Chemical analysis and dentin integration of bioactive cements. Jennifer Fernandez, DMD, MDS Resident, College of Dentistry, University of
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1 Chemical analysis and dentin integration of bioactive cements Jennifer Fernandez, DMD, MDS Resident, College of Dentistry, University of Tennessee, 875 Union Ave, Memphis TN, 38163, (901) , Jennifer Lou, DDS - Assistant Professor, College of Dentistry, University of Tennessee, 875 Union Ave, Memphis TN, 38163, (901) , Jlou3@uthsc.edu Brian R. Morrow, MS - Research Coordinator, College of Dentistry, University of Tennessee, 875 Union Ave, Memphis TN, 38163, (901) , morrow@uthsc.edu Franklin Garcia-Godoy, DDS, MS, PhD, PhD - Professor, Senior Executive Associate Dean for Research, College of Dentistry, University of Tennessee, 875 Union Ave, Memphis TN, 38163, (901) , godoy@uthsc.edu Correspondence: Dr. Franklin Garcia-Godoy, College of Dentistry, University of Tennessee Health Science Center, 875 Union Ave, Memphis TN, 38163, USA. fgarciagodoy@gmail.com
2 Chemical analysis and dentin integration of bioactive cements ABSTRACT: Purpose: This in vitro investigation evaluated and compared hydroxyapatite (HA) formation and the integration of two bioactive cements (BioCem and Ceramir), and a resin-modified glass-ionomer (RMGI) cement (RelyX) into dentin tubules. Methods: Dentin discs were cemented per manufacturers instructions with either BioCem Bioactive Cement, RelyX Luting Plus Automix Cement or Ceramir Crown and Bridge Cement and immersed in phosphate buffered saline (PBS) at 37 C for 1, 3, 7, and 21 days. The treated dentin discs were then dehydrated and analyzed to verify HA formation via scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS). Results: Formation of cement tags integrating into dentinal tubules was observed via SEM for both BioCem and Ceramir. Chemical analyses of the tags via EDS confirmed the calcium phosphate (CA/P) ratio to be consistent with that of calcium-ha (Ca/P = 1.67). CA/P for BioCem ranged from 2.8 at 24 hours to a at 7-day equilibrium. Ceramir tags were not detected until 7 days, at which time Ca/P was 2, increasing to 3.4 at 21 days and not reaching equilibrium during the study period. RelyX RMGI produced no tags between the cement and the dentin, and thus produced no evidence of HA formation. Clinical significance: Formation of HA at the material dentin interface could reduce post-operative sensitivity, guards against secondary caries, and seals margin against microleakage and failure. Chemical microanalysis of the cement and surrounding dentin indicated that BioCem and Ceramir may provide
3 important remineralization contributors, including phosphate, calcium, and fluoride, with BioCem providing a natural seal at the tooth-cement interface within 24 hours.
4 Introduction Bioactive cement s ability to either form or initiate the formation of hydroxyapatite (HA) is a defining aspect of this contemporary class of cements. The HA formation sequence and its subsequent integration into dentin, however, is not necessarily the same for each of these new bioactive cements. One of the most important aspects of bioactive dental materials is their ability to enhance mineralization due to their calcium and phosphate contents. This bioactivity also enhances the bond between the material and living tissues. 1 It is necessary to define HA formation through the stoichiometry of apatite as: Ca10(PO4)6(OH)2 Having a Ca/P ratio of 10:6 normally expressed as The Ca/P ratio is often used in the discussion of calcium phosphate phases. 2 This in-vitro investigation evaluated and compared the bioactive effects and HA formation and ability to integrate into dentin of two bioactive cements (BioCem a and Ceramir b ), and a resin-modified glass-ionomer (RMGI) cement (RelyX c ). Materials and Methods Extracted, lesion free, human adult molars were used to obtain the specimens. Crowns were sectioned horizontally into discs using a diamond well
5 disc, beginning at the occlusal surface and ending at the commencement of the pulpal chamber. Specimens were divided into three treatment groups, 4 of which were used at each of the 4 time points. Dentin discs were cemented per manufacturer s instructions with either BioCem Bioactive Cement, RelyX Luting Plus Automix Cement or Ceramir Crown and Bridge Cement and immersed in phosphate buffered saline (PBS) at 37 C for 24 hours, 72 hours, 7 days, and 21 days. Dentin discs were prepared and cement applied per manufacturer s instructions with either BioCem Bioactive Cement, RelyX Luting Plus Automix Cement or Ceramir Crown and Bridge Cement and immersed in phosphate buffered saline (PBS) at 37 C for 1, 3, 7, and 21 days. The treated dentin discs were then dehydrated and analyzed to verify HA formation via scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS). Analysis Samples were evaluated in a transverse plane to the dentinal tubule for resin tag formation. Chemical analysis for the weight percentages of calcium and phosphorous were performed on the tags, inside the tubules and intertubular dentin. In addition to the chemical analysis, the ratio of calcium to phosphate was also considered.
6 It is necessary to define HA formation through the stoichiometry of apatite as: Ca10(PO4)6(OH)2. Having a Ca/P ratio of 10:6 normally expressed as The Ca/P ratio is often used in the discussion of calcium phosphate phases. 2 Results Formation of cement tags integrating into dentinal tubules was observed via SEM for both BioCem and Ceramir. Chemical analyses of the tags via EDS confirmed the calcium phosphate (Ca/P) ratio to be consistent with that of calcium-ha (Ca/P = 1.67). Ca/P for BioCem ranged from 2.8 at 24 hours to a at 7-day equilibrium. (Figs. 5,6). Ceramir tags were not detected until 7days, at which time Ca/P was 2, increasing to 3.4 at 21 days and not reaching equilibrium during the study period. (Figs. 5, 7). RelyX RMGI produced no tags between the cement and the dentin, and thus produced no evidence of HA formation. (Figs. 2,8). Discussion A certain set of prerequisites needs to be fulfilled in order to get HA precipitation on a material surface; the correct ions must be present in high enough
7 concentrations, the ph must be in the correct alkaline range, and a negatively charged surface is necessary RelyX is known to lack bioactive properties. Resin-based materials are acidic or neutral, but so far not alkaline, and they do not show extended ion leakage. RMGIs are a well-established and characterized dental material with hydrophilic components/properties and thus offer a reasonable comparison to the experimental bioactive cements tested Ceramir is comprised of calcium aluminate and GI components. It depends on naturally occurring phosphate compounds ( free phosphates ) supplied by the body through the tooth structure, and made available in a reactive state at the cement/tooth interface. On the other hand, BioCem provides both the phosphate polymeric chemistry and the calcium base already in the cement and available at the time of reaction and initial bonding with two ionic species that Ceramir does not claim to have, calcium and phosphate. In theory BioCem should form hydroxyapatite at the time of cementation, available to integrate with and bond to tooth structure very quickly. RelyX is known to lack bioactive properties. Resin-based materials are acidic or neutral, but so far not alkaline, and they do not show extended ion leakage. RMGIs are a well-established and characterized dental material with hydrophilic components/properties and thus offer a reasonable comparison to the experimental bioactive cements tested
8 In summary, chemical microanalysis of the cement and surrounding dentin indicated that BioCem and Ceramir may provide important remineralization contributors, including phosphate, calcium, and fluoride, with BioCem providing a natural seal at the tooth-cement interface within 24 hours, reducing postoperative sensitivity. In conclusion, bioactive cements offer numerous biological benefits that RMGI or resin cements are simply incapable of offering. Both bioactive cements form tags within the dentin and have the capability of releasing and reabsorbing ions in the oral environment. Chemical microanalysis of the cement and surrounding dentin indicated that BioCem and Ceramir may provide important remineralization contributors, including phosphate, calcium, and fluoride, with BioCem providing a natural seal at the tooth-cement interface within 24 hours. BioCem demonstrated the formation of tags into the dentin and with an increasing amount of calcium and phosphate available for HA formation. Future work should investigate the ionic release characterization, ph affects, and biofilm formation of these bioactive materials.
9 Because the demineralization process is basically a removal of calcium and phosphate ions from tooth structures, the new bioactive cements with their potential to create surface apatite (HA) on the dentin surfaces would be an added benefit to dentistry. Chemical microanalysis of the cement and surrounding dentin indicate that BioCem may provide important remineralization contributors, including phosphate, calcium, and fluoride, and provide a natural seal at the tooth-cement interface within 24 hours. Study weaknesses Due to dentin-cement separation during preparation for SEM imaging, there were not sufficient specimens to run a statistical analysis. Specimens were etched to provide the most favorable environment for HA tag formation. Etching is not typical in cementation protocols. a. NuSmile, Houston, TX, USA. b. Doxa AB, Uppsala, Sweden. c. 3M ESPE, St. Paul, MN, USA. Disclosure statement: This research was supported by NuSmile. The authors declared no conflict of interest. Dr. Fernandez was a pediatric dentistry resident, College of Dentistry, University of Tennessee Health Science Center, Memphis, Tennessee, USA, and in private practice in St. Petersburg, Florida, USA. Dr. Lou is an adjunct assistant professor, Department of Pediatric Dentistry, Mr. Morrow is Research Associate, Department
10 of Bioscience Research, and Dr. Garcia-Godoy is Professor and Chair, Department of Bioscience Research, College of Dentistry, University of Tennessee Health Science Center, Memphis, Tennessee, USA, References 1. Kokubo T, Takadama H. How useful is SBF in predicting in vivo bone bioactivity? Biomaterials 2006;27: Kim HM, Himeno T,Kokubo T, Nakamura T. Process and kinetics of bonelike apatite formation on sintered hydroxyapatite in a simulated body fluid,. Biomaterials 2005;26: Kim HM. Ceramic bioactivity and related biomimetic strategy. Current Opinion in Solid State and Materials Science 2003;7: Gandolfi MG, Taddei P, Tinti A, Prati C. Apatite-forming ability (bioactivity) of ProRoot MTA. Int Endod J 2010;43: Jefferies S. Bioactive and biomimetic restorative materials: A comprehensive review. Part II. J Esthet Restor Dent 2014;26: Cenci MS, Pereira-Cenci T, Cury JA, Ten Cate JM. Relationship between gap size and dentine secondary caries formation assessed in a microcosm biofilm model. Caries Res 2009;43: Diercke A, Lussi A, Kersten T, Seemann R. Isolated development of inner (wall) caries like lesions in a bacterial-based in vitro model. Clin Oral Investing 2009;13: Rey C, Combes C, Drouet C, Grossin D. Bioactive ceramics: Physical chemistry. In Ducheyne P et al. Comprehensive biomaterials. Elsevier, Oxford, UK, 2011; Lööf J, Svahn F, Jarmar T, Engqvist H, Pameijer CH. A comparative study of the bioactivity of three materials for dental applications. Dent Mater 2008;24: Lööf J, Engqvist H, Ahnfelt NO, Lindqvist K, Hermansson L. Mechanical properties of a permanent dental restorative material based on calcium aluminate. J Mater Sci: Mater Med 2003;14:
11 Fig, 1. Dentin disks specimen preparation. Fig. 2. RelyX.
12 Fig. 3. Ceramir. Fig. 4. BioCem.
13 Fig. 5.
14 Fig. 6. BioCem Fig. 7. Ceramir Fig. 8. RelyX
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