Quantifying the amount of carbonate removed in bovine enamel after irradiating with 9.3 micron CO2 laser

Quantifying the amount of carbonate removed in bovine enamel after irradiating with 9.3 micron CO2 laser | 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 Quantifying the amount of carbonate removed in bovine enamel after irradiating with 9.3 micron CO 2 laser Vijayashankar Ramareddy, David Siegel, Charles Kerbage This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5632668/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 High power pulsed 9.3 µm CO 2 laser irradiation at sub ablative fluences removes carbonate groups in Carbonated Hydroxyapatite (CHA) resulting in formation of pure Hydroxyapatite (HA) which is less soluble in acidic environments. The amount of carbonate removed was quantified using Fourier Transform Infrared Spectroscopy and a theory based on Arrhenius damage integral was applied to obtain activation energy for the laser mediated chemical reaction. We show Activation energy is 70 ± 10 kJ/mol and fluence corresponding to 50% carbonate removed is 0.84 J/cm 2 . 9.3 µm CO2 laser Demineralization inhibition Arrhenius damage threshold Activation energy Caries Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Enamel is the hardest tissue in human body, and it covers the outmost part of the teeth. The primary constituent of enamel is Hydroxyapatite (HA), a mineral consisting of calcium and phosphate. Human enamel consists of 84% mineral by volume 1 , and with age, some of the phosphate groups in HA are replaced by carbonate groups resulting in formation of Carbonated Hydroxyapatite (CHA) 2 . CHA is more soluble in acids resulting in demineralization and caries. Caries is one of the most common dental problems affecting over 90% of adults in the age 20–64 years 3 . Current preventative methods focus on Fluoride, where the fluoride in dentifrices and mouthwashes slowly form Fluorapatite (FA), which is least soluble in acids. However, Fluoride in neurotoxic 4 , and alternative treatments are preferable. Lasers make an excellent choice since lasers produce no side effects. Lasers have been shown to produce pure HA from carbonated HA via a chemical rection that removes carbonate group when temperatures of the enamel raised above 300 C. The ultrashort pulses of lasers can raise temperatures on teeth surface to the required temperatures to remove carbonate group. However, if the laser is not highly absorbed in hydroxyapatite, a large amount of energy is required to achieve the required chemical changes to form pure HA, which results in adverse effects such as rising pulpal temperatures above safe thresholds. A suitable laser will have high absorption in enamel, so only a small amount of energy is required to affect the surface layer of the enamel without laser penetrating deep inside or scattering light away from the enamel. Phosphate groups in Hydroxyapatite have high absorption near 9.3 µm CO 2 lasers making this a special wavelength of interest to dental community 5 . When used at sub ablative powers, the 9.3 µm CO 2 laser irradiation can produce added effects of the current treatment protocols of hypersensitivity 6 , and bonding 7 . In particular, numerous studies have demonstrated the ability of the 9.3 µm CO 2 laser on preventing demineralization both in adult and primary teeth safely without rising pulpal temperature 8 – 14 and clinical study demonstrated improved carries resistance of enamel when irradiated with 9.3 µm CO 2 laser 15 with sub ablative powers. The effects of lasers and dentifrices on dental hard tissue in extracted teeth can be quantified using TMR 16 and microhardness 17 , 18 methods, which involves creating lesions post treatment. Fourier Transform Infrared Spectroscopy (FTIR) can be used to assess chemical changes produced by irradiation 19 – 24 . FTIR spectrum shows distinct changes in the spectra before and after treatment with lasers, which can be used to quantify the laser mediated carbonate removal chemical reaction in enamel. FTIR has been successfully used to measure the effect of carbonate removal in adult and primary enamel 11 , 25 . FTIR is simple and can be used immediately after irradiation. The objective of this research is to quantify the amount of carbonate removed in CHA using FTIR, and to estimate Activation Energy using a theory based on Arrhenius damage integral. MATERIALS AND METHODS A polished bovine enamel block was divided into six regions, and each region was irradiated with different Fluences. Baseline FTIR spectra were recorded in each region before irradiation. Six measurements were made in each region at different locations within the region. The FTIR spectra showed distinct peaks corresponding to carbonate group between 1600–1300 cm − 1 and Phosphate group between 1200–700 cm − 1 . The area under each peak was calculated using trapezoidal rule, and the percentage of carbonate removed was calculated using the formula 25 $$\:\%CR=100\left(1-\frac{\left({A}_{CO3}/{A}_{PO4}\right)\:after}{\left({A}_{CO3}/{A}_{PO4}\right)\:before}\right)$$ 1 Where, A CO3 and A PO4 represent areas under the carbonate and Phosphate groups as illustrated in Fig. 1 . The baseline for the peaks were obtained by fitting a straight line to two points corresponding to the average of 5 cm - 1 at the beginning and end of the peak. Laser settings Laser irradiation was performed using a 9.3 µm CO 2 laser (Solea, Convergent Dental, Inc., Waltham, MA). Different pulse fluences were generated by adjusting the pulse length. Irradiation was performed in non-contact mode with a handpiece that delivered a 1 mm collimated laser beam. The laser beam was patterned using a set of scanning mirrors to generate a spotsize corresponding to 6.2 mm 2 area in 0.38s. A 0.5s cooling time was allowed with air flow at 8 psi after each pattern. No water cooling was used. Sample was irradiated with very little overlap between the laser patterns. RESULTS AND DISCUSSION Bovine block was imaged using light microscope at 700X optical magnification in each region to visualize the morphological changes in enamel surface after irradiating with different pulse fluences as shown in Fig. 1 . For low fluences below 0.76 J/cm 2 /pulse, the effect of laser irradiation on the polished bovine enamel is not visible, while at medium fluence corresponding to 0.9 J/cm 2 /pulse, spots of melting is visible as shown in Fig. 1 (c). For fluences above 1.04 J/cm 2 /pulse, melting is progressively more pronounced as shown in Fig. 1 (d)-(f). Despite the microscopic melting, the irradiation does not produce adverse effects in human enamel for the fluences used in this study 9 , 11 . Figure 2 shows FTIR spectra corresponding to the regions shown in Fig. 1 . Area under carbonate peak progressively decreases and in comparison, the Phosphate peak progressively widens. For the fluences corresponding to nearly fully melted and recast enamel, a significant band broadening was observed indicating formation of Amorphous Calcium Phosphate 26 – 28 , which remineralizes when in contact with saliva 29 . In addition, for high fluences used in this study, FTIR spectrum shows a near complete removal of carbonate groups while forming smooth surfaces as illustrated in Figs. 1 f and 2 f. Smooth teeth surfaces are preferable for low plaque accumulation and reduce the occurrence of caries 30 . The effect of laser irradiation on tissue can be described using Arrhenius damage integral 31 , 32 , which is given in terms of activation energy (E a. ), at a Temperature T produced by laser irradiation, by 31 $$\:\varOmega\:=A\:{\int\:}_{0}^{\tau\:}{e}^{-\frac{{E}_{a}}{RT}}dt$$ 2 where, t is the exposure time related to pulse width, R is gas constant, A is the frequency factor, and \(\:\varOmega\:\) represents the tissue damage for soft tissue. In this study, we use this integral to quantify the amount of carbonate removed in enamel. For the pulse fluences used in this study, the temperature rise is expected in the range 400-1000C, and for these temperatures, 1/T was expanded using Taylor series as a function of scaled fluence, and the series was truncated after second order term. This results in error functions for the amount of carbonate removed as $$\:\varOmega\:/{\varOmega\:}_{0}=\:\left\{erf\left(b\left[\left(F-{F}_{0}\right)\right]\right)+erf\left(b{F}_{0}\right)\right\}$$ 3 where, F 0 is the Fluence corresponding to 50% carbonate removed and b is a constant, proportional to the activation energy. Figure 3 shows the percentage of carbonate removed as calculated using Eq. 1 at fluences used in Figs. 1 and 2 . Error bars correspond to standard deviation from six FTIR measurements in each region. The dashed line in Fig. 3 is a fit to Eq. 3 . Using the fits, the fluence corresponding to 50% carbonate removal, and activation energy were calculated to be 0.84 J/cm 2 and 70 ± 10 kJ/mol respectively. The large activation energy indicates the chemical reaction of carbonate removal is nearly impossible without additional energy. To supply a large energy safely, a laser should have high absorption in CHA and have ultrashort pulse so the high temperatures can only be realized in ultrashort intervals. We note that this is the first attempt to the best of our knowledge, to determine the activation energy of carbonate removal chemical reaction. The activation energies of other processes of enamel over temperature range used in this study have been reported to be in the same range. Bachmann et al 33 measured the removal of tightly bound water in enamel by heating to 700-1000C requires an activation energy of 60 kJ/mol. Jokanovic, V et al. 34 measured the activation energy of 85–100 kJ/mol for the process of sintering of HA in the range of 900 to 1100C. While the standard method of calculating activation energy involves reverting Eq. 2 , we use a slightly different approach, where we fit the equation to measurements and obtain activation energy. In addition, we obtain the fluence corresponding to 50% carbonate removed, which is 0.84 J/cm 2 . This value is small compared to other wavelengths due to the high absorption of HA at this wavelength. We used a polished bovine enamel for the study to simulate an ideal sample. The results are expected to hold for human samples. While FTIR method employed in this study can provide the chemical groups present on the surface layer of the enamel within the depths of 1–4 µm, Badreddine A et al. 11 use FTIR spectra of primary enamel and show 9.3 µm CO 2 laser irradiation can remove carbonate groups down to the depths of 15 µm with progressively less carbonate removed. The study presented in this article is expected to hold for the depth up to 15 µm. While penetration depth for 9.3 µm CO 2 laser is 2 µm, the deeper effect may be attributed to the parameters used in the study, that include air to cool, and pulse widths much longer than the thermal relaxation time. In conclusion, we have obtained the FTIR spectra of irradiated enamel to quantify carbonate removed in 9.3 µm CO 2 laser mediated chemical reaction at different pulse fluences. We calculated activation energy to be 70 kJ/mol and the 50% carbonate removal fluence of 0.84 J/cm 2 . Declarations Author Contribution VR Contributed to design, data acquisition, interpretation developing the theory and drafted the manuscript. DS Contributed to data acquisition and critically revised the manuscript. CK contributed to conception and critically revised the manuscript. All authors discussed the results and contributed to the final manuscript. Data Availability Data can be provided with a reasonable request. FUNDIND DECLARATION There was no funding for the research ETHICAL STATEMENT The human teeth samples utilized were extracted by licensed Oral Surgeons as part of the patient’s oral care treatment plan. Each patient signed an informed consent indicating their extracted teeth may be utilized for research & development purposes. INFORMED CONSENT Each patient signed an informed consent indicating their extracted teeth may be utilized for research & development purposes. No PHI or identifying information was disclosed. CONSENT FOR PUBLICATION Not applicable References Curzon ME, Featherstone JD (1983) Chemical composition of enamel. In: Lazzari EP (ed) CRC Handbook of Experimental Aspects of Oral Biochemistry. CRC, Boca Raton, FL, pp 123–135 Kuczumow A et al (2022) Measurements of Energetic States Resulting from Ion Exchanges in the Isomorphic Crystals of Apatites and Bioapatites. Molecules 27:8913 NICDC (2022) Dental Caries (Tooth Decay) in Adults (Ages 20 to 64 Years). https://www.nidcr.nih.gov/research/data-statistics/dental-caries/adults#dental-caries-in-the-permanent-adult-teeth Adkins EA, Brunst KJ (2021) Impacts of Fluoride Neurotoxicity and Mitochondrial Dysfunction on Cognition and Mental Health: A Literature Review. Int J Environ Res Public Health 18 Xue VW et al (2021) Effects of 9,300 nm Carbon Dioxide Laser on Dental Hard Tissue: A Concise Review. Clin Cosmet Investig Dent 13:155–161 Ramareddy V, Kerbage C (2024) An SEM study on the effect of 9.3-µm CO2 laser on dentinal tubules for hypersensitivity treatment. Lasers Med Sci 39:200 Rechmann P, Sherathiya K, Kinsel R, Vaderhobli R, Rechmann BM (2017) T. Influence of irradiation by a novel CO2 9.3-µm short-pulsed laser on sealant bond strength. Lasers Med Sci 32:609–620 Rechmann P et al (2016) Caries inhibition with a CO2 9.3 µm laser: An in vitro study. Lasers Surg Med 48:546–554 Badreddine AH et al (2021) Demineralization Inhibition by High-Speed Scanning of 9.3 µm CO 2 Single Laser Pulses Over Enamel. Lasers Surg Med 53:703–712 Zhao IS et al (2021) Use of a novel 9.3-µm carbon dioxide laser and silver diamine fluoride: Prevention of enamel demineralisation and inhibition of cariogenic bacteria. Dent Mater 37:940–948 Badreddine A, Ramareddy V, Kerbage C (2023) Effectiveness of carbonate removal and demineralization inhibition in primary teeth using a 9.3-µm carbon dioxide laser. JADA Foundational Sci 2:100017 Featherstone JDB, Barrett-Vespone NA, Fried D, Kantorowitz Z, Seka W (1998) CO2 Laser Inhibition of Artificial Caries-like Lesion Progression in Dental Enamel. J Dent Res 77:1397–1403 Rechmann P et al (2011) Caries inhibition in vital teeth using 9.6-µm CO2-laser irradiation. J Biomed Opt 16:071405 Rechmann P, Charland DA, Rechmann BMT, Le CQ, Featherstone JD (2013) B. In-vivo occlusal caries prevention by pulsed CO 2 ‐laser and fluoride varnish treatment—A clinical pilot study. Lasers Surg Med 45:302–310 Rechmann P, Kubitz M, Chaffee BW, Rechmann BM (2021) T. Fissure caries inhibition with a CO2 9.3-µm short-pulsed laser—a randomized, single-blind, split-mouth controlled, 1-year clinical trial. Clin Oral Investig 25:2055–2068 Jong E, de Bosch J, J. J., Noordmans J (1987) Optimised microcomputer-guided quantitative microradiography on dental mineralised tissue slices. Phys Med Biol 32:887–899 Featherstone JDB, ten Cate JM, Shariati M, Arends J (1983) Comparison of Artificial Caries-Like Lesions by Quantitative Microradiography and Microhardness Profiles. Caries Res 17:385–391 Kielbassa AM, Wrbas K-T, Schulte-Mönting J, Hellwig E (1999) Correlation of transversal microradiography and microhardness on in situ-induced demineralization in irradiated and nonirradiated human dental enamel. Arch Oral Biol 44:243–251 Corrêa-Afonso AM, Bachmann L, de Almeida CG, Corona SAM, Borsatto MC (2012) FTIR and SEM analysis of CO2 laser irradiated human enamel. Arch Oral Biol 57:1153–1158 Sharma S, Hegde MN, Ramesh S (2022) Fourier Transformation Infrared Spectroscopic Analysis of Enamel Following Different Surface Treatments: An Invitro Study. Cryst (Basel) 12:1619 da Silva Tagliaferro EP, Rodrigues LKA, Soares LES, Martin AA, Nobre-dos-Santos M (2009) Physical and Compositional Changes on Demineralized Primary Enamel Induced by CO 2 Laser. Photomed Laser Surg 27:585–590 Orilisi G et al (2020) Effect of a Sodium Fluoride-Releasing Rubber Cup on Hydroxyapatite Crystallinity of Human Enamel: FTIR Spectroscopy Analysis. Dent Res Oral Health 03 Yang WH, Xi XF, Li JF, Cai KY (2013) Comparison of Crystal Structure Between Carbonated Hydroxyapatite and Natural Bone Apatite with Theoretical Calculation. Asian J Chem 25:3673–3678 Lafon JP, Champion E, Bernache-Assollant D (2008) Processing of AB-type carbonated hydroxyapatite Ca10 – x(PO4)6 – x(CO3)x(OH)2 – x–2y(CO3)y ceramics with controlled composition. J Eur Ceram Soc 28:139–147 Zuerlein MJ, Fried D, Featherstone JD (1999) Modeling the modification depth of carbon dioxide laser-treated dental enamel. Lasers Surg Med 25:335–347 Querido W et al (2020) Fourier transform infrared spectroscopy of developing bone mineral: from amorphous precursor to mature crystal. Analyst 145:764–776 Uskoković V (2020) Visualizing different crystalline states during the infrared imaging of calcium phosphates. Vib Spectrosc 108:103045 Gadaleta SJ, Paschalis EP, Betts F, Mendelsohn R, Boskey AL (1996) Fourier transform infrared spectroscopy of the solution-mediated conversion of amorphous calcium phosphate to hydroxyapatite: New correlations between X-ray diffraction and infrared data. Calcif Tissue Int 58:9–16 Ferreira Zandoná AG, Ritter AV, Eidson RS Dental Caries. in Sturdevant’s Art and Science of Operative Dentistry 40–94 (Elsevier, 2019). 10.1016/B978-0-323-47833-5.00002-2 Quirynen M, Bollen CM (1995) L. The influence of surface roughness and surface-free energy on supra‐ and subgingival plaque formation in man. J Clin Periodontol 22:1–14 Lukač M, Lozar A, Perhavec T, Bajd F (2019) Variable heat shock response model for medical laser procedures. Lasers Med Sci 34:1147–1158 Lukač M et al (2024) Influence of tissue desiccation on critical temperature for thermal damage during Er:YAG laser skin treatments. Lasers Surg Med 56:107–118 Bachmann L, Gomes ASL, Zezell DM (2004) Bound Energy of Water in Hard Dental Tissues. Spectrosc Lett 37:565–579 Jokanović V et al (2008) Kinetics and sintering mechanisms of hydro-thermally obtained hydroxyapatite. Mater Chem Phys 111:180–185 Additional Declarations No competing interests reported. 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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-5632668","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":399575981,"identity":"99086279-23be-443c-8aa6-695fb59a7c1a","order_by":0,"name":"Vijayashankar Ramareddy","email":"data:image/png;base64,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","orcid":"","institution":"Convergent Dental","correspondingAuthor":true,"prefix":"","firstName":"Vijayashankar","middleName":"","lastName":"Ramareddy","suffix":""},{"id":399575982,"identity":"7eff2fd0-682f-4951-92a3-5dc9ad2e6edd","order_by":1,"name":"David Siegel","email":"","orcid":"","institution":"Convergent Dental","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Siegel","suffix":""},{"id":399575983,"identity":"a5085ec3-d90e-4b4d-8571-c80e6339715b","order_by":2,"name":"Charles Kerbage","email":"","orcid":"","institution":"Convergent Dental","correspondingAuthor":false,"prefix":"","firstName":"Charles","middleName":"","lastName":"Kerbage","suffix":""}],"badges":[],"createdAt":"2024-12-12 15:08:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5632668/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5632668/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73339334,"identity":"2ef96c92-60d3-485b-9aea-c92156ea4b51","added_by":"auto","created_at":"2025-01-09 05:07:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2525555,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic view of the bovine enamel irradiated with 9.3 mm CO\u003csub\u003e2\u003c/sub\u003e laser. (a)-(f) are 700X images of each region of Bovine sample irradiated with different fluences increasing from (a)-(f)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5632668/v1/4f05f741612430a99b9d01d8.png"},{"id":73339074,"identity":"ea8f47ec-7a94-42b9-af8f-38459274dd67","added_by":"auto","created_at":"2025-01-09 04:59:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":75032,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of bovine enamel irradiated with 9.3 mm CO\u003csub\u003e2\u003c/sub\u003e laser at different pulse fluences increasing from (a)-(f). Carbonate (1600 – 1300 cm\u003csup\u003e-1\u003c/sup\u003e) and phosphate (1200 – 700 cm\u003csup\u003e-1\u003c/sup\u003e) bands are highlighted in Green and red respectively. The bases of these bands were identified by a straight line between two points corresponding to the averages of 5 cm\u003csup\u003e-1\u003c/sup\u003e each at the beginning and end of the bands. Areas enclosed within the green and red curves were used to quantify the percentage of carbonate removed.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5632668/v1/feb910a42f153608c3fb96b1.png"},{"id":73341360,"identity":"5c34045a-5b2b-4d83-9e40-248bd2ac9041","added_by":"auto","created_at":"2025-01-09 05:32:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":51239,"visible":true,"origin":"","legend":"\u003cp\u003eResults of carbonate removal and fit to Arrhenius damage integral. F\u003csub\u003e0\u003c/sub\u003e is fluence corresponding to 50% carbonate removed.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5632668/v1/80a1d4fcbfd34e6cb7a670c1.png"},{"id":73341361,"identity":"f00d5953-d1fc-4872-811f-651346dbf3a9","added_by":"auto","created_at":"2025-01-09 05:32:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3117767,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5632668/v1/b21de6dd-d615-4ad4-b4a6-19a7744069b9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eQuantifying the amount of carbonate removed in bovine enamel after irradiating with 9.3 micron CO\u003csub\u003e2\u003c/sub\u003e laser\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eEnamel is the hardest tissue in human body, and it covers the outmost part of the teeth. The primary constituent of enamel is Hydroxyapatite (HA), a mineral consisting of calcium and phosphate. Human enamel consists of 84% mineral by volume\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, and with age, some of the phosphate groups in HA are replaced by carbonate groups resulting in formation of Carbonated Hydroxyapatite (CHA)\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. CHA is more soluble in acids resulting in demineralization and caries. Caries is one of the most common dental problems affecting over 90% of adults in the age 20\u0026ndash;64 years\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCurrent preventative methods focus on Fluoride, where the fluoride in dentifrices and mouthwashes slowly form Fluorapatite (FA), which is least soluble in acids. However, Fluoride in neurotoxic\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, and alternative treatments are preferable. Lasers make an excellent choice since lasers produce no side effects.\u003c/p\u003e \u003cp\u003eLasers have been shown to produce pure HA from carbonated HA via a chemical rection that removes carbonate group when temperatures of the enamel raised above 300 C. The ultrashort pulses of lasers can raise temperatures on teeth surface to the required temperatures to remove carbonate group. However, if the laser is not highly absorbed in hydroxyapatite, a large amount of energy is required to achieve the required chemical changes to form pure HA, which results in adverse effects such as rising pulpal temperatures above safe thresholds. A suitable laser will have high absorption in enamel, so only a small amount of energy is required to affect the surface layer of the enamel without laser penetrating deep inside or scattering light away from the enamel.\u003c/p\u003e \u003cp\u003ePhosphate groups in Hydroxyapatite have high absorption near 9.3 \u0026micro;m CO\u003csub\u003e2\u003c/sub\u003e lasers making this a special wavelength of interest to dental community\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. When used at sub ablative powers, the 9.3 \u0026micro;m CO\u003csub\u003e2\u003c/sub\u003e laser irradiation can produce added effects of the current treatment protocols of hypersensitivity\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, and bonding\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In particular, numerous studies have demonstrated the ability of the 9.3 \u0026micro;m CO\u003csub\u003e2\u003c/sub\u003e laser on preventing demineralization both in adult and primary teeth safely without rising pulpal temperature\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12 CR13\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e and clinical study demonstrated improved carries resistance of enamel when irradiated with 9.3 \u0026micro;m CO\u003csub\u003e2\u003c/sub\u003e laser\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e with sub ablative powers.\u003c/p\u003e \u003cp\u003eThe effects of lasers and dentifrices on dental hard tissue in extracted teeth can be quantified using TMR\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and microhardness\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e methods, which involves creating lesions post treatment. Fourier Transform Infrared Spectroscopy (FTIR) can be used to assess chemical changes produced by irradiation\u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. FTIR spectrum shows distinct changes in the spectra before and after treatment with lasers, which can be used to quantify the laser mediated carbonate removal chemical reaction in enamel. FTIR has been successfully used to measure the effect of carbonate removal in adult and primary enamel\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. FTIR is simple and can be used immediately after irradiation.\u003c/p\u003e \u003cp\u003eThe objective of this research is to quantify the amount of carbonate removed in CHA using FTIR, and to estimate Activation Energy using a theory based on Arrhenius damage integral.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eA polished bovine enamel block was divided into six regions, and each region was irradiated with different Fluences. Baseline FTIR spectra were recorded in each region before irradiation. Six measurements were made in each region at different locations within the region. The FTIR spectra showed distinct peaks corresponding to carbonate group between 1600\u0026ndash;1300 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and Phosphate group between 1200\u0026ndash;700 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The area under each peak was calculated using trapezoidal rule, and the percentage of carbonate removed was calculated using the formula\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\%CR=100\\left(1-\\frac{\\left({A}_{CO3}/{A}_{PO4}\\right)\\:after}{\\left({A}_{CO3}/{A}_{PO4}\\right)\\:before}\\right)$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere, A\u003csub\u003eCO3\u003c/sub\u003e and A\u003csub\u003ePO4\u003c/sub\u003e represent areas under the carbonate and Phosphate groups as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The baseline for the peaks were obtained by fitting a straight line to two points corresponding to the average of 5 cm\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e at the beginning and end of the peak.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eLaser settings\u003c/h2\u003e \u003cp\u003eLaser irradiation was performed using a 9.3 \u0026micro;m CO\u003csub\u003e2\u003c/sub\u003e laser (Solea, Convergent Dental, Inc., Waltham, MA). Different pulse fluences were generated by adjusting the pulse length. Irradiation was performed in non-contact mode with a handpiece that delivered a 1 mm collimated laser beam. The laser beam was patterned using a set of scanning mirrors to generate a spotsize corresponding to 6.2 mm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e area in 0.38s. A 0.5s cooling time was allowed with air flow at 8 psi after each pattern. No water cooling was used. Sample was irradiated with very little overlap between the laser patterns.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003eBovine block was imaged using light microscope at 700X optical magnification in each region to visualize the morphological changes in enamel surface after irradiating with different pulse fluences as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. For low fluences below 0.76 J/cm\u003csup\u003e2\u003c/sup\u003e/pulse, the effect of laser irradiation on the polished bovine enamel is not visible, while at medium fluence corresponding to 0.9 J/cm\u003csup\u003e2\u003c/sup\u003e/pulse, spots of melting is visible as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(c). For fluences above 1.04 J/cm\u003csup\u003e2\u003c/sup\u003e/pulse, melting is progressively more pronounced as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(d)-(f). Despite the microscopic melting, the irradiation does not produce adverse effects in human enamel for the fluences used in this study\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows FTIR spectra corresponding to the regions shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Area under carbonate peak progressively decreases and in comparison, the Phosphate peak progressively widens. For the fluences corresponding to nearly fully melted and recast enamel, a significant band broadening was observed indicating formation of Amorphous Calcium Phosphate\u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, which remineralizes when in contact with saliva\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In addition, for high fluences used in this study, FTIR spectrum shows a near complete removal of carbonate groups while forming smooth surfaces as illustrated in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef. Smooth teeth surfaces are preferable for low plaque accumulation and reduce the occurrence of caries\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe effect of laser irradiation on tissue can be described using Arrhenius damage integral\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, which is given in terms of activation energy \u003cem\u003e(E\u003c/em\u003e\u003csub\u003e\u003cem\u003ea.\u003c/em\u003e\u003c/sub\u003e), at a Temperature \u003cem\u003eT\u003c/em\u003e produced by laser irradiation, by\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:\\varOmega\\:=A\\:{\\int\\:}_{0}^{\\tau\\:}{e}^{-\\frac{{E}_{a}}{RT}}dt$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere, t is the exposure time related to pulse width, \u003cem\u003eR\u003c/em\u003e is gas constant, A is the frequency factor, and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\varOmega\\:\\)\u003c/span\u003e\u003c/span\u003e represents the tissue damage for soft tissue. In this study, we use this integral to quantify the amount of carbonate removed in enamel. For the pulse fluences used in this study, the temperature rise is expected in the range 400-1000C, and for these temperatures, \u003cem\u003e1/T\u003c/em\u003e was expanded using Taylor series as a function of scaled fluence, and the series was truncated after second order term. This results in error functions for the amount of carbonate removed as\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:\\varOmega\\:/{\\varOmega\\:}_{0}=\\:\\left\\{erf\\left(b\\left[\\left(F-{F}_{0}\\right)\\right]\\right)+erf\\left(b{F}_{0}\\right)\\right\\}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e is the Fluence corresponding to 50% carbonate removed and b is a constant, proportional to the activation energy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the percentage of carbonate removed as calculated using Eq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e at fluences used in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Error bars correspond to standard deviation from six FTIR measurements in each region. The dashed line in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e is a fit to Eq.\u0026nbsp;\u003cspan refid=\"Equ3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Using the fits, the fluence corresponding to 50% carbonate removal, and activation energy were calculated to be 0.84 J/cm\u003csup\u003e2\u003c/sup\u003e and 70\u0026thinsp;\u0026plusmn;\u0026thinsp;10 kJ/mol respectively.\u003c/p\u003e \u003cp\u003eThe large activation energy indicates the chemical reaction of carbonate removal is nearly impossible without additional energy. To supply a large energy safely, a laser should have high absorption in CHA and have ultrashort pulse so the high temperatures can only be realized in ultrashort intervals. We note that this is the first attempt to the best of our knowledge, to determine the activation energy of carbonate removal chemical reaction. The activation energies of other processes of enamel over temperature range used in this study have been reported to be in the same range. Bachmann \u003cem\u003eet al\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e measured the removal of tightly bound water in enamel by heating to 700-1000C requires an activation energy of 60 kJ/mol. Jokanovic, V et al.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e measured the activation energy of 85\u0026ndash;100 kJ/mol for the process of sintering of HA in the range of 900 to 1100C.\u003c/p\u003e \u003cp\u003eWhile the standard method of calculating activation energy involves reverting Eq.\u0026nbsp;\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, we use a slightly different approach, where we fit the equation to measurements and obtain activation energy. In addition, we obtain the fluence corresponding to 50% carbonate removed, which is 0.84 J/cm\u003csup\u003e2\u003c/sup\u003e. This value is small compared to other wavelengths due to the high absorption of HA at this wavelength. We used a polished bovine enamel for the study to simulate an ideal sample. The results are expected to hold for human samples.\u003c/p\u003e \u003cp\u003eWhile FTIR method employed in this study can provide the chemical groups present on the surface layer of the enamel within the depths of 1\u0026ndash;4 \u0026micro;m, Badreddine A et al.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e use FTIR spectra of primary enamel and show 9.3 \u0026micro;m CO\u003csub\u003e2\u003c/sub\u003e laser irradiation can remove carbonate groups down to the depths of 15 \u0026micro;m with progressively less carbonate removed. The study presented in this article is expected to hold for the depth up to 15 \u0026micro;m. While penetration depth for 9.3 \u0026micro;m CO\u003csub\u003e2\u003c/sub\u003e laser is 2 \u0026micro;m, the deeper effect may be attributed to the parameters used in the study, that include air to cool, and pulse widths much longer than the thermal relaxation time.\u003c/p\u003e \u003cp\u003eIn conclusion, we have obtained the FTIR spectra of irradiated enamel to quantify carbonate removed in 9.3 \u0026micro;m CO\u003csub\u003e2\u003c/sub\u003e laser mediated chemical reaction at different pulse fluences. We calculated activation energy to be 70 kJ/mol and the 50% carbonate removal fluence of 0.84 J/cm\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eVR Contributed to design, data acquisition, interpretation developing the theory and drafted the manuscript. DS Contributed to data acquisition and critically revised the manuscript. CK contributed to conception and critically revised the manuscript. All authors discussed the results and contributed to the final manuscript.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eData can be provided with a reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDIND DECLARATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was no funding for the research\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICAL STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human teeth samples utilized were extracted by licensed Oral Surgeons as part of the patient\u0026rsquo;s oral care treatment plan. Each patient signed an informed consent indicating their extracted teeth may be utilized for research \u0026amp; development purposes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eINFORMED CONSENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach patient signed an informed consent indicating their extracted teeth may be utilized for research \u0026amp; development purposes. No PHI or identifying information was disclosed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT FOR PUBLICATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCurzon ME, Featherstone JD (1983) Chemical composition of enamel. In: Lazzari EP (ed) CRC Handbook of Experimental Aspects of Oral Biochemistry. CRC, Boca Raton, FL, pp 123\u0026ndash;135\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuczumow A et al (2022) Measurements of Energetic States Resulting from Ion Exchanges in the Isomorphic Crystals of Apatites and Bioapatites. Molecules 27:8913\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNICDC (2022) Dental Caries (Tooth Decay) in Adults (Ages 20 to 64 Years). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.nidcr.nih.gov/research/data-statistics/dental-caries/adults#dental-caries-in-the-permanent-adult-teeth\u003c/span\u003e\u003cspan address=\"https://www.nidcr.nih.gov/research/data-statistics/dental-caries/adults#dental-caries-in-the-permanent-adult-teeth\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdkins EA, Brunst KJ (2021) Impacts of Fluoride Neurotoxicity and Mitochondrial Dysfunction on Cognition and Mental Health: A Literature Review. Int J Environ Res Public Health 18\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXue VW et al (2021) Effects of 9,300 nm Carbon Dioxide Laser on Dental Hard Tissue: A Concise Review. Clin Cosmet Investig Dent 13:155\u0026ndash;161\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamareddy V, Kerbage C (2024) An SEM study on the effect of 9.3-\u0026micro;m CO2 laser on dentinal tubules for hypersensitivity treatment. Lasers Med Sci 39:200\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRechmann P, Sherathiya K, Kinsel R, Vaderhobli R, Rechmann BM (2017) T. Influence of irradiation by a novel CO2 9.3-\u0026micro;m short-pulsed laser on sealant bond strength. Lasers Med Sci 32:609\u0026ndash;620\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRechmann P et al (2016) Caries inhibition with a CO2 9.3 \u0026micro;m laser: An in vitro study. Lasers Surg Med 48:546\u0026ndash;554\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBadreddine AH et al (2021) Demineralization Inhibition by High-Speed Scanning of 9.3 \u0026micro;m CO 2 Single Laser Pulses Over Enamel. Lasers Surg Med 53:703\u0026ndash;712\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao IS et al (2021) Use of a novel 9.3-\u0026micro;m carbon dioxide laser and silver diamine fluoride: Prevention of enamel demineralisation and inhibition of cariogenic bacteria. Dent Mater 37:940\u0026ndash;948\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBadreddine A, Ramareddy V, Kerbage C (2023) Effectiveness of carbonate removal and demineralization inhibition in primary teeth using a 9.3-\u0026micro;m carbon dioxide laser. JADA Foundational Sci 2:100017\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeatherstone JDB, Barrett-Vespone NA, Fried D, Kantorowitz Z, Seka W (1998) CO2 Laser Inhibition of Artificial Caries-like Lesion Progression in Dental Enamel. J Dent Res 77:1397\u0026ndash;1403\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRechmann P et al (2011) Caries inhibition in vital teeth using 9.6-\u0026micro;m CO2-laser irradiation. J Biomed Opt 16:071405\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRechmann P, Charland DA, Rechmann BMT, Le CQ, Featherstone JD (2013) B. In-vivo occlusal caries prevention by pulsed CO \u003csub\u003e2\u003c/sub\u003e ‐laser and fluoride varnish treatment\u0026mdash;A clinical pilot study. Lasers Surg Med 45:302\u0026ndash;310\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRechmann P, Kubitz M, Chaffee BW, Rechmann BM (2021) T. Fissure caries inhibition with a CO2 9.3-\u0026micro;m short-pulsed laser\u0026mdash;a randomized, single-blind, split-mouth controlled, 1-year clinical trial. Clin Oral Investig 25:2055\u0026ndash;2068\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJong E, de Bosch J, J. J., Noordmans J (1987) Optimised microcomputer-guided quantitative microradiography on dental mineralised tissue slices. Phys Med Biol 32:887\u0026ndash;899\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeatherstone JDB, ten Cate JM, Shariati M, Arends J (1983) Comparison of Artificial Caries-Like Lesions by Quantitative Microradiography and Microhardness Profiles. Caries Res 17:385\u0026ndash;391\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKielbassa AM, Wrbas K-T, Schulte-M\u0026ouml;nting J, Hellwig E (1999) Correlation of transversal microradiography and microhardness on in situ-induced demineralization in irradiated and nonirradiated human dental enamel. Arch Oral Biol 44:243\u0026ndash;251\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorr\u0026ecirc;a-Afonso AM, Bachmann L, de Almeida CG, Corona SAM, Borsatto MC (2012) FTIR and SEM analysis of CO2 laser irradiated human enamel. Arch Oral Biol 57:1153\u0026ndash;1158\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma S, Hegde MN, Ramesh S (2022) Fourier Transformation Infrared Spectroscopic Analysis of Enamel Following Different Surface Treatments: An Invitro Study. Cryst (Basel) 12:1619\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eda Silva Tagliaferro EP, Rodrigues LKA, Soares LES, Martin AA, Nobre-dos-Santos M (2009) Physical and Compositional Changes on Demineralized Primary Enamel Induced by CO \u003csub\u003e2\u003c/sub\u003e Laser. Photomed Laser Surg 27:585\u0026ndash;590\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrilisi G et al (2020) Effect of a Sodium Fluoride-Releasing Rubber Cup on Hydroxyapatite Crystallinity of Human Enamel: FTIR Spectroscopy Analysis. Dent Res Oral Health 03\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang WH, Xi XF, Li JF, Cai KY (2013) Comparison of Crystal Structure Between Carbonated Hydroxyapatite and Natural Bone Apatite with Theoretical Calculation. Asian J Chem 25:3673\u0026ndash;3678\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLafon JP, Champion E, Bernache-Assollant D (2008) Processing of AB-type carbonated hydroxyapatite Ca10\u0026thinsp;\u0026ndash;\u0026thinsp;x(PO4)6\u0026thinsp;\u0026ndash;\u0026thinsp;x(CO3)x(OH)2\u0026thinsp;\u0026ndash;\u0026thinsp;x\u0026ndash;2y(CO3)y ceramics with controlled composition. J Eur Ceram Soc 28:139\u0026ndash;147\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZuerlein MJ, Fried D, Featherstone JD (1999) Modeling the modification depth of carbon dioxide laser-treated dental enamel. 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Mater Chem Phys 111:180\u0026ndash;185\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"9.3 µm CO2 laser, Demineralization inhibition, Arrhenius damage threshold, Activation energy, Caries","lastPublishedDoi":"10.21203/rs.3.rs-5632668/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5632668/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHigh power pulsed 9.3 \u0026micro;m CO\u003csub\u003e2\u003c/sub\u003e laser irradiation at sub ablative fluences removes carbonate groups in Carbonated Hydroxyapatite (CHA) resulting in formation of pure Hydroxyapatite (HA) which is less soluble in acidic environments. The amount of carbonate removed was quantified using Fourier Transform Infrared Spectroscopy and a theory based on Arrhenius damage integral was applied to obtain activation energy for the laser mediated chemical reaction. We show Activation energy is 70\u0026thinsp;\u0026plusmn;\u0026thinsp;10 kJ/mol and fluence corresponding to 50% carbonate removed is 0.84 J/cm\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e","manuscriptTitle":"Quantifying the amount of carbonate removed in bovine enamel after irradiating with 9.3 micron CO2 laser","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-09 04:59:27","doi":"10.21203/rs.3.rs-5632668/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":"cf6e1090-b103-464d-8f07-9c63478ab788","owner":[],"postedDate":"January 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-09T04:59:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-09 04:59:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5632668","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5632668","identity":"rs-5632668","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}