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 periodontitis. Micro-CT (µCT) 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 µCT 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.
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° 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 ≈ 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 (µCT) and scanning electron microscopy (SEM) are used for structural analysis. µCT 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 µCT 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.
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.
1. Extraction of mandibular incisors in a mouse dental fluorosis model
2. Extraction of maxillary alveolar bones in a mouse ligature-induced periodontal bone resorption (L-PBR) model
Figure 1: Representative μCT images of enamel in control and fluoride-treated mice incisors. (A) Representative μCT sagittal image of mandibular incisor. (B-D) μCT coronal images of control incisor (NaF 0 ppm). (E-G) μCT coronal images of incisor treated with NaF (125 ppm). Representative enamel mineral density (EMD) is shown (g/cm3). Please click here to view a larger version of this figure.
3. Embedding samples in resin
Figure 2: Flow of resin-embedding and polishing procedure. (A) Dehydrated and degreased incisor. (B) Dehydrated and degreased alveolar bone in L-PBR. (C, D) Incisors and alveolar bone immersed in resin. (E, F) By cutting off the resin, it is easier to polish the target tissue surface. (G, H) Resin corners rounded for the polishing process. Abbreviations: L-PBR = ligature-induced periodontal bone resorption. Please click here to view a larger version of this figure.
4. Polishing of specimens
NOTE: Polishing of specimens is done manually using waterproof abrasive papers (from rough to finer) on an advanced grinder-polisher under water flooding.
5. Vickers microhardness test
NOTE: Indentation of a mirror finish surface specimen is done using a microhardness tester. Testing is performed with a load of 25 g for 10 s with a Vickers tip.
Figure 3: Evaluation regions of microhardness in mandibular incisor. (A) Mirror finish surface sample containing mandibular incisor. (B) Indentations in each region; cervical, middle, and tip (NaF 0 ppm). (C) Three enamel layers; from DEJ, Inner, Middle, and Outer enamel. Abbreviations: D = dentin, E = enamel, DEJ = dentin enamel junction Please click here to view a larger version of this figure.
Figure 4: Vickers microhardness of enamel treated with or without NaF. The microhardness of dentin and three enamel layers were evaluated in each region, cervical, middle, and tip region. (A-C) Control and (D-F) NaF (125 ppm) treatment. Data are presented as mean ± SD. Significant differences were evaluated by one-way ANOVA with Tukey's post-hoc test. p values < 0.05 were considered statistically significant. **p < 0.005, ***p < 0.0005, ****p < 0.0001 Please click here to view a larger version of this figure.
Dental fluorosis model: Figure 1 shows representative μCT 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 enamel (Figure 1E-G), only one sample out of five was evaluated for EMD in the cervical region (0.835 g/cm3; Figure 1E and Supplementary Figure 1). The EMD in all regions decreased compared to the control (Figure 1F,G). The low EMD levels were consistent with the low enamel microhardness values. As shown in Figure 3, six points were indented at dentin and three layers of enamel (inner, middle, and outer) in the cervical, middle, and tip regions. In the control, each enamel layer's microhardness was lower than dentin in the cervical region (Figure 4A). In the middle and tip regions, enamel microhardness of each layer was significantly higher than that of dentin (Figure 4B,C). Among the three enamel layers, microhardness increased from the inner to the outer enamel in each middle and tip region (Figure 4B,C). Dentin had a microhardness value around 100 HV with small variations in cervical, middle and tip regions, whereas enamel microhardness was significantly different in regions and in enamel three layers. These results suggest that enamel microhardness significantly differs depending on indentation sites (regions and enamel layers). In the fluoride-treated tooth, contrary to the control, enamel microhardness was less than dentin even in the middle region (Figure 4E). In the tip region, microhardness significantly decreased from the inner to the outer enamel layer (Figure 4F). These gradient differences of microhardness among enamel layers are difficult to evaluate by μCT images.
L-PBR model: Figure 5A shows μCT images of alveolar bone in the ligature-induced periodontal bone resorption (L-PBR) model. The representative bone mineral density (BMD) (mean of the mesial and distal sides of alveolar bone around second molar) was 0.76 g/cm3 in control bone and 0.61 g/cm3 in L-PBR. Bone resorption levels were quantified by the distance from the cement enamel junction (CEJ) to the alveolar bone crest (ABC). The CEJ-ABC length was significantly increased in L-PBR compared to the control bone (Figure 5B). Figure 6 shows microhardness indentation sites and corresponding μCT images. From the alveolar bone crest, five indentations were done in each medial and distal side (total 10 sites) in the control bone between 1st and 2nd molar indicated by the white square (Figure 6A). The 3 indentations in each mesial and distal side (total 6 sites) were measured in the L-PBR (Figure 6B). Vickers microhardness values (HV) were the means of indentations of alveolar bones between 1st and 2nd molar (Figure 6B; White square), and between 2nd and 3rd molar (Figure 6B; Blue square). Alveolar bone BMD and HV values showed a lower tendency in L-PBR (affected by periodontal diseases) compared to control (healthy) alveolar bone.
Figure 5: Representative μCT image and bone loss in the L-PBR model. (A) Representative μCT images in the L-PBR model (Control and L-PBR group). The representative bone mineral density (BMD; mean of the mesial and distal sides of alveolar bone around second molar) is shown (g/cm3). (B) Distance from the mesial and distal CEJ of the maxillary second molar to the alveolar bone crest in the root apical direction. Data are presented as mean ± SD. Significant differences were evaluated by t-test. p values < 0.05 were considered statistically significant. **** p < 0.0001. Abbreviations: L-PBR = ligature-induced periodontal bone resorption. CEJ = cement enamel junction, ABC = alveolar bone crest. Please click here to view a larger version of this figure.
Figure 6: Representative microhardness results in the L-PBR model. Representative buccal side of indentation sites (left) and the corresponding μCT image (right) of (A) control alveolar bone, and (B) L-PBR. White squares show indentation areas in the alveolar bone between M1 and M2. Blue squares show indentation areas in the alveolar bone between M2 and M3. Microhardness values (HV) are the means of indentations in white and blue square areas. M1: 1st molar, M2: 2nd molar, M3: 3rd molar. Abbreviations: L-PBR = ligature-induced periodontal bone resorption. Please click here to view a larger version of this figure.
Supplementary Figure 1: μCT image of enamel treated with NaF (coronal section). NaF (125 ppm) caused enamel hypo-mineralization, which was significantly observed in the cervical and middle regions. Only one sample (sample no. 1) out of five barely showed enamel in the cervical and middle regions. Please click here to download this File.
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 the specimen in resin embedding, keeping the evaluation surface parallel to the ground, serial polishing of evaluation surface to obtain mirror finish, and indentation regions and sites set by reference point. During the resin embedding and polishing procedures, it is important to check that the evaluation surface is consistently parallel to the ground and the surface is intact by eye or under a microscope. Although it is optional, μCT analysis is encouraged to determine indentation sites.
In the dental fluorosis model, NaF (125 ppm) treatment made it difficult to identify enamel structure from the cervical to middle regions by μCT. Only the tip region enamel could be distinguished from dentin (Figure 1 and Supplementary Figure 1). Therefore, to evaluate enamel microhardness in the dental fluorosis model, the tip region indentation is appropriate. In accordance, previous studies evaluated the tip region of incisor enamel in dental fluorosis models6,7. In the periodontal disease model, 3D observation by μCT helps identify the bone resorption on both buccal and palatal sides (Figure 5). This is critical for understanding the amount of bone loss and the anatomical position of the alveolar bone to determine consistent indentation sites for microhardness.
A previous study demonstrated a positive correlation between microhardness and mineral density17. Our results of EMD by μCT and enamel microhardness (HV; Figure 1 and Figure 4) are concordant with the study. These results suggest that the approximate tendency of microhardness can be anticipated by μCT non-destructively. However, the gradient microhardness differences among the enamel three layers (Figure 4B,C,F) are difficult to identify as EMD gradients by μCT analysis. In this regard, microhardness testing could be considered higher resolution than μCT to clarify pathological conditions. Also, this protocol can be applied to other dental hard tissues, including dentin. Using the same specimen, multifaceted evaluation (SEM, SEM-EDX, micro-XRF and Raman spectroscopy) can be incorporated into the experimental flow prior to microhardness indentations18. Since indentations damage samples, start with a non-destructive test.
One of the critical limitations of microhardness testing is that the value tends to be affected by several factors during sample preparation and indentation. To minimize subjective factors, it is necessary to optimize indentation sites and to standardize measurement protocols that are appropriate for each pathological condition or disease model. In this study, we demonstrated an enamel microhardness protocol for a dental fluorosis model. However, modification and/or optimization of the protocol may be necessary for other enamel hypoplasia, e.g., amelogenesis imperfecta (AI) model because pathology differs in each disease model. In the periodontal disease model, alveolar bone is the main target tissue. L-PBR models are highly applicable in terms of the application of genetic modification techniques in mice. To date, many studies on L-PBR models have been published19,20 . However, to the best of our knowledge, none of the studies has ever addressed microhardness of alveolar bone in mouse periodontal disease models. This can be attributed to several factors. The relationship between alveolar bone microhardness and periodontal disease is not yet clear. The microhardness test is technically difficult to perform in the mouse alveolar bone, especially in bone resorption lesions (because of difficulties to set indentation sites due to bone destruction). It is reasonable to assume that the latter is the factor why microhardness has not been evaluated in periodontal disease models, despite microhardness value is validated as a mechanical parameter in femur and other bones21. This standardized protocol can evaluate the mechanical properties of alveolar bone affected by periodontal disease and/or disease recovery model.
In this report, we demonstrate the standardized protocol to evaluate enamel and alveolar bone microhardness in a mouse oral disease model. This opens the door for future evaluation of enamel and periodontal bone loss/regeneration to develop novel preventive and therapeutic strategies for enamel malformation and periodontal disease.
The authors have nothing to disclose.
Research reported in this publication was supported by JSPS KAKENHI JP21K09915 (MO) and the National Institute of General Medical Sciences; T34GM145509 (MM) and the National Institute of Dental and Craniofacial Research; R01DE025255 and R21DE032156 (XH); R01DE029709, R21DE028715 and R15DE027851 (TK); R01DE027648 and K02DE029531 (MS).
Braided Silk Suture 6-0 | Teleflex | ||
Canica Small Animal Surgery System | Kent Scientific Corporation | SURGI 5001 | |
CarbiMet PSA 120/P120 | Buehler | 30080120 | |
CarbiMet PSA 60/P60 | Buehler | 36080060 | |
CarbiMet PSA 600/P1200 | Buehler | 36080600 | |
Castroviejo Micro Needle hilder | F.S.T | 12060-01 | |
Epofix cold setting embeding Resin | Electron Microscopey Science | CAT-1237 | |
Fisherbrand 112xx Series Advanced Ultrasonic Cleaner | Fisher Brand | FB11201 | |
Fluoride-free Rodent diet | Bio Serv | F1515 | AIN-76A, 1/2" Pellets |
in-vivo microCT Skyscan 1176 | Bruker | ||
Isomet 1000 Precison saw | Buehler | MA112180 | |
Lapping film 0.3µm | Maruto instrument co, LTD. Japan | 26-4203 | Alternative A3-0.3 SHT, 3M USA |
Lapping film 1µm | Maruto instrument co, LTD. Japan | 26-4206 | Alternative A3-1 SHT, 3M USA |
Lapping film 12µm | Maruto instrument co, LTD. Japan | 26-4211 | Alternative A3-12 SHT, 3M USA |
Lapping film 3µm | Maruto instrument co, LTD. Japan | 26-4204 | Alternative A3-3 SHT, 3M USA |
Lapping film 9µm | Maruto instrument co, LTD. Japan | 26-4201 | Alternative A3-9 SHT, 3M USA |
Leica wild microscope | Leica | LEIC M690 | |
Metaserv 2000 Variable speed Grinder polisher | Buehler | No: 557-MG1-1160 | |
MicroCut PSA 1200/P2500 | Buehler | 36081200 | |
MicroCut PSA P4000 | Buehler | 36084000 | |
Microhardness tester, ALPHA-MHT-1000Z | PACE Technologies | ||
SamplKups 1 inch | Buehler | No: 209178 | |
Sodium Fluoride | Fisher Scientific | S299-100 | |
West cott Stitch Scissor | JEDMED | Cat. #25-1180 | |
ZooMed Repti Thern Undertank heater (U.T.H) | Zoo Med Laboratories, Inc. | RH-4 |