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Indentation Modulus of the Alveolar Process in Dogs

¹ Section of Orthodontics, College of Dentistry, The Ohio State University, 4088 E Postle Hall, 305 W. 12th St., Columbus, OH 43210, USA; and
² Center for Biostatistics, The Ohio State University, 320 W. 10th Ave., Columbus, OH 43210, USA

^* corresponding author, huja.1{at}osu.edu

	ABSTRACT

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One mechanism of bone adaptation is alteration in tissue level material properties. We hypothesized that alteration in the indentation modulus of the alveolar process is an adaptive response to the localized mechanical environment. Forty-eight specimens representing anterior and posterior regions of the maxilla and mandible were obtained from 6 mature male beagle dogs. The indentation properties of the alveolar bone proper and more distant osteonal cortical bone were estimated. The bone types were further divided into 3 regions (coronal, middle, and apical), with 27 indents being made in each region of tooth-supporting bone. There was a significant difference (p < 0.001) in the indentation moduli of the jaws (maxilla/mandible), location (anterior/posterior), and bone type (alveolar bone proper vs. cortical bone). However, statistical interactions exist which preclude the simple interpretation of results. The distribution of relative stiffness provides a better understanding of bone adaptations in the alveolar process.

KEY WORDS: bone • remodeling • adaptation • indentation modulus • alveolar process

	INTRODUCTION

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A gradient of cortical bone remodeling has been observed in the mandible (Tricker et al., 2002). The rate of cortical bone remodeling can be as high as 34%/yr in the mandibular alveolar process and tapers down to 10%/yr in the region of the inferior border of the mandible. These remodeling rates are significantly higher than the 2%/yr observed in the tibial cortical bone from the same animals. Because of the small volume of bone tissue in the alveolar process, a high rate of localized bone turnover may result in similar gradients of material properties within the alveolar process (Huja et al., 2006). For example, the coronal regions may have a lower stiffness than more apical regions. In addition, there is evidence to suggest that mandibular bone density differs from that of other cranial bones (Dechow et al., 1993). However, the elastic properties of the alveolar process of the jaws are unknown. The tooth receives load during mastication and other oral functions. One study in dogs recorded bite forces with a range of 13–1394 N (Lindner et al., 1995). In a beagle dog, the maxillary and mandibular second premolars are not directly in occlusal contact with any other teeth. However, the maxillary fourth premolar and mandibular first molar are in occlusal contact during biting and are the shearing teeth (Greaves, 1995). This functional arrangement of contacting and non-contacting teeth suggests possible differences in bite forces borne by the anterior and posterior regions of the canine jaw, which may result in altered bone density and material properties. Finally, there is evidence to suggest that Sharpey’s fiber diameter varies in different regions of the alveolar bone (Johnson, 1992), and that occlusal function changes the morphology of fibers, inserting into the alveolar bone (Short and Johnson, 1990). This interfacial bone may have altered physical properties that have not been investigated previously.

Based on the remodeling data within the alveolar process/jaws, functional anatomy, and histology of different regions of the jaws, we hypothesized that: (a) a gradient of indentation modulus exists, with the lowest stiffness being in the coronal regions of the alveolar process; (b) the mandibular alveolar process has a higher indentation modulus than in the maxilla; (c) the bone supporting the posterior teeth has a higher indentation modulus than in the anterior teeth; and (d) the alveolar bone proper has a lower indentation modulus than the cortical bone within the alveolar process.

	MATERIALS & METHODS

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Institutional Animal Care Committee approval was obtained. The jaws were obtained from 6 skeletally mature (mean age, 13 mos; SD, 11 days) dogs immediately after death. The alveolar processes surrounding 4 specific landmark teeth were obtained. The landmark teeth were used as a guide for sampling bone at different locations and regions. Bilaterally, the maxillary second, maxillary fourth premolar, mandibular second premolar, and mandibular first molar and their supporting bone were obtained (4 teeth x 2 jaws x 6 dogs = 48 teeth) from each dog.

Sample Preparation
Bone sections were made bucco-lingually through the jaws and aligned with the center of the longitudinal axis of a landmark tooth crown and root, with the use of a diamond-coated saw (Exakt 310 CP, Exakt Technologies, Oklahoma City, OK, USA) under water lubrication. Approximately 4-mm-thick bone sections were obtained from the jaws and associated with the 4 landmark teeth. For each of the landmark teeth except the maxillary fourth premolar, the bone associated with the anterior root and crown was sectioned. However, for the maxillary fourth premolar, the posterior root and crown was chosen, since they are the functional antagonists of the anterior root of the corresponding lower molar. The sectioned tooth/bone blocks were frozen at –20°C in saline-soaked gauze for a brief storage period (Martin and Sharkey, 2001). The tooth/bone block specimens were thawed just prior to being tested. The tooth/bone block was glued into a well of a custom-made polycarbonate specimen-holder. The sectioned specimens were wet-polished, sonicated in de-ionized water for 30 sec, and tested immediately thereafter (Huja et al., 1998).

Nanoindentation
The sample preparation and test procedure for indentation of moist specimens has been described in detail in the literature (Huja et al., 2006). Briefly, a polycarbonate specimen-holder containing the polished tooth/bone block was inserted and secured into a sample tray of the indentation system (Nano-XP, MTS, Oakridge, TN, USA). Fifteen indents were made in the alveolar bone proper, and 12 indents were made on osteonal cortical bone (Figs. 1a, 1b) in each region for bone supporting the landmark teeth. Specimens were tested at room temperature at a loading rate of 10 nm/sec, with each indent being made at least 30 µm away from the adjacent indent. For each measurement, the bone was loaded with a Berkovich tip, according to established protocols (Hoffler et al., 1997; Rho et al., 1999; Huja et al., 2006), to a 500-nm depth. A 30-second hold period was imposed at the peak depth. The unloading rate equaled the loading rate. The mean load at peak depth was ~ 3 mN for indents. The method of Oliver and Pharr (1992) was used to calculate the reduced elastic modulus. Poisson’s ratio for bone was assumed to be 0.3. The indenter was calibrated with the use of a fused silica specimen. The mean indentation modulus obtained for silica was 70.9 ± 0.4 GPa, which is close to the known value of 72 GPa.

View larger version (60K): [in this window] [in a new window]   Figure 1. The alveolar process and regions/locations for indentation testing. (a) Schematic of bucco-lingual section of mesial root of a premolar and its alveolar process. The alveolar process is divided into 3 regions—coronal, middle, and apical—along the root length of the tooth. Each third is numbered 1 through 6, with 1 being the coronal region on the buccal side and 6 being the coronal region of the lingual side (for the mandibular alveolar process) or the palatal side (for the maxillary alveolar process). Dark lines near (< 200 µm) the periodontal ligament indicate the location of indents in alveolar bone proper (also know as alveolar bone/cribriform plate, bundle bone); stippled boxes indicate approximate locations of indents in cortical bone. (b) Photomicrograph of the alveolar process and surrounding dental structures in the coronal region. Two rectangular boxes indicate approximate sites for making 5 indentations in bone. The box on the left is closer to the periodontal ligament and indicates the location of indents in alveolar bone proper. The box on the right indicates the center of the osteonal cortical bone. This photomicrograph clearly depicts differences in bone morphology and heterogeneity in the alveolar process. Bar indicates 100 µm.

	RESULTS

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In total, 7947 indents were attempted on the 47 tooth-supporting alveolar processes. One tooth-supporting bone (maxillary second premolar) was incorrectly sectioned and could not be used for indentation. Approximately 10% (860) of the total indent cycles were either aborted automatically by the machine or were not within 30 nm of the 500-nm target depth. This is not uncommon in the testing of biological specimens, and these values were eliminated from further analysis. The mean and standard deviation of each jaw, location, region, and bone type are reported in Table 1. From the mixed-model results, overall differences were found for indentation modulus between the jaws (maxilla vs. mandible), location (anterior vs. posterior), and bone types (alveolar vs. cortical), but not for regions 1 through 6 (Table 2 and Fig. 2a). Moreover, significant two-way interactions were seen between (a) region and bone type (p < 0.001) and (b) jaw and bone type (p = 0.029). In addition, a three-way interaction was seen among region, bone type, and jaw (p = 0.04).

View larger version (12K): [in this window] [in a new window]   Figure 2. Indentation modulus of the jaws. (a) Indentation modulus (LSM, 95% CI) of alveolar bone proper and cortical bone, depicting two-way interaction. There was a significant difference (p < 0.0001) in the overall indentation modulus of the alveolar bone proper and cortical bone. Cortical bone region 1 was different (p = 0.0143) from cortical bone regions 2 and 3. However, cortical bone regions 4, 5, and 6 were not different (p = 0.97) from each other. Alveolar bone proper region 1 was not different from alveolar bone proper in regions 2 and 3. Alveolar bone proper region 4 was not different from alveolar bone proper in regions 5 and 6. The sample size, mean, and SE for the 6 regions (from 1 to 6) in the alveolar bone proper were: 47, 9.8, 0.5; 47, 10.3, .5; 47, 11.3, 0.5; 47, 10.7, 0.5; 47, 10.2, 0.5; and 47, 9.9, 0.5. Similarly, the sample size, mean, and SE for the cortical bone were: 46, 11.1, 0.5; 46, 12.0, 0.6; 47, 12.5, 0.5; 47, 12.5, 0.5; 47, 12.8, 0.5; and 47, 12.8, 0.5. (b) Mean indentation modulus of both jaws and bone types plotted separately to highlight significant three-way interactions. The difference in cortical bone and alveolar bone proper for each jaw is depicted and would not be predicted by the graphs in Fig 2a. The maxillary and mandibular cortical bone are significantly (p = 0.002) different for all 6 regions. The mandibular cortical bone and alveolar bone proper show a trend toward having different indentation modulus; however, this is not the case in the maxilla (e.g., region 3). Also, the mandibular alveolar bone proper has a higher indentation modulus than maxillary cortical bone, emphasizing the difference in the indentation modulus of the maxilla and mandibular jaw bone. The sample size, mean, and SE for the 6 regions (from 1 to 6) for each cell were as follows: for cortical bone in the mandible, (24, 13.7, 0.7), (24, 14.4, 0.7), (24, 14.5, 0.7), (24, 15.0, 0.7), (24, 15.1, 0.7), and (24, 15.4, 0.7); for alveolar bone in the mandible, (24, 11.5, 0.6), (24, 11.0, 0.7), (24, 11.5, 0.7), (24, 11.9, 0.6), (24, 11.7, 0.7), and (24, 11.3, 0.6); for cortical bone in the maxilla, (22, 8.5, 0.7), (22, 9.5, 0.8), (23, 10.4, 0.7), (23, 10.0, 0.7), (23, 10.5, 0.7), and (23, 10.2, 0.7); and for alveolar bone in the maxilla, (23, 8.4, 0.6), (23, 9.6, 0.7), (23, 11.2, 0.7), (23, 9.6, 0.7), (23, 8.7, 0.7), and (23, 8.5, 0.7).

	DISCUSSION

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Until recently, it has not been possible to measure the indentation modulus of small volumes of bone tissue. This is probably the reason for the lack of information in the literature on the indentation modulus of the alveolar process. The nanoindentation procedure to measure indentation modulus was chosen, due to the relative ease of specimen preparation, its high resolution in selection of the area of measurement, and its relatively nondestructive nature. Other methods to estimate the material properties of small volumes of tissue include scanning acoustic microscopy (Bumrerraj and Katz, 2001) and microtesting of bone specimens (Rho et al., 1993).

The theoretical basis and limitations of nanoindentation have been described in the literature (Oliver and Pharr, 1992; Hoffler et al., 1997; Hay and Pharr, 2000). While the assumption of isotropic behavior was made during ultra-low load indentation, nanoindentation procedures have been able to estimate the indentation properties of a variety of mineralized tissues (Kinney et al., 1996; Roy et al., 2001; Swadener et al., 2001; Tesch et al., 2001). It is important to polish the tissue specimens, since surface roughness will alter indentation property measurements at shallow depths (Bobji and Biswas, 1998). While not the focus of this study, with our polishing protocol, the average surface roughness of osteonal cortical bone, measured by atomic force microscopy (DualScope C-21, Danish Micro Engineering, Herlev, Denmark), ranged from 10-18 nm. Our indentation modulus values for cortical bone are similar to those reported in the literature (Rho et al., 1997). Based on the results of this study, we are able to provide quantitative data on the indentation modulus of bone within the alveolar process.

Our results demonstrated that the relative indentation modulus of the maxilla is two-thirds that of the mandible. The difference in the mean indentation modulus values, of ~ 3.5 GPa between the jaws, can have biologic and clinical implications. For example, a difference in stiffness of the bone tissue could have implications in surgical procedures performed in the two jaws. In addition, the indentation modulus of the posterior tooth-supporting bone was approximately 20% higher than that of the anterior tooth-supporting bone. The posterior teeth and supporting bone are likely to experience more strains during biting. This is especially the case in dogs, where the 2nd premolars do not come into direct contact in function. We were unable to detect a distinct gradient of indentation modulus for the 6 regions, while there was a trend toward a lower indentation modulus in the coronal region. Analysis of our data suggests that differences exist in the indentation modulus (a) between the maxilla and mandible, (b) between anterior and posterior tooth-supporting bone, and (c) between the alveolar bone proper and cortical bone within the alveolar process. However, simple interpretation of data is precluded because of statistical two- and three-way interactions among the jaw, region, and bone type. In addition, examination of the interaction effects demonstrated that (a) the higher value of cortical bone across the 6 regions was mainly due to the mandible cortical bone values, and (b) the higher indentation modulus for region 3 of the alveolar bone proper was mainly due to the peak observed in the maxillary alveolar bone proper.

It is clear that alveolar bone proper has a lower indentation modulus than cortical bone (Fig. 2a). However, when the data were separated by jaw for these two bone types, the cortical bone within the maxilla had a lower mean indentation modulus than the alveolar bone proper of the mandible (Fig 2b). There was a large variability (Table 1) in the data, and morphologic differences in bone types were apparent (Fig. 1b). Bone was inhomogeneous at a nanometer resolution. A limitation of this study and of many measurement techniques is that they cannot routinely account for this inhomogeneity.

Bone remodeling can be a potential reason for a change in tissue level properties of bone (Huja et al., 2006). While one study (Tricker et al., 2002) reported bone formation rates for the alveolar process in the mandible, no corresponding information exists about the maxillary alveolar process. In addition, there are no reports of bone formation rate in the 6 regions of the alveolar process that were used in this study. However, if a distinct gradient of bone formation rate did exist in the alveolar process, it could alter the region’s indentation modulus. In this study, we made indents to obtain an estimate of the indentation modulus as a reflection of regional bone tissue age. Given that the indentation modulus of the maxilla was lower than that of the mandible, it can be hypothesized that the bone formation rate of the maxilla is higher than that of the mandible. However, bone adaptation typically involves changes in bone mass, architecture, and material properties. While worthy of further examination, a simple and direct relationship between material properties and remodeling may not exist, since change in bone mass and architecture may precede changes in material properties.

Indents on alveolar bone proper were primarily located on non-osteonal lamellar bone. These indents were located at approximately 100-150 µm from the periodontal ligament. The Sharpey’s fibers from the periodontal ligament embedded in this alveolar bone; however, the exact extent of penetration and regional density of these fibers in alveolar bone proper in dogs is unknown. However, high-voltage electron microscopy demonstrates an extremely complex arrangement of the fibers within a mineralized bone matrix (Short and Johnson, 1990). We found that alveolar bone had a lower indentation modulus than did surrounding osteonal cortical bone. This difference could be attributed to new bone formation in the alveolar bone proper, and to the density of the fibers at the specific locations and the bone type, all of which were not directly measured in this study. Also, there are regions of the alveolar bone proper that do not contain fibers; however, regional differences in fiber density around rat teeth have been recorded (Johnson, 1992). This study could not detect significant differences in regional indentation modulus for the alveolar bone proper. It is possible that, while differences do exist, the study design and its resolution did not allow for detection. Coordinated morphologic and mechanical analyses of specific features of the alveolar bone proper are required before we can understand this complex interfacial structure.

Prior to this study, there was no estimate of the indentation properties of the bone in the alveolar process. This study provides evidence of differences in physical properties between the jaws, between anterior and posterior locations within the jaws, and within the alveolar process. Further studies on the functional significance of these differences in physical properties are required before we can understand the structure-function relationships in the alveolar process.

	ACKNOWLEDGMENTS

 
Funding from NIDCR Grant R03 DE015233 and the College of Dentistry is acknowledged.

Received January 4, 2006; Last revision October 26, 2006; Accepted November 7, 2006

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