Research reported in this publication was supported by JSPS KAKENHI JP21K09915 (MO) and the National Institute of General Medical Sciences;&#160;T34GM145509 (MM) and the National Institute of Dental and&#160;Craniofacial Research; R01DE025255 and R21DE032156&#160;(XH); R01DE029709, R21DE028715 and R15DE027851&#160;(TK); R01DE027648 and K02DE029531 (MS).

All procedures described in this protocol have been performed in accordance with guidelines and regulations for the use of vertebrate animals approved by the Institutional Animal Care Use Committee (IACUC) at Augusta University and at Nova Southeastern University which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Note that Dr. Suzuki was employed by Augusta University where the mouse dental fluorosis experiments were completed.
<p class="jove_titl...

Microhardness Protocol for Evaluating Rodent Oral Disease

<ol>
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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+adaptation+of+bone-pdl-tooth+complex:+Rattus-norvegicus+as+a+model+system.">Age-related adaptation of bone-pdl-tooth complex: Rattus-norvegicus as a model system.</a> PLoS One. 7 (4), e35980 (2012).</li><li>Johnson, W. M., Rapoff, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microindentation+in+bone:+Hardness+variation+with+five+independent+variables.">Microindentation in bone: Hardness variation with five independent variables.</a> J Mater Sci Mater Med. 18 (4), 591-597 (2007).</li><li>Kweon, Y. 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Microhardness is performed to evaluate mechanical properties of hard tissues like tooth and bone. To date, divergent microhardness measurement methods have been reported. Most of the measurement information, especially sample preparations and the indentation sites are likely to be insufficient. This study focused on the microhardness protocol for enamel and alveolar bone in dental fluorosis and periodontal diseases models. To obtain consistent and accurate results, the critical steps in this protocol are orientation of t...

Hardness is one of the mechanical properties (e.g., elasticity, hardness, viscoelasticity, and fracture behavior) and is commonly used to characterize the ability to resist compression deformation and fracture of a local area of a material. The static indentation hardness test is the most used method, including Vickers hardness and Knoop hardness1. The Vickers hardness test is implemented by pressing a diamond indenter into the surface under a fixed testing load. The indenter is pyramid-shaped, with a square base and an angle of 136&#176; between opposite faces. The length of both diagonals formed on the test surface is measured, and the average is used to calculate the hardness, which is determined by the ratio F/A (where F is the force and A is the surface area of the indentation). The Vickers microhardness number (HV=F/A) is usually expressed in kilograms-force (kgf) per mm2 indentation, with 1 HV &#8776; 0.1891 F/d2 (N/mm2). The Knoop hardness also consists of a diamond square pyramid indenter formed by two unequal opposite angles. The Knoop hardness number (HK) equals the ratio of applied load to the projected contact area. Hardness tests are classified into micro-indentation (microhardness) tests and macro-indentation tests, depending on the force applied to the test material. Micro-indentation tests typically use loads in the range 0.01-2 N (about 1-203 gf); meanwhile, macro-indentation tests use over 10 N (10119 gf)1.
To evaluate features of dental hard tissues in oral diseases, including tooth and alveolar bone, micro-CT (&#181;CT) and scanning electron microscopy (SEM) are used for structural analysis. &#181;CT provides 3D imaging information (volume and mineral density)2, and SEM produces microstructure images (enamel prism and bone lacuna-canalicular)3. Complementarily to structural analysis by &#181;CT and SEM, microhardness is one of the informative parameters to evaluate how structural changes alter the mechanical properties of tooth and alveolar bone in oral diseases, e.g., enamel malformation and periodontal bone resorption. The Vickers microhardness value of human enamel (HV = 283-374) is about 4 to 5 times higher than that of dentin (HV = 53-63)4,5. In rodent dental fluorosis models, enamel microhardness significantly decreases in mouse incisors treated with fluoride (HV = 136) compared to control enamel (HV = 334)6,7. This suggests that fluorosed enamel is softer and weaker with lower mineral content and higher protein content than found in non-fluorosed enamel. Microhardness is used to evaluate bone mechanical properties. Several previous studies have examined the mechanical behavior of human bone from different anatomic sites, including long bone microhardness8,9,10. The mean microhardness of human fluorosed femurs showed a significant decrease (HV = 222.4) compared to non-fluorosed femurs (HV = 294.4)11. Despite being a useful parameter, there is a scarcity of literature describing microhardness (either Vickers12 or Knoop13,14) of alveolar bone in oral diseases.
To date, divergent microhardness measurement methods have been reported. Since microhardness values vary15 depending on sample preparation (polishing and flat surface) and indentation site, diverse protocols can cause discrepancies among studies. Standardization of the microhardness testing protocol is essential for consistent and accurate evaluation in oral disease models. In the present study, we demonstrate a standardized protocol for microhardness analysis in tooth and alveolar bone in mouse dental fluorosis model and periodontal bone resorption model.

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			<td>Maruto instrument co, LTD. Japan</td>
			<td>26-4204</td>
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			A3-3 SHT, 3M USA</td>
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			<td>Maruto instrument co, LTD. Japan</td>
			<td>26-4201</td>
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			A3-9 SHT, 3M USA</td>
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microhardness measurements on tooth and alveolar bone in rodent oral disease models

The mechanical property, microhardness, is evaluated in dental enamel, dentin, and bone in oral disease models, including dental fluorosis and periodontitis. Micro-CT (&#181;CT) provides 3D imaging information (volume and mineral density) and scanning electron microscopy (SEM) produces microstructure images (enamel prism and bone lacuna-canalicular). Complementarily to structural analysis by &#181;CT and SEM, microhardness is one of the informative parameters to evaluate how structural changes alter mechanical properties. Despite being a useful parameter, studies on microhardness of alveolar bone in oral diseases are limited. To date, divergent microhardness measurement methods have been reported. Since microhardness values vary depending on the sample preparation (polishing and flat surface) and indentation sites, diverse protocols can cause discrepancies among studies. Standardization of the microhardness protocol is essential for consistent and accurate evaluation in oral disease models. In the present study, we demonstrate a standardized protocol for microhardness analysis in tooth and alveolar bone. Specimens used are as follows: for the dental fluorosis model, incisors were collected from mice treated with/without fluoride-containing water for 6 weeks; for ligature-induced periodontal bone resorption (L-PBR) model, alveolar bones with periodontal bone resorption were collected from mice ligated on the maxillary 2nd molar. At 2 weeks after the ligation, the maxilla was collected. Vickers hardness was analyzed in these specimens according to the standardized protocol. The protocol provides detailed materials and methods for resin embedding, serial polishing, and indentation sites for incisors and alveolar. To the best of our knowledge, this is the first standardized microhardness protocol to evaluate the mechanical properties of tooth and alveolar bone in rodent oral disease models.

Author Spotlight: Establishing an Accurate Microhardness Testing Protocol for Craniofacial Tissues

Microhardness Measurements on Tooth and Alveolar Bone in Rodent Oral Disease Models

In this research, we focused on establishing accurate and consistent microhardness testing. Microhardness testing is a traditional method, but fear not, the methodology is not sufficiently described in previous publications. As we demonstrated in the result, provide immediate minimal microhardness reduction depends on the intention sites of the interim, like inner, middle and outer layers.Our results suggests that the intention site is important to obtain accurate and consistent results. The scientific gap is that the previous studies did not sufficiently describe the microhardness testing methods, especially the variation sites. Due to this, microhardness result were not consistent between studies.To fill this gap, we established this protocol for accurate and effective evaluation. Our established protocol will advance mineral tissue testing in enamel, denti, and earlier bone in periodontal disease, bone origination, and oral cancer bone invasion models. We will focus on how environmental factors affect dental craniofacial pathophysiology.For example, chloride and organ rolling, PFAS. PFAS is manmade forever hazardous chemicals. Our microhardness protocol will contribute to evaluating how PFAS affect mechanical properties in mineral tissues in tooth development, periodontal bone resorption, and oral cancer bone invasion.

Dental fluorosis model: Figure 1 shows representative &#956;CT images of incisors in control and fluoride-treated mice. In the control (Figure 1B-D), the cervical region showed lower enamel mineral density (EMD) of 1.188 g/cm3 (Figure 1B) compared to the middle (1.924 g/cm3) and tip (1.819 g/cm3; Figure 1C,D). In the fluoride-treated ena...

Watch this Scientific Journal Video about Author Spotlight: Establishing an Accurate Microhardness Testing Protocol for Craniofacial Tissues at JoVE.com

Microhardness is a mechanical property and an informative parameter for evaluating hard tissue pathophysiology. Here, we demonstrate a standardized protocol (sample preparation, polishing, flat surface, and indentation sites) for microhardness analysis in tooth and alveolar bone in rodent oral disease models, namely, dental fluorosis, and ligature-induced periodontal bone resorption.

The mechanical property, microhardness, is evaluated in dental enamel, dentin, and bone in oral disease models, including dental fluorosis and ...

microhardness-measurements-on-tooth-alveolar-bone-rodent-oral-disease