodrigues MC, Lussi A, Carvalho TS, Buzalaf MAR, (2022) Preventive effect of chitosan gel containing CaneCPI-5 against enamel erosive wear in situ. Clin Oral Investig 26(11):6511-6519. https://doi.org/10.1007/s00784-022-04600-z. Ji L, Orthmann A, Cornacchia L, Peng J, Sala G, Scholten E (2022) Effect of different molecular characteristics on the lubrication behavior of polysaccharide solutions. Carbohydr Polym 297:120000. https://doi.org/10.1016/j.carbpol.2022.120000. Vinke J, Kaper HJ, Vissink A, Sharma PK (2020) Dry mouth: saliva substitutes which adsorb and modify existing salivary condition films improve oral lubrication. Clin Oral Investig 24(11):4019-4030. https://doi.org/10.1007/s00784-020-03272-x. Gudipaneni RK, Kumar R V, Jesudass G, Peddengatagari S, Duddu Y (2014) Short term comparative evaluation of antimicrobial efficacy of tooth paste containing lactoferrin, lysozyme, lactoperoxidase in children with severe early childhood caries: a clinical study. J Clin Diagn Res 8(4):ZC18-20. https://doi.org/10.7860/JCDR/2014/8161.4232. Gookizadeh A, Emami H, Najafizadeh N, Roayaei M (2012) Clinical evaluation of BIOXTRA in relieving signs and symptoms of dry mouth after head and neck radiotherapy of cancer patients at Seyed-al-Shohada Hospital, Isfahan, Iran. Adv Biomed Res 1:72. https://doi.org/10.4103/2277-9175.102976. Souza BM, Fernandes Neto C, Salomão PMA, Vasconcelos LRSM, Andrade FB, Magalhães AC (2018) Analysis of the antimicrobial and anti-caries effects of TiF4 varnish under microcosm biofilm formed on enamel. J Appl Oral Sci 26:e20170304. https://doi.org/10.1590/1678-7757-2017-0304. Tables Table 1. Mean ± SD of CFU counting (log 10 CFU/ mL) of Total Microorganisms, Lactobacillus sp . (10 -5 ), Total streptococci and Streptococcus mutans/Streptococcus sobrinus (10 -4 ) of Microcosm Biofilm Produced from Different Sources of Biofilm on Distinct Dentin Substrates. total microorganisms Lactobacillus sp. total streptococci S. mutans/ S. sobrinus Irritated dentin and irritated biofilm 6.66 ± 0.23 Aa 7.15 ± 0.37 Aa 6.82± 0.35 Aa 6.91 ± 0.33 Aa Irritated dentin and healthy biofilm 6.43 ± 0.48 Aa 6.86 ± 0.37 Aa 6.81 ± 0.22 Aa 6.88 ± 0.13 Aa Healthy dentin and irritated biofilm 6.67 ± 0.37 Aa 7.13 ± 0.38 Aa 7.28 ± 0.35 Bb 7.08 ± 0.30 Ab Healthy dentin and healthy biofilm 6.44 ± 0.36 Aa 6.72 ± 0.39 Ab 6.95 ± 0.31 Aa 6.72 ± 0.28 Aa Different capital letters in the same column indicate a significant difference between dentin types. Different lowercase letters in the same column indicate a significant difference between biofilm types. 2 way-ANOVA/Bonferroni (total microorganisms: interaction p=0.988, biofilm type p=0.037, dentin type p=0.969; Lactobacillus spp. : interaction p=0.573, biofilm type p=0.003, dentin type p=0.471; total streptococci : interaction p=0.085, biofilm type p=0.060, dentin type p=0.002; mutans streptococci : interaction p=0.045, biofilm type p=0.020, dentin type p=0.950). Table 2. Mean ± SD of CFU Counting (log 10 CFU/ mL) of Total Microorganisms and Lactobacillus sp. (10 -5 ) and Median (interquartile range) of Total streptococci and Streptococcus mutans/Streptococcus sobrinus (10 -4 ) of Microcosm Biofilm Treated with Different Saliva Formulations Total microorganisms Lactobacillus sp. total streptococci S. mutans/ S. sobrinus Saliva A (inorganic) 6.69 ± 0.22 B 7.20 ± 0.32 B 7.54 (0.34) BC 7.30 (0.25) C Saliva A + 1mg/mL hemoglobin 6.69 ± 0.32 B 7.29 ± 0.22 B 7.65 (0.16) C 7.42 (0.34) C Saliva A + 0.1mg/mL cystatin 6.80 ± 0.32 B 7.30 ± 0.33 B 7.61 (0.46) BC 7.28 (0.30) C Saliva A + 1mg/mL hemoglobin + 0.1mg/mL cystatin 6.64 ± 0.38 B 7.22 ± 0.37 B 7.53 (0.19) BC 7.11 (0.37) BC BioXtra ® (positive control) 6.21± 0.23 A 6.83 ± 0.19 A 6.88 (0.56) A 6.63 (0.64) A Water (negative control) 6.61 ± 0.31 B 7.11 ± 0.28 AB 7.22 (0.59) AB 6.75 (0.54) AB Different letters show statistical difference between treatments. ANOVA/Tukey: total microorganisms (p=0.0003); Lactobacillus spp. (p=0.0022). Kruskal-Wallis/Dunn: total streptococci (p<0.0001); mutans streptococci (p<0.0001). Table 3. Mean ± SD of the Integrated Mineral Loss (ΔZ, vol%. μm), Lesion Depth (LD, μm) and the Mean Mineral Loss (R, vol%) of Irradiated and Sound Dentin Submitted to Demineralization from Different Biofilm Sources. D Z (vol%. m m) LD ( m m) R (vol%) Irritated dentin and irritated biofilm 3,186 ± 676 123 ± 27 26.1 ± 2.8 Aa Irritated dentin and healthy biofilm 3,494 ± 516 126 ± 25 28.1 ± 3.3 Aa Healthy dentin and irritated biofilm 3,174 ± 914 114 ± 26 25.6 ± 2.2 Aa Healthy dentin and healthy biofilm 3,302 ± 601 134 ± 27 23.7 ± 2.9 Ba Different capital letters in the same column indicate a significant difference between dentin types. Different lowercase letters in the same column indicate a significant difference between biofilm types. Absence of letters shows no statistical difference. 2 way-ANOVA/Tukey (ΔZ: interaction p=0.655; biofilm type p=0.281; dentin type p=0.613; LD: interaction p=0.275; biofilm type p=0.131; dentin type p=0.990; R: interaction p=0.021; biofilm type p=0.960; dentin type p=0.0042). Table 4. Median (interquartile range) of the Integrated Mineral Loss (ΔZ, vol%. μm) and Mean ± SD of the Lesion Depth (LD, μm) and the Mean Mineral Loss (R, vol%) of Irradiated Dentin Submitted to Microcosm Biofilm Treated with Different Saliva Formulations D Z (vol%. m m) LD ( m m) R (vol%) Saliva A (inorganic) 2,390 (485) C 120 ± 17 C 20.2 ± 4.2 B Saliva A + 1mg/mL hemoglobin 2,240 (358) BC 113 ± 20 C 19.4 ± 2.1 B Saliva A + 0.1mg/mL cystatin 2,025 (1258) BC 112 ± 23 C 19.9 ± 4.4 B Saliva A + 1mg/mL hemoglobin + 0.1mg/mL cystatin 2,395 (1408) BC 131± 30 C 21.1 ± 4.4 B BioXtra ® (positive control) 445 (420) A 35 ± 15 A 10.8 ± 2.5 A Water (negative control) 1,520 (390) AB 81 ± 18 B 17.3 ± 3.3 B Different letters show statistical difference between treatments. Kruskal-Wallis/Dunn: ΔZ (p<0.0001). ANOVA/Tukey: LD (p<0.0001); R (p<0.0001). Additional Declarations No competing interests reported. Supplementary Files Onlinesource1flowchart.tif Onlinesource2biofilm.tif Onlinesource3TMR.tifGoogleDrive.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3787488","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":263260210,"identity":"8e10787e-76b6-4953-96fa-c46c76668525","order_by":0,"name":"Beatriz Martines de Souza","email":"","orcid":"","institution":"University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Beatriz","middleName":"Martines","lastName":"de Souza","suffix":""},{"id":263260211,"identity":"960243e1-2635-4c1a-81ab-7760b7f48c12","order_by":1,"name":"Aline Silva Braga","email":"","orcid":"","institution":"University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Aline","middleName":"Silva","lastName":"Braga","suffix":""},{"id":263260213,"identity":"71ffcb00-0214-42c7-ab5e-e512b64335bc","order_by":2,"name":"Mariele Vertuan","email":"","orcid":"","institution":"University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Mariele","middleName":"","lastName":"Vertuan","suffix":""},{"id":263260215,"identity":"aa71e624-954f-4966-ae47-e4a29f738714","order_by":3,"name":"Susan Sassaki","email":"","orcid":"","institution":"University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Susan","middleName":"","lastName":"Sassaki","suffix":""},{"id":263260218,"identity":"80892ad5-126d-44ed-821c-da0703692019","order_by":4,"name":"Tamara Teodoro Araújo","email":"","orcid":"","institution":"University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Tamara","middleName":"Teodoro","lastName":"Araújo","suffix":""},{"id":263260219,"identity":"ef9c1a07-afe6-4762-8a45-d0e281601974","order_by":5,"name":"Paulo Sergio da Silva Santos","email":"","orcid":"","institution":"University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Paulo","middleName":"Sergio da Silva","lastName":"Santos","suffix":""},{"id":263260221,"identity":"34a57caf-9bdb-4ee0-8812-96b9424652e9","order_by":6,"name":"Marilia Afonso Rabelo Buzalaf","email":"","orcid":"","institution":"University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Marilia","middleName":"Afonso Rabelo","lastName":"Buzalaf","suffix":""},{"id":263260222,"identity":"6cb11446-8709-4ba5-8cd7-91b73baa149c","order_by":7,"name":"Ana Carolina Magalhães","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAiElEQVRIiWNgGAWjYHACxgcg0oAULcwGJGthkyBNC3/76bRqnj93GMylDxCpReJM7rbbvG3PGCz7Eoi15gYvUEvDYQaDM8TqkAdqKeb5Q4oWA6AWZh42UrQYnsndLDm37RmPZQ+xWuSOn9344c2fO3LmPMRqgYIDpGoAaiFZxygYBaNgFIwgAABkMibm0+ynXQAAAABJRU5ErkJggg==","orcid":"","institution":"University of São Paulo","correspondingAuthor":true,"prefix":"","firstName":"Ana","middleName":"Carolina","lastName":"Magalhães","suffix":""}],"badges":[],"createdAt":"2023-12-21 14:31:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3787488/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3787488/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49077421,"identity":"d9f94e50-d656-4256-9c4d-71d57bb2e0de","added_by":"auto","created_at":"2024-01-02 19:06:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5618776,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative TMR images of dentin sample from each of the following groups:(1) Irritated dentin and irritated biofilm; (2) Irritated dentin and healthy biofilm; (3) Sound dentin and irritated biofilm; (4) Sound dentin and healthy biofilm.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3787488/v1/42743ccf65117995da1bac2d.png"},{"id":49077419,"identity":"ba6d54aa-1932-4847-bfc7-8b8060321cc9","added_by":"auto","created_at":"2024-01-02 19:06:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":10564884,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative TMR images of irradiated dentin sample with irradiated biofilm from each of the following treatments: (A) Artificial Saliva A; (B) Saliva A + 1 mg/mL hemoglobin; (C) Saliva A + 0.1 mg/mL sugarcane cystatin; (D) Saliva A + 1 mg/mL hemoglobin + 0.1 mg/mL sugarcane cystatin; (E) BioXtra\u003csup\u003e®\u003c/sup\u003e (positive control); (F) Deionized Water (negative control).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3787488/v1/9a0dfd1a7e74416425bc5784.png"},{"id":50447101,"identity":"f5985341-146a-4b6f-8db0-1a9b42f93d1b","added_by":"auto","created_at":"2024-01-31 16:16:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1394787,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3787488/v1/d07de387-eedf-47f2-9e0e-ff9fe15fec56.pdf"},{"id":49077422,"identity":"34732b84-d7bc-47cd-a811-9f6ebaca93ef","added_by":"auto","created_at":"2024-01-02 19:06:25","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4929788,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinesource1flowchart.tif","url":"https://assets-eu.researchsquare.com/files/rs-3787488/v1/2319bbcb928276683718c7f4.tif"},{"id":49077424,"identity":"74501e61-9d00-4e2d-8bac-a97506ce08aa","added_by":"auto","created_at":"2024-01-02 19:06:26","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14195690,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinesource2biofilm.tif","url":"https://assets-eu.researchsquare.com/files/rs-3787488/v1/5396aa085d584af9275cc46a.tif"},{"id":49078046,"identity":"412435ed-6291-4712-b673-f272e6423e83","added_by":"auto","created_at":"2024-01-02 19:14:25","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":118874,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinesource3TMR.tifGoogleDrive.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3787488/v1/4933d4dc99b57d1c219f235a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Influence of irradiated dentin, biofilm and different artificial saliva formulations on root dentin caries development","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDespite the technological advances related to radiotherapy (conformational and modulated) to minimize secondary radiation to tissues adjacent to the tumor, this side effect still occurs even at a lower intensity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Considering that radiotherapy is the main treatment of the head and neck cancer (HNC), the side-effects may involve the oral tissues, such as salivary glands, oral mucosa and dental structures [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] with a negative impact on the health and quality of life of this population.\u003c/p\u003e \u003cp\u003eDentin has shown significant morphological changes after radiotherapy [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], which may compromise adhesive restorations performed after the cancer treatment period [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. There seems to be an increase in the expression and activity of MMP-2 and MMP-9 in irradiated dentin, suggesting greater susceptibility to degradation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In addition, it is expected reduced salivary flow, increased viscosity, reduced buffering capacity and changes in concentrations of electrolytes and proteins with antimicrobial function in saliva. All these alterations can increase the individual susceptible to the development of dental caries [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe caries lesions incidence in patients submitted to head and neck radiotherapy is about 16% higher after the first year, reaching up to 74% higher after 7 years of treatment [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Interventions to prevent this type of dental disease are needed and very important to provide an improved quality of life to the patients.\u003c/p\u003e \u003cp\u003e Besides the strategies to control dental caries, such as sugar consumption control and oral hygiene with fluoride toothpaste, the use of artificial saliva has been an important strategy applied to control the symptoms of hyposalivation in these patients, especially with respect to oral dryness. There are several types of artificial saliva or saliva substitutes on the market, some containing only minerals and humectants, as well as, more complex formulas containing some proteins [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the vast majority of artificial saliva options do not have the ability to control caries development [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], especially in root dentin.\u003c/p\u003e \u003cp\u003eCystatin, a typical saliva protein, and hemoglobin, a typical blood protein, were identified in saliva proteomics, showing to be acid-resistant and, consequently, to have a significant anti-caries role [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Sugarcane cystatin, known as Cane CPI-5, has been used as a substitute for human cystatin, due to its low cost, a good affinity for hydroxyapatite, high acid resistance [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and antibiofilm effect \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Hemoglobin also has a strong affinity to hydroxyapatite [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and good results against the initial erosion \u003cem\u003ein vitro\u003c/em\u003e as with Cane CPI-5 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, the aim of this study was to test the influence of radiation on the development of root dentin caries as well as of new formulations of artificial saliva containing Cane CPI-5 and hemoglobin on the development of root caries lesions, with the objective of reducing the development of radiation caries, focusing on its possible use by patients who are undergoing radiotherapy treatment of the HNC.\u003c/p\u003e \u003cp\u003eThe null hypothesis are: (1) there is no difference between sound and irradiated dentin in relation to carious lesion development using a microcosm biofilm model, regardless of the biofilm quality (from irradiated or nonirradiated patients); (2) There is no difference between the biofilm from irradiated patients and healthy patients in relation to carious lesion development using a microcosm biofilm, regardless of the type of the dentin (sound vs. irradiated); (3) there is no difference between experimental artificial saliva formulations compared to the negative control with respect to antibiofilm and anticaries effects.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cem\u003eBiofilm Collection\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study was previously approved by the local Ethics Committee (CAAE: 97497318.00000.5417). Written informed consent was obtained from all individual participants included in the study. Biofilm considered control was collected from two healthy patients (1 male: 60 years old with 26 teeth and 1female: 56 years old with 26 teeth) and pooled. The inclusion criteria followed previously reported protocols [20]. The collection of irradiated biofilms was performed from two donors (1 male: 65 years old with 20 teeth and 1 female: 57 years old with 24 teeth) who received the total head and neck 3D radiotherapy (final dose: 70 Gy), 5 months previously to the study, following inclusion criteria previously determined and described by de Souza et al. [20]. The biofilms were diluted in 0.9% saline solution (proportion 2 mg: 1 ml), and then vortexed (30 s) and sonicated (5% amplitude, 3 pulses of 10 s each). Thereafter, 1 ml aliquots (70% biofilm solution and 30% glycerol) were prepared and stored at -80\u0026ordm;C [20, 21].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTooth Specimen Preparation and treatment groups\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study was first approved by ethics committee on animal research (CEUA, Number: 004/2018). The bovine teeth were donated by\u0026nbsp;food manufacturing industry (Frigol S.A, Len\u0026ccedil;\u0026oacute;is Paulista, S\u0026atilde;o Paulo, SP, Brazil). One hundred and twenty bovine root samples (4 mm x 4 mm) were prepared and minimum polished (5 s), to allow bacterial adhesion [20]. Of these, 96 dentin samples were irradiated with a total dose of 70 Gy while 24 were not irradiated (sound dentin). Two parts of each third of the surface were covered with red nail polish (Love-Risqu\u0026eacute;\u003csup\u003e\u0026reg;\u003c/sup\u003e, Guarulhos, SP, Brazil) to allow later appropriate analysis of tooth demineralization by transverse microradiography - TMR. Thereafter, the samples were sterilized by exposure to ethylene oxide [20] and randomly distributed to the groups, considering roughness values (measured by using a contact\u0026nbsp;profilometer - Mahr Perthometer, G\u0026ouml;ttingen, Germany and the software MarSurf XCR-20 -Mahr Perthometer, G\u0026ouml;ttingen, Germany) as criteria (mean: 0.28 \u0026plusmn; 0.02 \u0026micro;m).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eForty-eight dentin samples (n=24 irradiated and n=24 non-irradiated) were submitted to two distinct microcosm biofilm formations (using irradiated biofilm or healthy biofilm), totalizing four groups (n=12/group): (1)\u0026nbsp;Irradiated dentin and irradiated biofilm; (2) Irradiated dentin and healthy biofilm; (3) Sound dentin and irradiated biofilm; (4) Sound dentin and healthy biofilm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile seventy-two samples of irradiated dentin were submitted to microcosm biofilm formation using biofilm from irradiated patients and divided into 6 treatments with artificial saliva (n=12/group):\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(A) Artificial Saliva A: pH 5.6 \u0026ndash;\u0026nbsp;NaHCO\u003csub\u003e3\u003c/sub\u003e (0.219%); K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e (0.127%); CaCI\u003csub\u003e2\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO (0.0654%); MgCl\u003csub\u003e2\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO (0.0125%); KCl (0.082%); Methylparaben (0.01%); Propylparaben (0.01%); Carboxymethylcellulose (0.8%);\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(B)\u0026nbsp;Saliva A + 1 mg/mL hemoglobin (human hemoglobin, Sigma Aldrich, Saint Louis, MI, USA, H7379) (pH 5.4) [19];\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(C)\u0026nbsp;Saliva A + 0.1 mg/mL sugarcane cystatin (Cane CPI-5) (pH 5.5) [16];\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(D)\u0026nbsp;Saliva A + 1 mg/mL hemoglobin + 0.1 mg/mL sugarcane cystatin (Cane CPI-5) (pH 5.5);\u003c/p\u003e\n\u003cp\u003e(E)\u0026nbsp;BioXtra\u003csup\u003e\u0026reg;\u003c/sup\u003e (positive control) [active components: lysozyme; lactoferrin, lactoperoxidase; colostrum extrac. Other ingredients: water, Propylene Glycol, Xylitol, Sodium Monofluorophosphate (1500 ppm F\u003csup\u003e-\u003c/sup\u003e)., Poloxamer 407, Hydroxyethylcellulose, Aroma, Aloe Barbadensis Leaf Juice, EDTA, Lactic Acid, Sodium Benzoate, Limonene, Linalool, CI42090.\u0026nbsp;Lifestream Pharma, Seneffe, Belgium] (pH 6.3);\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(F) Deionized Water (negative control) (pH 7.1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll the experiments were done in biological triplicate (n=4/replicate, n final=12). The flowchart of the study is in Online Resource 1. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMicrocosm biofilm formation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe biofilm-glycerol stock was diluted in McBain artificial saliva [22], at a ratio of 1:50 (inoculum) [20]. The microcosm biofilm was grown on the dentin samples placed into the 24-well plates, for five days. On the first day, each dentin sample was exposed to 1.5 ml of inoculum for 8 hours. After this time, the inoculum was removed, the samples were washed with PBS (1.5 ml, 5 s) and received 1.5 ml fresh medium (McBain Saliva with 0.2% of sucrose) until completing 16 hours. \u0026nbsp;From the second to the fifth day, the medium with sucrose was changed once a day and the plates were incubated at 5% CO\u003csub\u003e2\u003c/sub\u003e and 37\u0026deg;C [17, 20]. Treatments with artificial saliva were performed between medium changes, once/day for 1 minute, during the following 4 days of microcosm biofilm formation (schematic diagram of biofilm microcosm in Online Resource 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eColony-forming unit (CFU) counting\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAfter 5 days, the microbial suspension from each well plate, obtained by adding 1 ml of 0.89% NaCl solution over the formed biofilm, was sonicated (Sonifier Cell Disruptor B-30, Branson) for 30 s at 20 W. Bacterial suspensions were diluted (10\u003csup\u003e\u0026minus;4\u003c/sup\u003e or 10\u003csup\u003e-5\u003c/sup\u003e) and spread on Petri dishes (25 \u0026mu;L/dish) and then, the dishes incubated under 5% CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eand 37 \u0026deg;C for 48 h [17, 20]. The four agar culture media used were [20]: 1) brain heart infusion agar (BHI; Difco, Detroit, USA) for total microorganisms (dilution factor 10\u003csup\u003e-5\u003c/sup\u003e); (2) mitis salivarius agar (MSA; Neogen, Indaiatuba, Brazil) for total \u003cem\u003estreptococci (\u003c/em\u003edilution factor 10\u003csup\u003e-4\u003c/sup\u003e); (3) SB-20M for \u003cem\u003emutans streptococci\u003c/em\u003e (\u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eS. sobrinus\u003c/em\u003e) (dilution factor 10\u003csup\u003e-4\u003c/sup\u003e); and (4) MRS (Kasvi, Curitiba, Brazil) for \u003cem\u003eLactobacillus\u003c/em\u003e \u003cem\u003esp\u003c/em\u003e. (dilution factor 10\u003csup\u003e-5\u003c/sup\u003e). \u0026nbsp; After 48h, the CFU numbers were counted and used, together with the dilution factor, to calculate the total CFU for each type of microorganism per group. The data were transformed in log\u003csub\u003e10\u003c/sub\u003e CFU/mL [20].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTransverse microradiography (TMR) - demineralization analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDentin samples were cleaned\u0026nbsp;with sterile gauze, transversally sectioned and polished (100-120 \u0026micro;m thickness). The procedure of exposure to X-ray\u0026nbsp;(20kV and 20 mA, Softex, Tokyo, Japan), development and analysis of the microradiography plates was performed as previously described [20], using the TMR from Inspektor Research System (Amsterdam, Netherlands) (schematic diagram of the sample preparation for TMR in Online Resource 3). The mineral content was calculated assuming 50 vol% of mineral content for sound dentin and that the lesion depth ends when dentin contains around of 47.5% of mineral volume [20]. The integrated mineral loss (\u0026Delta;Z, %vol. \u0026micro;m), the average mineral loss over the lesion depth (mean mineral loss, R, %vol) and the lesion depth (LD, \u0026micro;m) were calculated [20].\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe data were submitted to statistical analysis using the Graph Pad Prism 7.04 program (San Diego, USA). (p\u0026lt;0.05).\u0026nbsp;The normal distribution and homogeneity were checked using Kolmogorov\u0026ndash;Smirnov and Bartlett tests, respectively. For the comparison between biofilm (irradiated or healthy) x dentin (irradiated or sound) 2-way ANOVA/Bonferroni test (CFU counting) and 2-way ANOVA/Sidak test (TMR parameters) were done. For comparison between treatments with artificial saliva, ANOVA/Tukey test was applied for CFU counting (total microorganisms and\u0026nbsp;\u003cem\u003eLactobacillus\u0026nbsp;\u003c/em\u003espp) and for TMR parameters (R and LD) and Kruskal-Wallis/Dunn for CFU counting (total streptococci and \u003cem\u003emutans streptococci\u003c/em\u003e) and for the Integrated Mineral Loss (\u0026Delta;Z). Statistical sample size (n=12) was based on previous study using irradiated dentin and microcosm biofilm model [20].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eCFU counting\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThere was no difference between the type of dentin and biofilm on the growth of total microorganisms. Regarding total streptococci, sound dentin had a higher number of CFU compared to irradiated dentin, in the case of irradiated biofilm, but no difference was seen in the case of healthy biofilm. For \u003cem\u003eLactobacillus sp.\u0026nbsp;\u003c/em\u003eand \u003cem\u003emutans streptococci,\u003c/em\u003e there was no difference between the type of dentin (irradiated or sound), while the irradiated biofilm had a higher number of CFU compared to healthy biofilm for sound dentin. For irradiated dentin, no difference was found (Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWith respect to the artificial saliva formulations comparisons, for total \u003cem\u003estreptococci\u003c/em\u003e and \u003cem\u003emutans streptococci\u003c/em\u003e, treatments with experimental saliva, especially those containing hemoglobin, had a higher CFU counting than the negative control (water), differing significantly (Table 2). The positive control (BioXtra\u003csup\u003e\u0026reg;\u003c/sup\u003e) significantly reduced the CFU counting for all analyzed species compared to the experimental saliva (sample power of 97%, calculated from http://powerandsamplesize.com/Calculators ); however, it did not differ from negative control (water) with respect to \u003cem\u003eLactobacillus sp.\u003c/em\u003e, \u003cem\u003emutans streptococci\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;\u003c/em\u003etotal \u003cem\u003estreptococci\u0026nbsp;\u003c/em\u003egrowth. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTMR analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNo differences were found between the type of biofilm and type of dentin regarding the integrated mineral loss (\u0026Delta;Z). For lesion depth (LD) and mean mineral loss (R), sound dentin presented lower demineralization compared to the irradiated one (Table 3). Figure 1 shows representatives TMR images from each group.\u003c/p\u003e\n\u003cp\u003eThe experimental saliva formulations were not able to reduce the caries lesion development under this experimental model. Only the positive control (BioXtra\u003csup\u003e\u0026reg;\u003c/sup\u003e) was able to reduce LD and R significantly compared to the experimental saliva and the negative control (Table 4, Figure 2).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIrradiated biofilm has no influence on dentin caries lesion formation while irradiation of dentin increases the susceptibility to demineralization under the tested model. Therefore, the first null hypothesis was rejected and the second one accepted. There are changes in the composition and structure of root dentin at exposure to doses greater than 60 Gy with reduction of the mineral/organic matrix ratio [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], in addition, to possible changes in the composition of the organic and collagen content [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The mineral content also appears to be altered due to the radiation, with a decrease in the calcium and phosphate ratio, changing dentin hardness value [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The cited alterations in the mineral content and mainly in the organic content can make dentin more susceptible to demineralization [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious study has shown no difference in the formation of the microcosm biofilm with respect to the inoculum source, whether it comes from saliva or from dental biofilm [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Considering that hyposalivation is one of the main consequences of head and neck radiotherapy, and that this condition can persist even 6 months after the end of treatment [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], we chose to collect dental biofilm in the present study.\u003c/p\u003e \u003cp\u003eChange in the oral microbiota of patients submitted to doses greater than 30 Gy has been detected [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], with a high level of \u003cem\u003eLactobacillus\u003c/em\u003e (58%) and \u003cem\u003emutans streptococci\u003c/em\u003e (35%) even 1 year after radiotherapy [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In the present study, there is a small difference in the results of CFU counting, with a slight increase of cariogenic bacteria in the irradiated biofilm, although these differences were not significant to influence the demineralization parameters, which may be due to the continuous sucrose exposure during microcosm biofilm formation. Thus, the irradiated biofilm in this experimental model appears to exert no influence on the formation of the initial dentin caries lesion. A recent work [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] demonstrated that the initial bacteriological profile of the inoculum did not interfere in the formation of the initial caries lesion under microcosm biofilm model, once the conditions for biofilm growth have been standardized \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. If a less cariogenic model would have been applied, the distinction between the sources of microorganism could have been different. More specific techniques, such as biofilm microbiome, would be relevant for understanding eventual microbiological differences between dental biofilm sources (from irradiated vs. healthy patient).\u003c/p\u003e \u003cp\u003eThe experimental artificial saliva with proteins (Cane CPI-5 and hemoglobin) did not present promising results in the reduction of demineralization or not even in the reduction of the CFU counting when compared to water (negative control); thus, the third null hypothesis was accepted. In fact, the negative control group, with deionized water washing, showed better results than the tested experimental saliva. This fact could be explained by the washing effect of water, which could have disorganized the biofilm or/and remove the acid product, since this group was subjected to washing procedure twice (PBS and water). This also helps to justify differences in dentin demineralization between negative control vs. irradiated dentin/irradiated biofilm (not compared statistically), since irradiated dentin/irradiated biofilm group was not exposed to any type of washing.\u003c/p\u003e \u003cp\u003eCane CPI-5 has shown promising results in preventing erosive wear (enamel and dentin) \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and \u003cem\u003ein situ\u003c/em\u003e at a concentration of 0.1 mg/ml [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Regarding the prevention of enamel caries, a recent study also under microcosm biofilm showed that different concentrations of Cane CPI-5 (0.05, 0.1 and 0.5 mg/ml) were effective in significantly reducing the integrated mineral loss when compared to the negative control (PBS) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Furthermore, the lowest concentrations (0.05 and 0.1 mg/ml) were the most effective in reducing the CFU counting for cariogenic bacteria (\u003cem\u003eLactobacillus sp\u003c/em\u003e, total \u003cem\u003estreptococci\u003c/em\u003e and \u003cem\u003emutans streptococci\u003c/em\u003e) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. When the Cane CPI-5 concentration was increased to 1 mg/ml, the protective antibacterial effect was lost [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The concentrations that seem to be most effective, both for preventing tooth erosion and for enamel caries control, are 0.05 and 0.1 mg/ml. However, we did not find similar results for dentin in our study, using a concentration of 0.1 mg/ml. Lower concentrations of Cane CPI-5 (0.025 and 0.05 mg/ml) have already been tested in microcosm biofilm for root dentin and they were not able to reduce the CFU counting for any bacterial group evaluated neither to reduce demineralization compared to negative control (PBS) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHemoglobin at a concentration of 1 mg/ml showed similar results to Cane CPI-5 in preventing erosive tooth wear \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In addition, the association of hemoglobin (1 mg/ml) and Cane CPI-5 (0.1 mg/ml) was the one that presented the best protective result against erosive tooth wear \u003cem\u003ein vitro\u003c/em\u003e when compared to the isolated proteins [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Thus, the combination of both proteins could be more effective in preventing dental caries as well. No previous studies evaluated hemoglobin and the association between hemoglobin and Cane CPI-5 for preventing dental caries. However, our study did not show additive effect of the combination.\u003c/p\u003e \u003cp\u003eDifferently from enamel, dentin has a higher organic content that, once exposed to demineralization, is subjected to the activation of matrix metalloproteinase (MMP) responsible for degrading its organic content. MMPs are considered important mediators of the degradation of the dentin organic matrix, being collagenases and gelatinases the most associated with dentin caries [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Radiotherapy has been associated with the increased expression and activation of MMPs in some tissues [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In addition, there is a greater presence of cariogenic bacteria in dentinal lesions compared to enamel lesions [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] as well as the presence of proteolytic bacteria [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], which could have digested the tested proteins added in experimental saliva and their derivates. All these factors may have influenced the low efficacy of the experimental saliva, and perhaps for the specific condition (dentin\u0026thinsp;+\u0026thinsp;radiotherapy) the concentration of Cane CPI-5 and hemoglobin should have been higher to those tested in the present study.\u003c/p\u003e \u003cp\u003eIt is also important to consider that, despite the good results of proteins in previous studies with enamel, both Cane CPI-5 (pH 7.88) and hemoglobin were tested diluted in deionized water [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Only Pel\u0026aacute; et al. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] evaluated Cane CPI-5 incorporated into a chitosan gel (prepared with acetic acid), pH 5.5, against erosive tooth wear. When these proteins are added in a more complex vehicle in terms of the number of reagents, such as the artificial saliva used in the present work, many variables may influence their effect, such as the final pH of the product and possible interactions between components, both affecting the protein bioavailability. Accordingly, carboxymethylcellulose, which has a negative charge, could have interacted with the tested proteins, whose charge is positive [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Carboxymethylcellulose is added into artificial saliva since it plays an important role in improving oral lubrication [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. More studies need to be carried out focusing on the maintenance of the protective action of proteins and their stability, minimizing possible interactions within dental products as gels, mousses, mouthwashes, and toothpastes. It was not possible to adjust the pH of the experimental saliva (pH values between 5.4 and 5.6) to be similar to the positive control \u0026ndash; BioXtra (pH 6.3), since it could alter the stability and reactivity of the saliva.\u003c/p\u003e \u003cp\u003eThe only saliva that had a protective potential was the positive control (BioXtra\u0026reg;), which has proteins lysozyme, lactoferrin and lactoperoxidase as active ingredients, with broad-spectrum bacteriostatic activity [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] and it is used to relieve symptoms of dry mouth after radiotherapy [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. No study has evaluated saliva BioXtra\u0026reg; with respect to antimicrobial or anticaries effect. Formulations of toothpaste and gel (with the same active ingredients of artificial saliva) have shown to reduce the number of \u003cem\u003eLactobacillus sp.\u003c/em\u003e and \u003cem\u003eS. mutans\u003c/em\u003e in previous clinical studies [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In addition, sodium monofluorophosphate (1,500 ppm F\u003csup\u003e\u0026minus;\u003c/sup\u003e) is present in its composition, described as an inactive ingredient. Although described as an inactive ingredient, in our experimental model, fluoride into the artificial saliva might have reduced demineralization and increased remineralization [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The influence of the presence of fluoride in BioXtra\u0026reg; saliva should be studied in the future. As the work had focus on artificial saliva, we did not include F solution as a control group. This shall be done in the future to better understand the results.\u003c/p\u003e \u003cp\u003eIn conclusion, irradiated biofilm has no influence, while the type of dentin has impact on dentin caries development; none of the experimental saliva was able to reduce its occurrence. Considering that no previous study had compared the impact of tooth irradiation and biofilm from irradiated patients on the dentin caries formation, our study highlighted the impact of the irradiation in increasing the susceptibility of dentin to demineralization. Other important finding is that BioXtra\u0026reg; saliva is able to reduce dentin caries lesion development, while the experimental solutions were not, which should be confirmed in future studies.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The authors B.M.S. and A.C.M. received\u0026nbsp;by S\u0026atilde;o Paulo Research Foundation (FAPESP) (grant number\u0026nbsp;2019/07241-0 and 2019/21797-0, respectively).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e All the authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval:\u003c/strong\u003e\u0026nbsp; This study was approved by ethics committee on animal research of \u0026nbsp;the Bauru School of Dentistry, University of S\u0026atilde;o Paulo, Brazil (CEUA, Number: 004/2018). All procedures performed in studies involving human participants were in accordance with the ethical standards of the Institutional and/or National Research Committee (Ethics Committee of the Bauru School of Dentistry, University of S\u0026atilde;o Paulo, Brazil, number\u0026nbsp;97497318.00000.5417) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e Informed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eAll data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eJensen SB, Pedersen AM, Vissink A,\u0026nbsp;Andersen E, \u0026nbsp;Brown CG, \u0026nbsp;Davies AN, Dutilh J, Fulton JS, Jankovic L, Lopes NN, Mello AL, Muniz LV, Murdoch-Kinch CA, Nair RG, Nape\u0026ntilde;as JJ, Nogueira-Rodrigues A, Saunders D, Stirling B, von B\u0026uuml;ltzingsl\u0026ouml;wen I, Weikel DS, Elting LS, Spijkervet FK, Brennan MT (2010)\u0026nbsp;Salivary gland hypofunction/xerostomia section, oral care study group, multinational association of supportive care in cancer (MASCC)/international society of oral oncology (ISOO). A systematic review of salivary gland hypofunction and xerostomia induced by cancer therapies: prevalence, severity and impact on quality of life. Support Care Cancer 18(8):1039-1060. https://doi.org/10.1007/s00520-010-0827-8.\u003c/li\u003e\n \u003cli\u003ePereira IF, Firmino RT, Meira HC, Do Egito Vasconcelos BC, De Souza Noronha VRA, Santos VR (2019) Radiation-induced oral mucositis in brazilian patients: Prevalence and associated factors. In vivo 33(2):605-609. https://doi.org/10.21873/invivo.11517.\u003c/li\u003e\n \u003cli\u003eVelo MMAC, Farha ALH, da Silva Santos PS,\u0026nbsp;Shiota A, Sansavino SZ, Souza AT, Hon\u0026oacute;rio HM, Wang L (2018)\u0026nbsp;Radiotherapy alters the composition, structural and mechanical properties of root dentin in vitro. Clin Oral Investig 22(8):2871-78. https://doi.org/\u0026nbsp;10.1007/s00784-018-2373-6.\u003c/li\u003e\n \u003cli\u003eRodrigues RB, Soares CJ, Junior PCS, Lara VC, Arana-Chavez VE, Novais VR (2018) Influence of radiotherapy on the dentin properties and bond strength. Clin Oral Investig (2):875-883. https://doi.org/10.1007/s00784-017-2165-4.\u003c/li\u003e\n \u003cli\u003eQueiroz AM, Bonilla CMC, Palma-Dibb RG,\u0026nbsp;Oliveira HF, Nelson-Filho P, Silva LAB, Lucisano MP, Paula-Silva FWG (2019) Radiotherapy Activates and Protease Inhibitors Inactivate Matrix Metalloproteinases in the Dentinoenamel Junction of Permanent Teeth. Caries Res 53(3):253-259. https://doi.org/10.1159/000492081.\u003c/li\u003e\n \u003cli\u003eMakkonen T(1988) Studies on oral complications of head and neck cancer radiotherapy. Proc Finn Dent Soc 84 Suppl 4-5:1-111.\u003c/li\u003e\n \u003cli\u003eHamilton SN, Arshad O, Kwok J,\u0026nbsp;Tran E, Fuchsia Howard A, Serrano I, Goddard K (2019)\u0026nbsp;Documentation and incidence of late effects and screening recommendations for adolescent and young adult head and neck cancer survivors treated with radiotherapy. Support Care Cancer 27(7):2609-2616. https://doi.org/10.1007/s00520-018-4559-5.\u003c/li\u003e\n \u003cli\u003eSiala W, Mnejja W, Khabir A,\u0026nbsp;Ben Mahfoudh K, Boudawara T, Ghorbel A, Frikha M, Daoud J (2019)\u0026nbsp;Toxicit\u0026eacute; neurologique tardive apr\u0026egrave;s traitement des carcinomes nasopharyng\u0026eacute;s (Late neurotoxicity after nasopharyngeal carcinoma treatment).\u0026nbsp;Cancer Radiother 13(8):709-714.French. https://doi.org/10.1016/j.canrad.2009.05.006.\u003c/li\u003e\n \u003cli\u003eMeyer-Lueckel H, Umland N, Hopfenmuller W, Kielbassa AM (2004) Effect of mucin alone and in combination with various dentifrices on in vitro Remineralization. Caries Res 38(5):478-483. https://doi.org/10.1159/000079630.\u003c/li\u003e\n \u003cli\u003eKielbassa AM, Oeschger U, Schulte-Monting J, Meyer-Lueckel H (2005) Microradiographic study on the effects of salivary proteins on in vitro demineralization of bovine enamel. J Oral Rehabil 32(2):90-96. https://doi.org/10.1111/j.1365-2842.2004.01392.x.\u003c/li\u003e\n \u003cli\u003eSmith G, Smith AJ, Shaw L, Shaw MJ (2001) Artificial saliva substitutes and mineral dissolution. J Oral Rehabil 28(8):728-731. https://doi.o rg/10.1046/j.1365-2842.2001.00803.x.\u003c/li\u003e\n \u003cli\u003eMeyer-Lueckel H, Schulte-M\u0026ouml;nting J, Kielbassa AM (2002) The effect of commercially available saliva substitutes on predemineralized bovine dentin in vitro. Oral Dis 8(4):192-198. https://doi.org/10.1034/j.1601-0825.2002.01762.x.\u003c/li\u003e\n \u003cli\u003eF\u0026aacute;bi\u0026aacute;n TK, Hermann P, Beck A, Fej\u0026eacute;rdy P, F\u0026aacute;bi\u0026aacute;n G (2012) Salivary defense proteins: their network and role in innate and acquired oral immunity. Int J Mol Sci 13(4):4295-4320. https://doi.org/10.3390/ijms13044295.\u003c/li\u003e\n \u003cli\u003eDelecrode TR, Siqueira WL, Zaidan FC,\u0026nbsp;Bellini MR, Moffa EB, Mussi MC, Xiao Y, Buzalaf\u0026nbsp;MA (2015) Identification of acid-resistant proteins in acquired enamel pellicle. J Dent 43(12):1470-1475. https://doi.org/10.1016/j.jdent.2015.10.009.\u003c/li\u003e\n \u003cli\u003eVentura TMDS, Cassiano LPS, Souza E Silva CM,\u0026nbsp;Taira EA, Leite AL, Rios D, Buzalaf MAR\u0026nbsp;(2017) The proteomic profile of the acquired enamel pellicle according to its location in the dental arches. Arch Oral Biol 79:20-29. https://doi.org/10.1016/j.archoralbio.2017.03.001.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSantiago AC, Khan ZN, Miguel MC, Gironda CC, Soares-Costa A, Pel\u0026aacute; VT, Leite AL, Edwardson JM, Buzalaf MAR, Henrique-Silva F (2017) A new sugarcane cystatin strongly binds to dental enamel and reduces erosion. J Dent Res 96(9):1051-1057. https://doi.org/10.1177/0022034517712981.\u003c/li\u003e\n \u003cli\u003eAraujo TT, Camiloti GD, Valle AD, Silva NDG, Souza BM, Carvalho TS, C\u0026acirc;mara JVF, Shibai PYT, Henrique-Silva F, Cruvinel T, Magalh\u0026atilde;es AC, Buzalaf MAR (2021) A sugarcane cystatin (CaneCPI-5) alters microcosm biofilm formation and reduces dental caries. Biofouling 37(1):109-116. https://doi.org/10.1080/08927014.2021.1881065.\u003c/li\u003e\n \u003cli\u003eYu YD, Zhu YJ, Qi C, Jiang YY, Li H, Wu J (2017) Hydroxyapatite nanorod-assembled porous hollow polyhedra as drug/protein carriers. J Colloid Interface Sci 496:416-424. https://doi.org/10.1016/j.jcis.2017.02.041.\u003c/li\u003e\n \u003cli\u003eMartini T, Rios D, Dionizio A, Cassiano LPS, Taioqui Pel\u0026aacute; V, Silva CMS, Taira EA, Ventura TM, Magalh\u0026atilde;es AC, Carvalho TS, Baumann T, Lussi A, de Oliveira RB, Palma-Dibb RG, Buzalaf MAR (2020) Salivary hemoglobin protects against erosive tooth wear in gastric reflux patients. Caries Res 54(5-6):466-474. https://doi.org/10.1159/000507110.\u003c/li\u003e\n \u003cli\u003ede Souza BM, Silva MS, Braga AS, Bueno PSK, da Silva Santos PS, Buzalaf MAR, Magalh\u0026atilde;es AC (2021) Protective effect of titanium tetrafluoride and silver diamine fluoride on radiation-induced dentin caries in vitro. Sci Rep 11(1):6083. https://doi.org/10.1038/s41598-021-85748-8.\u003c/li\u003e\n \u003cli\u003eSignori C, van de Sande FH, Maske TT, de Oliveira EF, Cenci MS (2016) Influence of the Inoculum Source on the Cariogenicity of in vitro Microcosm Biofilms. Caries Res 50(2):97-103. https://doi.org/10.1159/000443537.\u003c/li\u003e\n \u003cli\u003eMcBain AJ (2009) Chapter 4: In vitro biofilm models: an overview. Adv Appl Microbiol. 69:99-132. https://doi.org/10.1016/S0065-2164(09)69004-3.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ede Miranda RR, Silva ACA, Dantas NO, Soares CJ, Novais VR (2019) Chemical analysis of in vivo-irradiated dentine of head and neck cancer patients by ATR-FTIR and Raman spectroscopy. Clin Oral Investig 23(8):3351-3358. https://doi.org/10.1007/s00784-018-2758-6.\u003c/li\u003e\n \u003cli\u003eCampi LB, Lopes FC, Soares LES, de Queiroz AM, de Oliveira HF, Saquy PC, de Sousa-Neto MD (2019) \u0026nbsp;Effect of radiotherapy on the chemical composition of root dentin. Head Neck 41(1):162-169. https://doi.org/10.1002/hed.25493.\u003c/li\u003e\n \u003cli\u003eDouchy L, Gauthier R, Abouelleil-Sayed H, Colon P, Grosgogeat B, Bosco J (2022) The effect of therapeutic radiation on dental enamel and dentin: A systematic review. Dent Mater 38(7):e181-e201. https://doi.org/10.1016/j.dental.2022.04.014.\u003c/li\u003e\n \u003cli\u003eGaetti-Jardim E Jr, Jardim ECG, Schweitzer CM, da Silva JCL, Oliveira MM, Masocatto DC, dos Santos CM (2018) Supragingival and subgingival microbiota from patients with poor oral hygiene submitted to radiotherapy for head and neck cancer treatment. Arch Oral Biol 90:45-52. https://doi.org/10.1016/j.archoralbio.2018.01.003.\u003c/li\u003e\n \u003cli\u003eAl-Nawas B, Gr\u0026ouml;tz KA (2006) Prospective study of the long-term change of the oral flora after radiation therapy. Support Care Cancer 14(3):291-296. https://doi.org/10.1007/s00520-005-0895-3.\u003c/li\u003e\n \u003cli\u003eEliasson L, Carl\u0026eacute;n A, Almst\u0026aring;hl A, Wikstr\u0026ouml;m M, Lingstr\u0026ouml;m P (2006) Dental plaque pH and micro-organisms during hyposalivation. J Dent Res 85(4):334-338. https://doi.org/10.1177/154405910608500410.\u003c/li\u003e\n \u003cli\u003eViana CS, Maske TT, Signori C, Van de Sande FH, Oliveira EF, Cenci MS (2021) Influence of caries activity and number of saliva donors: mineral and microbiological responses in a microcosm biofilm model. J Appl Oral Sci 29:e20200778. https://doi.org/10.1590/1678-7757-2020-0778.\u003c/li\u003e\n \u003cli\u003eSantos LA, Martini T, C\u0026acirc;mara JVF, Reis FN, Ortiz AC, Camiloti GD, Levy FM, Shibao PyT, Honorio HM, Henrique-Silva F, Pieretti JC, Seabra AB, Cardoso CAB, Buzalaf MAR (2021) Solutions and gels containing a sugarcane-derived cystatin (CaneCPI-5) reduce enamel and dentin erosion in vitro. Caries Res 55(6):594-602. https://doi.org/10.1159/000520261.\u003c/li\u003e\n \u003cli\u003eGironda CC, Pel\u0026aacute; VT, Henrique-Silva F, Delbem ACB, Pessan JP, Buzalaf MAR (2022) New insights into the anti-erosive property of a sugarcane-derived cystatin: different vehicle of application and potential mechanism of action. J Appl Oral Sci 30:e20210698. https://doi.org/10.1590/1678-7757-2021-0698.\u003c/li\u003e\n \u003cli\u003ePel\u0026aacute; VT, Lunardelli JGQ, Tokuhara CK, Gironda CC, Silva NDG, Carvalho TS, Santiago AC, Souza BM, Moraes F, Henrique-Silva F, Magalh\u0026atilde;es AC, Oliveira RC, Buzalaf MAR (2021) Safety and In Situ Antierosive Effect of CaneCPI-5 on Dental Enamel. J Dent Res 100(12):1344-1350. https://doi.org/10.1177/00220345211011590.\u003c/li\u003e\n \u003cli\u003ePel\u0026aacute; VT, Braga AS, Camiloti GD,Lunardelli JGQ, Pires JG, Toyama D, Santiago AC, Henrique-Silva F, Magalh\u0026atilde;es AC, Buzalaf MAR (2021) Antimicrobial and anti-caries effects of a novel cystatin from sugarcane on saliva-derived multi-species biofilms. Swiss Dent J 2021;131(5):410-416.\u003c/li\u003e\n \u003cli\u003eFraz\u0026atilde;o C\u0026acirc;mara JV, Araujo TT, Mendez DAC, da Silva NDG, Medeiros FF, Santos LA, de Souza Carvalho T, Reis FN, Martini T, Moraes SM, Shibao PYT, Groisman S, Magalh\u0026atilde;es AC, Henrique-Silva F, Buzalaf MAR (2021) Effect of a sugarcane cystatin on the profile and viability of microcosm biofilm and on dentin demineralization. Arch Microbiol 203(7):4133-4139. https://doi.org/10.1007/s00203-021-02403-6.\u003c/li\u003e\n \u003cli\u003ePel\u0026aacute; VT, Buzalaf MAR, Niemeyer SH, Baumann T, Henrique-Silva F, Toyama D, Crusca E, Marchetto R, Lussi A, Carvalho TS (2021) Acquired pellicle engineering with proteins/peptides: Mechanism of action on native human enamel surface. J Dent 107:103612. https://doi.org/10.1016/j.jdent.2021.103612.\u003c/li\u003e\n \u003cli\u003eGomes-Silva W, Prado Ribeiro AC, de Castro Junior G, Salvajoli JV, Rangel Palmier N, Lopes MA, Rocha MM, de Goes MF, Brand\u0026atilde;o TB, Santos-Silva AR (2017)\u0026nbsp;Head and neck radiotherapy does not increase gelatinase (metalloproteinase-2 and -9) expression or activity in teeth irradiated in vivo. Oral Surg Oral Med Oral Pathol Oral Radiol 124(2):175-182. https://doi.org/10.1016/j.oooo.2017.04.009.\u003c/li\u003e\n \u003cli\u003eRichards VP, Alvarez AJ, Luce AR, et al. Microbiomes of Site-Specific Dental Plaques from Children with Different Caries Status. Infect Immun 2017;85(8):e00106-17. https://doi.org/10.1128/IAI.00106-17.\u003c/li\u003e\n \u003cli\u003eBhaumik D, Salzman E, Davis E, Bedenbaugh M, Mitchell ML, Burne RA, Nascimento MM (2022) Plaque Microbiome in Caries-Active and Caries-Free Teeth by Dentition. JDR Clin Trans Res 23800844221121260. https://doi.org/10.1177/23800844221121260.\u003c/li\u003e\n \u003cli\u003eTakahashi N, Nyvad B (2016) Ecological hypothesis of dentin and root caries. Caries Res 50(4):422-431. https://doi.org/10.1159/000447309.\u003c/li\u003e\n \u003cli\u003ePel\u0026aacute; VT, Brito L, Taira EA, Henrique-Silva F, Pieretti JC, Seabra AB, de Almeida Baldini Cardoso C, de Souza EP , Groisman S, Rodrigues MC, Lussi A, Carvalho TS, Buzalaf MAR,\u0026nbsp;(2022) Preventive effect of chitosan gel containing CaneCPI-5 against enamel erosive wear in situ. Clin Oral Investig 26(11):6511-6519. https://doi.org/10.1007/s00784-022-04600-z.\u003c/li\u003e\n \u003cli\u003eJi L, Orthmann A, Cornacchia L, Peng J, Sala G, Scholten E (2022) Effect of different molecular characteristics on the lubrication behavior of polysaccharide solutions. Carbohydr Polym 297:120000. https://doi.org/10.1016/j.carbpol.2022.120000.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eVinke J, Kaper HJ, Vissink A, Sharma PK (2020) Dry mouth: saliva substitutes which adsorb and modify existing salivary condition films improve oral lubrication. Clin Oral Investig 24(11):4019-4030. https://doi.org/10.1007/s00784-020-03272-x.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eGudipaneni RK, Kumar R V, Jesudass G, Peddengatagari S, Duddu Y (2014) Short term comparative evaluation of antimicrobial efficacy of tooth paste containing lactoferrin, lysozyme, lactoperoxidase in children with severe early childhood caries: a clinical study. J Clin Diagn Res 8(4):ZC18-20. https://doi.org/10.7860/JCDR/2014/8161.4232.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eGookizadeh A, Emami H, Najafizadeh N, Roayaei M (2012) Clinical evaluation of BIOXTRA in relieving signs and symptoms of dry mouth after head and neck radiotherapy of cancer patients at Seyed-al-Shohada Hospital, Isfahan, Iran. Adv Biomed Res 1:72. https://doi.org/10.4103/2277-9175.102976.\u003c/li\u003e\n \u003cli\u003eSouza BM, Fernandes Neto C, Salom\u0026atilde;o PMA, Vasconcelos LRSM, Andrade FB, Magalh\u0026atilde;es AC (2018) Analysis of the antimicrobial and anti-caries effects of TiF4 varnish under microcosm biofilm formed on enamel. J Appl Oral Sci 26:e20170304. https://doi.org/10.1590/1678-7757-2017-0304.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Mean \u0026plusmn; SD of CFU counting (log\u003csub\u003e10\u003c/sub\u003e CFU/ mL) of Total Microorganisms, \u003cem\u003eLactobacillus sp\u003c/em\u003e. (10\u003csup\u003e-5\u003c/sup\u003e), Total \u003cem\u003estreptococci\u0026nbsp;\u003c/em\u003eand \u003cem\u003eStreptococcus mutans/Streptococcus sobrinus\u003c/em\u003e (10\u003csup\u003e-4\u003c/sup\u003e) of Microcosm Biofilm Produced from Different Sources of Biofilm on Distinct Dentin Substrates. \u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"675\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.444444444444443%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.25925925925926%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003etotal microorganisms\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.037037037037038%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eLactobacillus sp.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.77777777777778%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003etotal \u003cem\u003estreptococci\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.48148148148148%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. mutans/ S. sobrinus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.444444444444443%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIrritated dentin and irritated biofilm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.25925925925926%\"\u003e\n \u003cp\u003e6.66 \u0026plusmn; 0.23\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.037037037037038%\"\u003e\n \u003cp\u003e7.15 \u0026plusmn; 0.37\u003csup\u003e\u0026nbsp;Aa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.77777777777778%\"\u003e\n \u003cp\u003e6.82\u0026plusmn; 0.35\u003csup\u003e\u0026nbsp;Aa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.48148148148148%\"\u003e\n \u003cp\u003e6.91 \u0026plusmn; 0.33\u003csup\u003e\u0026nbsp;Aa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.444444444444443%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIrritated dentin and healthy biofilm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.25925925925926%\"\u003e\n \u003cp\u003e6.43 \u0026plusmn; 0.48\u003csup\u003e\u0026nbsp;Aa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.037037037037038%\"\u003e\n \u003cp\u003e6.86 \u0026plusmn; 0.37\u003csup\u003e\u0026nbsp;Aa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.77777777777778%\"\u003e\n \u003cp\u003e6.81 \u0026plusmn; 0.22\u003csup\u003e\u0026nbsp;Aa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.48148148148148%\"\u003e\n \u003cp\u003e6.88 \u0026plusmn; 0.13\u003csup\u003e\u0026nbsp;Aa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.444444444444443%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHealthy dentin and irritated biofilm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.25925925925926%\"\u003e\n \u003cp\u003e6.67 \u0026plusmn; 0.37\u003csup\u003e\u0026nbsp;Aa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.037037037037038%\"\u003e\n \u003cp\u003e7.13 \u0026plusmn; 0.38\u003csup\u003e\u0026nbsp;Aa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.77777777777778%\"\u003e\n \u003cp\u003e7.28 \u0026plusmn; 0.35\u003csup\u003e\u0026nbsp;Bb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.48148148148148%\"\u003e\n \u003cp\u003e7.08 \u0026plusmn; 0.30\u003csup\u003e\u0026nbsp;Ab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.444444444444443%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHealthy dentin and healthy biofilm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.25925925925926%\"\u003e\n \u003cp\u003e6.44 \u0026plusmn; 0.36\u003csup\u003e\u0026nbsp;Aa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.037037037037038%\"\u003e\n \u003cp\u003e6.72 \u0026plusmn; 0.39\u003csup\u003e\u0026nbsp;Ab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.77777777777778%\"\u003e\n \u003cp\u003e6.95 \u0026plusmn; 0.31\u003csup\u003e\u0026nbsp;Aa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.48148148148148%\"\u003e\n \u003cp\u003e6.72 \u0026plusmn; 0.28\u003csup\u003e\u0026nbsp;Aa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eDifferent capital letters in the same column indicate a significant difference between dentin types. Different lowercase letters in the same column indicate a significant difference between biofilm types. 2 way-ANOVA/Bonferroni (total microorganisms: interaction p=0.988, biofilm type p=0.037, dentin type p=0.969; \u003cem\u003eLactobacillus\u003c/em\u003e \u003cem\u003espp.\u003c/em\u003e: interaction p=0.573, biofilm type p=0.003, dentin type p=0.471; total \u003cem\u003estreptococci\u003c/em\u003e: interaction p=0.085, biofilm type p=0.060, dentin type p=0.002; \u003cem\u003emutans streptococci\u003c/em\u003e: interaction p=0.045, biofilm type p=0.020, dentin type p=0.950).\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Mean \u0026plusmn; SD of CFU Counting (log\u003csub\u003e10\u003c/sub\u003e CFU/ mL) of Total Microorganisms and \u003cem\u003eLactobacillus sp.\u003c/em\u003e (10\u003csup\u003e-5\u003c/sup\u003e) and Median (interquartile range) of Total \u003cem\u003estreptococci\u003c/em\u003e and \u003cem\u003eStreptococcus mutans/Streptococcus sobrinus\u003c/em\u003e (10\u003csup\u003e-4\u003c/sup\u003e) of Microcosm Biofilm Treated with Different Saliva Formulations\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"675\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.333333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.925925925925927%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal microorganisms\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.185185185185187%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eLactobacillus sp.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.925925925925927%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003etotal \u003cem\u003estreptococci\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.62962962962963%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. mutans/ S. sobrinus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.333333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSaliva A (inorganic)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.925925925925927%\"\u003e\n \u003cp\u003e6.69 \u0026plusmn; 0.22\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.185185185185187%\"\u003e\n \u003cp\u003e7.20 \u0026plusmn; 0.32\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.925925925925927%\"\u003e\n \u003cp\u003e7.54 (0.34)\u003csup\u003eBC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.62962962962963%\"\u003e\n \u003cp\u003e7.30 (0.25)\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.333333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSaliva A + 1mg/mL hemoglobin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.925925925925927%\"\u003e\n \u003cp\u003e6.69 \u0026plusmn; 0.32\u003csup\u003e\u0026nbsp;B\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.185185185185187%\"\u003e\n \u003cp\u003e7.29 \u0026plusmn; 0.22\u003csup\u003e\u0026nbsp;B\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.925925925925927%\"\u003e\n \u003cp\u003e7.65 (0.16)\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.62962962962963%\"\u003e\n \u003cp\u003e7.42 (0.34)\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.333333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSaliva A + 0.1mg/mL cystatin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.925925925925927%\"\u003e\n \u003cp\u003e6.80 \u0026plusmn; 0.32\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.185185185185187%\"\u003e\n \u003cp\u003e7.30 \u0026plusmn; 0.33\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.925925925925927%\"\u003e\n \u003cp\u003e7.61 (0.46)\u003csup\u003eBC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.62962962962963%\"\u003e\n \u003cp\u003e7.28 (0.30)\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.333333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSaliva A + 1mg/mL hemoglobin + 0.1mg/mL cystatin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.925925925925927%\"\u003e\n \u003cp\u003e6.64 \u0026plusmn; 0.38\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.185185185185187%\"\u003e\n \u003cp\u003e7.22 \u0026plusmn; 0.37\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.925925925925927%\"\u003e\n \u003cp\u003e7.53 (0.19)\u003csup\u003eBC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.62962962962963%\"\u003e\n \u003cp\u003e7.11 (0.37)\u003csup\u003eBC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.333333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBioXtra\u003csup\u003e\u0026reg;\u003c/sup\u003e (positive control)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.925925925925927%\"\u003e\n \u003cp\u003e6.21\u0026plusmn; 0.23\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.185185185185187%\"\u003e\n \u003cp\u003e6.83 \u0026plusmn; 0.19\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.925925925925927%\"\u003e\n \u003cp\u003e6.88 (0.56)\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.62962962962963%\"\u003e\n \u003cp\u003e6.63 (0.64)\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.333333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003eWater (negative control)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.925925925925927%\"\u003e\n \u003cp\u003e6.61 \u0026plusmn; 0.31\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.185185185185187%\"\u003e\n \u003cp\u003e7.11 \u0026plusmn; 0.28\u003csup\u003eAB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.925925925925927%\"\u003e\n \u003cp\u003e7.22 (0.59)\u003csup\u003eAB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.62962962962963%\"\u003e\n \u003cp\u003e6.75 (0.54)\u003csup\u003eAB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eDifferent letters show statistical difference between treatments. ANOVA/Tukey: total microorganisms (p=0.0003); \u003cem\u003eLactobacillus spp.\u003c/em\u003e (p=0.0022). Kruskal-Wallis/Dunn: total \u003cem\u003estreptococci\u003c/em\u003e (p\u0026lt;0.0001); \u003cem\u003emutans streptococci\u003c/em\u003e (p\u0026lt;0.0001).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u003c/strong\u003e Mean \u0026plusmn; SD of the Integrated Mineral Loss (\u0026Delta;Z, vol%. \u0026mu;m), Lesion Depth (LD, \u0026mu;m) and the Mean Mineral Loss (R, vol%) of Irradiated and Sound Dentin Submitted to Demineralization from Different Biofilm Sources.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"632\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.34177215189873%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.158227848101266%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cstrong\u003eZ\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(vol%.\u003c/strong\u003e\u003cstrong\u003em\u003c/strong\u003e\u003cstrong\u003em)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.354430379746834%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLD\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003em\u003c/strong\u003e\u003cstrong\u003em)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.145569620253166%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eR\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(vol%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.34177215189873%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIrritated dentin and irritated biofilm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.158227848101266%\"\u003e\n \u003cp\u003e3,186\u0026nbsp;\u0026plusmn; 676\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.354430379746834%\"\u003e\n \u003cp\u003e123 \u0026plusmn; 27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.145569620253166%\"\u003e\n \u003cp\u003e26.1 \u0026plusmn; 2.8\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.34177215189873%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIrritated dentin and healthy biofilm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.158227848101266%\"\u003e\n \u003cp\u003e3,494 \u0026plusmn; 516\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.354430379746834%\"\u003e\n \u003cp\u003e126 \u0026plusmn; 25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.145569620253166%\"\u003e\n \u003cp\u003e28.1 \u0026plusmn; 3.3\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.34177215189873%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHealthy dentin and irritated biofilm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.158227848101266%\"\u003e\n \u003cp\u003e3,174 \u0026plusmn; 914\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.354430379746834%\"\u003e\n \u003cp\u003e114 \u0026plusmn; 26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.145569620253166%\"\u003e\n \u003cp\u003e25.6 \u0026plusmn; 2.2\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.34177215189873%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHealthy dentin and healthy biofilm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.158227848101266%\"\u003e\n \u003cp\u003e3,302 \u0026plusmn; 601\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.354430379746834%\"\u003e\n \u003cp\u003e134 \u0026plusmn; 27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.145569620253166%\"\u003e\n \u003cp\u003e23.7 \u0026plusmn; 2.9\u003csup\u003eBa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eDifferent capital letters in the same column indicate a significant difference between dentin types. Different lowercase letters in the same column indicate a significant difference between biofilm types. Absence of letters shows no statistical difference.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;2 way-ANOVA/Tukey (\u0026Delta;Z: interaction p=0.655; biofilm type p=0.281; dentin type p=0.613; LD: interaction p=0.275; biofilm type p=0.131; dentin type p=0.990; R: interaction p=0.021; biofilm type p=0.960; dentin type p=0.0042).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.\u003c/strong\u003e Median (interquartile range) of the Integrated Mineral Loss (\u0026Delta;Z, vol%. \u0026mu;m) and Mean \u0026plusmn; SD of the Lesion Depth (LD, \u0026mu;m) and the Mean Mineral Loss (R, vol%) of Irradiated Dentin Submitted to Microcosm Biofilm Treated with Different Saliva Formulations\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"603\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"40.728476821192054%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.033112582781456%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cstrong\u003eZ\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(vol%.\u003c/strong\u003e\u003cstrong\u003em\u003c/strong\u003e\u003cstrong\u003em)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.205298013245034%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLD\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003em\u003c/strong\u003e\u003cstrong\u003em)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.033112582781456%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eR\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(vol%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"40.728476821192054%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSaliva A (inorganic)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.033112582781456%\"\u003e\n \u003cp\u003e2,390 (485)\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.205298013245034%\"\u003e\n \u003cp\u003e120 \u0026plusmn; 17\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.033112582781456%\"\u003e\n \u003cp\u003e20.2 \u0026plusmn; 4.2\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"40.728476821192054%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSaliva A + 1mg/mL hemoglobin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.033112582781456%\"\u003e\n \u003cp\u003e2,240 (358)\u003csup\u003eBC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.205298013245034%\"\u003e\n \u003cp\u003e113 \u0026plusmn; 20\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.033112582781456%\"\u003e\n \u003cp\u003e19.4 \u0026plusmn; 2.1\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"40.728476821192054%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSaliva A + 0.1mg/mL cystatin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.033112582781456%\"\u003e\n \u003cp\u003e2,025 (1258)\u003csup\u003eBC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.205298013245034%\"\u003e\n \u003cp\u003e112 \u0026plusmn; 23\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.033112582781456%\"\u003e\n \u003cp\u003e19.9 \u0026plusmn; 4.4\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"40.728476821192054%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSaliva A + 1mg/mL hemoglobin + 0.1mg/mL cystatin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.033112582781456%\"\u003e\n \u003cp\u003e2,395 (1408)\u003csup\u003eBC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.205298013245034%\"\u003e\n \u003cp\u003e131\u0026plusmn; 30\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.033112582781456%\"\u003e\n \u003cp\u003e21.1 \u0026plusmn; 4.4\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"40.728476821192054%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBioXtra\u003csup\u003e\u0026reg;\u003c/sup\u003e (positive control)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.033112582781456%\"\u003e\n \u003cp\u003e445 (420)\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.205298013245034%\"\u003e\n \u003cp\u003e35 \u0026plusmn; 15\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.033112582781456%\"\u003e\n \u003cp\u003e10.8 \u0026plusmn; 2.5\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"40.728476821192054%\"\u003e\n \u003cp\u003e\u003cstrong\u003eWater (negative control)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.033112582781456%\"\u003e\n \u003cp\u003e1,520 (390)\u003csup\u003eAB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.205298013245034%\"\u003e\n \u003cp\u003e81 \u0026plusmn; 18\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.033112582781456%\"\u003e\n \u003cp\u003e17.3 \u0026plusmn; 3.3\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eDifferent letters show statistical difference between treatments. Kruskal-Wallis/Dunn: \u0026Delta;Z (p\u0026lt;0.0001). ANOVA/Tukey: LD (p\u0026lt;0.0001); R (p\u0026lt;0.0001). \u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"biofilms, dental caries, head and neck neoplasms, radiotherapy","lastPublishedDoi":"10.21203/rs.3.rs-3787488/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3787488/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eTo evaluate the influence of radiation as well as of new formulations of artificial saliva on the development of root caries lesions.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eBovine root samples were divided into: irradiated (70 Gy) dentin or not; the type of biofilm (from irradiated or non-irradiated patients) and the type of artificial saliva (for the condition irradiated dentin/biofilm): Saliva A (inorganic); Saliva A\u0026thinsp;+\u0026thinsp;1mg/ml hemoglobin; Saliva A\u0026thinsp;+\u0026thinsp;0.1mg/ml cystatin; Saliva A\u0026thinsp;+\u0026thinsp;hemoglobin\u0026thinsp;+\u0026thinsp;cystatin; Bioextra (positive control) and water (negative control) (n\u0026thinsp;=\u0026thinsp;12/group). Biofilm was produced using human biofilm and McBain saliva (0.2% of sucrose, 37\u003csup\u003eo\u003c/sup\u003e C and 5% CO\u003csub\u003e2\u003c/sub\u003e); the treatments were done 1x/day, for 5 days. Colony-forming units (CFU) counting was performed; demineralization was quantified by transversal microradiography. Two-way ANOVA/Bonferroni or Sidak test for the comparison between biofilm x dentin and ANOVA/Tukey or Kruskal-Wallis/Dunn for comparing artificial saliva were done (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe type of biofilm had no influence on CFU and demineralization. Sound dentin under control biofilm presented the lowest \u003cem\u003eLactobacillus\u003c/em\u003e ssp. and \u003cem\u003eStreptococcus mutans\u003c/em\u003e CFU and the lowest mean mineral loss (R) (25.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2; 23.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9%) compared to irradiated dentin (26.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8; 28.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.004) for both types of biofilms (irradiated and no irradiated, respectively). Bioextra was the only one that reduced R (10.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5%) and LD (35\u0026thinsp;\u0026plusmn;\u0026thinsp;15\u0026micro;m) compared to water (17.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3%, 81\u0026thinsp;\u0026plusmn;\u0026thinsp;18\u0026micro;m, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIrradiation of dentin has impact on caries development; none of the experimental saliva was able to reduce its occurrence.\u003c/p\u003e","manuscriptTitle":"Influence of irradiated dentin, biofilm and different artificial saliva formulations on root dentin caries development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-02 19:06:20","doi":"10.21203/rs.3.rs-3787488/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e35bf643-42cf-4de0-9097-9c776c668a5c","owner":[],"postedDate":"January 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-31T16:08:48+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-02 19:06:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3787488","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3787488","identity":"rs-3787488","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}