Orthodontic mechanotransduction and the role of the P2X7 receptor

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Orthodontic mechanotransduction and the role of the P2X7 receptor Rodrigo F. Viecilli,a Thomas R. Katona,b Jie Chen,c James K. Hartsfield, Jr,d and W. Eugene Robertse Indianapolis, Ind Introduction: The P2X7 receptor plays a crucial role in bone biology and inflammation. Its main function is to promote necrotic tissue metabolism by ensuring a normal acute-phase inflammatory response. We used a mouse model to describe and compare orthodontic mechanotransduction in wild-type and P2X7 knockout mice. Methods: By using finite element analysis, mouse orthodontic mechanics were scaled to produce typical human stress levels. External root resorption, bone modeling, and bone remodeling were analyzed with fluorescent bone labels, Masson trichrome stain, and microcomputed tomography. Relationships between the biologic responses and the calculated stresses were statistically tested and compared between mouse types. Results: There were direct relationships between certain stress magnitudes and root resorption and bone formation. Hyalinization and root and bone resorption were different in the 2 types of mice. Conclusions: Orthodontic responses are related to the principal stress patterns in the periodontal ligament, and the P2X7 receptor plays a significant role in their mechanotransduction. (Am J Orthod Dentofacial Orthop 2009;135:694.e1-694e.16)

T

he sustained cellular activity associated with orthodontic tooth movement is well defined, but the initial mechanotransduction is obscure.1,2 The purpose of this study was to describe the role of the P2X7 receptor in the transduction of orthodontic loads into bone adaptation and external root resorption (ERR) responses. To experimentally determine the individual contributions of biologic and mechanical factors to orthodontic responses, both must be simultaneously controlled. Data on periodontal ligament (PDL) reactions to orthodontic treatment are restricted primarily to rodents. Extrapolating those results to humans requires appropriately scaled orthodontic force magnitudes. This prob-

From the Indiana University School of Dentistry and Purdue School of Engineering and Technology, Indianapolis, Ind. a Orthodontic resident, Biomechanics Laboratory, Department of Orthodontics and Orofacial Genetics. b Director and associate professor, Biomechanics Laboratory, Department of Orthodontics and Orofacial Genetics. c Associate professor and codirector, Biomechanics Laboratory, Department of Mechanical Engineering. d Adjunct professor, Department of Orthodontics and Orofacial Genetics. e Professor emeritus, Department of Orthodontics and Orofacial Genetics. Winner of the 2009 Milo Hellman Award. Supported by grant NIH/NIDCR-R01DE015767, the Jarabak Chair Endowment, and internal PhD Program funds. The authors report no commercial, proprietary, or financial interest in the products or companies described in this article. Reprint requests to: Rodrigo F. Viecilli, Biomechanics Laboratory, Department of Orthodontics and Orofacial Genetics, Indiana University School of Dentistry and Purdue School of Engineering and Technology, 1121 W Michigan St, DS 244, Indianapolis, IN 46202; e-mail, [email protected]. Submitted, April 2008; revised and accepted, October 2008. 0889-5406/$36.00 Copyright Ó 2009 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2008.10.018

lem was explored in a systematic review of optimal orthodontic force magnitudes.3 It is critical to distinguish between the loads (forces and moments) applied to a tooth by an orthodontic appliance vs the stresses that those loads produce in the structural components (roots, PDL, and bone) of that tooth. Stresses provide the stimulus for biologic responses. But, unfortunately, the simplest orthodontic force produces a complex stress field, even in the structures of an idealized single-rooted tooth. That stress field is immensely complicated by realistic force offsets, moments applied by the appliance, asymmetric or nonuniform anatomic shapes, or a multi-rooted tooth structure. Because of the 3-dimensional (3D) nature of stress fields surrounding the tooth, 1-dimensional or 2-dimensional (2D) (vs 3D) idealizations are essentially useless.4 When human-scale orthodontic load levels are applied to small rodents, the resulting structural stress range is orders of magnitude higher. The influence of tooth size on the load-stress relationship is not trivial, and it cannot be derived with simple calculations such as force divided by overall area. Tooth morphology is also an important complicating factor because it results in a great range of stresses. Similarly, the same orthodontic load levels applied to various human teeth—eg, canine vs mandibular incisor vs maxillary first molar—also produce different stress distributions and magnitudes. If a study aims to obtain a biomechanical animal model that is clinically relevant, it is necessary to first perform a finite element (FE) stress analysis on a human tooth subjected to a clinically realistic orthodontic load system to ascertain the associated stresses in its 694.e1

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structures. Then, with an FE model of the mouse tooth, the orthodontic load magnitude necessary to approximate the stress magnitude range in the human tooth can be determined. Thus, rather than matching orthodontic load levels, physical matching must be done on the calculated stress levels and directions. To match stress directions, the complex shapes of rodent teeth require histologic sectioning planes that consider the 3D nature of stresses and strains. It is incorrect to assume that compression and tension areas in the PDL are always along the line of action of the force, because, typically, tooth displacements and rotations occur in all 3 planes of space. Therefore, the directions of compression and tension must be calculated. Stress analysis with FE modeling is the engineering state-of-the-art method for defining the magnitudes and directions of compression and tension in complex structures. It is a reliable tool for determining the optimal orientation for histologic sections. Because of biomechanical principles familiar to orthodontists, the ideal root sectioning planes to look for in a mouse tooth should approximate the stress distributions associated with a single-rooted tooth (ie, a canine) tipping in 1 plane. If such a match is found, the other mouse roots become irrelevant. There are also biologic complications involved in controlling orthodontic experiments. Most studies are performed on animals with diverse genetics, resulting in highly variable biologic responses that are difficult to partition into biologic and mechanical variability. Despite few attempts to establish scientifically sound relationships between mechanical stimuli and orthodontic responses, the specific relationships are elusive, with controversies on the specific roles of mechanics and biology in individual variability.5,6 A clinically relevant biomechanical mouse model of orthodontic treatment was developed to overcome these difficulties and meet the explained requirements. Mice were used because, among other advantages, they share 99% of their genes with humans; thus, they are good animal models for studies of human biology. Mice from an inbred strain share 99.99% of their genetic material. By using a single strain, the role of genetic variation in the dispersion of data is practically eliminated. Because inbred mice have the same tooth and bone morphology, they can be modeled by a single FE model.7,8 By using stress calculations from an FE model of a human canine subjected to an orthodontic load, it is possible to calculate the orthodontic load that would cause similar peak stress levels in mice. Furthermore, with the mouse FE model representing all mice, it is possible to localize histologic planes in which the stress magnitudes and direc-

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tions approximate those of the human PDL compressive and tensile stress zones, and cut the roots consistently in the same spatial position. Knock-out (KO) mice are invaluable for studying the role of individual genes in eliciting a biologic response. KO mice differ from the inbred wild-type (WT) strain essentially by the deletion of a single (knocked-out) gene. In this study, the role of the P2X7 receptor gene (P2X7R) in the initiation of orthodontic tooth movement was studied. A previous study showed that the mice used in this study (P2X7R KO and C57B/6 WT) have similar dentoalveolar morphology and can, therefore, be represented by the same FE model.7 The P2X7 receptor is an adenosine triphosphate (ATP)-gated ionotropic channel and a key mediator of inflammation and bone adaptation responses.9 It can be activated (opened) after binding to extracellular ATP, which is a danger signal from cells under mechanical stress.10-12 Opening the channel causes the accumulation of intracellular calcium and the release of inflammatory mediators such as PGE2, IL-1a, and IL-1b, all fundamental in the control of bone physiology.13-17 Recent research has shown that this receptor has a major role in the metabolism of apoptotic and necrotic tissues. After mechanical trauma, damaged cells release ATP that leads to the activation of P2X7 in macrophages and other cell types, which in turn release IL-1 cytokines.18 This is a general transduction mechanism of mechanical stimulus into a biologic response. The released cytokines affect nonbone-marrow derived cells, which, in turn, release chemo-attractants for neutrophils and lymphocytes.19 The neutrophils can act quickly to eliminate apoptotic cells and prevent further necrosis. P2X7R KO-derived macrophages do not release IL-1 in response to ATP, resulting in an attenuated acute inflammatory response.13,20 Normal function of the P2X7R results in an optimal acute-phase response that extends the life cycle of neutrophils.21 An inefficient acute response can lead to an overwhelming chronic response: massive macrophage infiltration, abundant apoptotic and necrotic cells, and generalized tissue damage.13 P2X7R activation has already been shown to be an important mechanism in long-bone mechanotransduction: P2X7R mice tibia have reduced sensitivity to mechanical loading because of decreased secretion of PGE2.16 Thus, the P2X7R is an ideal candidate for mediating the orthodontic response, which involves metabolism of an associated necrotic-type tissue (hyalinized PDL tissue) and bone modeling or remodeling.22-27 These processes are mediated by the P2X7R in other physiologic systems. Moreover, ERR, an undesirable side effect of tooth movement, has been associated with the presence of macrophages in several

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Fig 1. Flowchart of the development of the biomechanical orthodontic mouse model to study mechanotransduction.

studies.26-28 In addition, a strong association has been previously reported between IL-1b (active form depends on P2X7R activation) and ERR in animal and clinical studies.29,30 The P2X7R also has direct clinical relevance because there are many functional human polymorphisms in the gene that can induce different levels of affinity of these receptors to ATP. One polymorphism, a null allele that occurs in 2% of the white population, is especially important because it results in reduced expression of the ion channel by 50%.31 It is hypothesized that variability in the expression of the P2X7R is an important factor in the individual variation to applied orthodontic loads when the mechanical environment is controlled. MATERIAL AND METHODS

The following steps (Fig 1) were carried out to develop the biomechanical mouse model of this study. 1.

2.

3.

An FE model of an average human maxillary canine subjected to orthodontic tipping was built to calculate the mechanical environment in the plane of maximum (generally tension) and minimum (generally compression) principal stresses. A prestudy was run to determine the line of action of the force produced by a spring ligated between a mouse maxillary left first molar and the central incisor. A mouse maxillary first molar FE model was built. By using the orthodontic force direction established in 2 above, possible histologic cutting planes were established so that the first and third principal stress

4.

5.

directions resembled the previously determined (in 1 above) human plane. Calculated principal stress directions were used to orient the 3D position of the cutting planes. Then the in-vivo mouse force magnitude that would allow appropriate comparisons between human and mice stress magnitudes in the histologic sections was calculated. The histological planes for root resorption (higher stresses) and bone formation analyses with bone labels (lower stresses) were determined. A special superelastic nickel-titanium spring that delivered the appropriate force magnitude was produced.

This animal study was approved by the Institutional Animal Care and Use Committee of the Indiana University School of Dentistry. Data were obtained from 86 male mice (55 C57B/6 WT, 31 P2X7R KO). The 16-week-old (6 3 days) mice were distributed into 7 groups: WT controls (19), WT controls 1 bone labels (9), WT force (18), WT force 1 bone labels (9), KO controls (12), KO force (10), and KO force 1 bone labels (9). The mice that received no bone labels were killed at 17 weeks and 3 days of age, and the labeled mice were killed at 17 weeks and 4 days for bone formation analysis. The animals were randomized, and the examiner (R.F.V.) was blinded with regard to the KO or WT strain by a coded system until the analysis was complete. The WT mice were purchased from Taconic Farms (Germantown, NY). The KO mice were obtained from a colony developed by the Orthopedic Biomechanics Laboratory at Indiana University School of

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Fig 3. A, Computer-aided design model of the jaw segment and a cut showing the PDL, the adjacent premolar socket, and the lateral incisor root; B, FE model.4 Fig 2. A, Segmentation, and B, 3D reconstruction of the mouse molar; C, isolated trabecular bone sphere for bone morphometry (reprinted from Viecilli et al8 with permission from Taylor & Francis [http://www.informaworld. com]); D, FE model of the mouse molar.7

Medicine. After arrival, the animals were allowed at least 1 month of acclimation to compensate for their different origins. They were killed by inhalation of carbon dioxide, and, after decapitation, the heads were washed and prepared for histologic and microcomputed tomographic (micro-CT) analysis. The mouse heads were put in cold neutral buffered formalin solution and stored in a refrigerator for 24 hours. Sections of the maxillae were extracted after dissection and fixed in the neutral buffered formalin solution in the refrigerator for an additional day. Then, they were placed in 70% ethanol solution and kept at ambient temperature. The sections were coated with paraffin to prevent dehydration and scanned in a micro-CT instrument (model 1072, Skyscan, Kontich, Belgium). Reconstruction of the maxillae in 2D transverse slices was performed by using NRecon (Skyscan) software. All scans and reconstructions were standardized with optimized settings as described previously.8 The reconstructed data were imported into Mimics software (Materialise, Aarstelar, Belgium), and the left molars were digitally separated from the PDL and the bone for quantification of tooth volume and move-

ment (Fig 2, A and B); 3D tooth movement was also quantified by measuring the minimum distance between the crowns in the nonlabeled force groups. CT Analyzer (Skyscan) software was used to perform 3D bone morphometry. The bone used for analysis was sampled from a 0.5-mm diameter sphere, chosen so that it was equidistant to all roots, and located at the midplane between the mesial root apex and the furcation, parallel to the occlusal plane (Fig 2, C). To segment the bone from the rest of the tissue in this spherical sample, a global gray value threshold was standardized for all specimens. Bone mineral density and Hounsfield unit calibrations were performed by using scans of water and hydroxyapatite rods (CIRS, Norfolk, Va) of dimensions similar to the specimens and known mineral densities of 0.75 and 0.25 g per cubic centimeter. Details of this methodology were described previously.8 The left maxillary molar of a mouse was used to construct the FE model. The segmentation and meshing were performed with Amira software (Visage Imaging, Dusseldorf, Germany). Bone, PDL, and tooth were segmented and smoothed to guarantee a mesh free of self-intersecting units. The tissue surfaces were meshed with 4-noded tetrahedrons. The resulting model had strong anatomic fidelity, keeping the characterized complex internal trabecular structure as part of the model (Fig 2, C). Nodes of each material were shared at the interfaces. The model was imported into IDEAS 10 (Structural Dynamics Research, Milford, Ohio) and

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Fig 4. A, Mouse spring placement procedure with custom prop (reprinted from Viecilli et al8 with permission from Taylor & Francis [http://www.informaworld.com]); B, spring in place; C, sagittal and D, occlusal views of the line of action of the force.

saved in IGES format to be imported into ANSYS 10 (Ansys Inc, Canonsburg, Pa), the FE analysis (FEA) software, where elements were converted into approximately 200,000 SOLID45 tetrahedrons. The material properties used were obtained from rodent tooth displacement experiments on autopsy specimens.32 The PDL was modeled as a nonlinear (bilinear) isotropic material. The model is shown in Figure 2, D. For the human FE model, a maxillary segment containing a maxillary right canine was constructed by using computer-aided design software (Dassault Systems, Concord, Mass). The computer-aided design file was then imported into ANSYS, where it was meshed with approximately 200,000 10-noded tetrahedrons for FEA. The material properties were also based on previously determined human canine displacement experiments, and the PDL was modeled as a nonlinear (bilinear) isotropic material.33 The modeling process is shown on Figure 3. A thorough stress analysis of this model was described previously.4 The boundary conditions for both models were fixed by assigning zero displacement in all directions to the mesial, distal, and apical bone surfaces of the model. To determine a clinically translatable force magnitude for the mouse maxillary first molar, a simulation was conducted by using the human FE model. A relatively high tipping force (1.2 N) was applied to the bracket to produce simple distal crown tipping of the canine. (An antirotation first-order couple was also

applied.) The mean nodal principal stress levels in all regions of the PDL were calculated to be –35 to 25 KPa. Then, orthodontic force was applied iteratively in the mouse FE model until this stress range was approximated in the mouse PDL. With 0.03 N (about 40 times smaller than the 1.2-N human force), the mean principal stress range in the total mouse molar PDL was –25 to 35 KPa. Thus, the appropriate force magnitudes to compare human with mouse responses were established despite the inherent morphologic differences between the teeth. Each of the 3 mouse maxillary first molar roots has a different stress magnitude range; generally, smaller roots experience higher magnitude stress ranges than do larger roots. Thus, by choosing different roots for the analysis of bone formation and root resorption, we can observe the effects of clinically relevant small and large stress magnitudes. A custom mouse closed-coil spring made of thin 0.003-in superelastic nickel-titanium wire, with a lumen of 0.019 in, was designed and then produced by G&H Wire (Greenwood, Ind). Spring measurements were made by using a 0.01-N resolution transducer (Orthomeasurements, Fairfield, Conn). To obtain a 0.03-N force with 2 mm of activation, the springs were cut to 3 mm of length and then tied with metallic ligatures to the maxillary left molar and incisor. At the incisor, it was also secured with resin composite. The spring placement procedure and the final average 3D line of action are shown in Figure 4. The FE calculated

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Fig 5. A, Occlusal view of idealized human canine PDL stress directions during tipping. The small red and white arrows represent maximum compressive and tensile stress directions, respectively. The line of action of the force (large white arrow) and the maximum PDL stress directions are coincident. B and C, Apical views of the mouse PDL stress directions during tipping, with the corresponding root identification; D, occlusal view of mouse molar. (DB, distobuccal; DP, distopalatal; M, mesial). Histologic cutting plane for ERR analysis (dashed black line in B, C, and D) follows the maximum compression PDL stress direction (red arrows), not the line of action of the force (white arrow in D).

maximum compression directions are not coincident with the line of action of the force because the tooth underwent 3D rotations and translations. From an engineering standpoint, maximum 3D compression and tension can be determined by calculating the 3 principal stresses, each acting in its specific mutually orthogonal direction. The 3 principal stresses are derived from the equivalent general state of stress (ie, 3 shear and 3 normal components) by rotating the local coordinate system used to describe them so that all shear stresses disappear, leaving only the 3 principal (normal) stresses. The 3 principal stresses are all tensile or compressive, or a combination. Histology

After the teeth from the control and force WT and KO groups were scanned in the micro-CT instrument, histologic sections were prepared to quantify the incidence of root resorption, assess the extent of hyalinized PDL tissue, identify the cellular types involved in its resorption, and measure bone formation.

The choice of root and section plane for the ERR study was determined by identifying the direction and magnitude of the FE calculated maximum compressive stress in the mouse PDL (ie, the third principal stress). This was localized to the PDL of the distobuccal root, the smallest of the 3 roots. In the PDL of this root, the compression magnitude is smallest and largest near the apex and furcation, respectively. This maximum compression level is equivalent to that in the human canine FE model. Figure 5 illustrates the rationale for the cutting-plane spatial position. A more detailed mechanical description of stress directions was given in a previous FE study.4 For bone-formation analysis, the bone-label groups were injected peritoneally after 2 days (calcein green, 30 mg/kg) and 9 days (alizarin complexone, 50 mg/ kg) of applied force. The distopalatal root was chosen for this examination because it had tipping displacement with stress levels consistent with those of human canine movement but of lower magnitude if compared with the distobuccal root, chosen for ERR analysis. For clinical reference, in the PDL of the distopalatal

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Fig 6. Static sections of the 3D FE model showing calculated initial (contour) and final (solid) positions of the teeth on the A, ERR analysis section plane and B, bone formation analysis section plane. Because the sections followed stress directions, the tooth has negligible out-of-plane movement. This is evidenced by little change observed in tooth and root shapes between the initial and final positions in these planes.

root, the maximum tensile stress (ie, the first principal stress in this case) would be equivalent to the calculated maximum tension in the human canine tipped with a 0.5N force. The cutting-plane position for bone formation was determined according to the maximum tensile stress directions in this PDL. The cutting plane used for boneformation analysis was approximately parallel to that for the ERR assessment, passing through the long axis of the distopalatal root. The movement tendencies predicted by the FE method for bone formation and the ERR histologic sectioning planes are shown in Figure 6. The maxillae were embedded in methylmethacrylate and slowly ground by using a series of papers up to 1200 grit on an ExaKt system (ExaKt Technologies, Oklahoma City, Okla) to obtain a section as close as possible to the plane of histologic analysis, traced on the acrylic surface as determined by the FE method. The sections were polished down to 8 to 10 mm (nonbone-labeled groups for ERR analysis) and 20 mm (bone-labeled groups) with papers of 2500 and 4000 grit. Thin mineralized sections were produced to preserve nuclear and cytoplasm shape and improve cell identification (nonlabeled groups). In the labeled groups, the thin 20-mm sections prevented layered superimposition of bone formation from different planes, which could produce an illusion of excessive bone modeling or remodeling due to layer superimposition. The nonbonelabeled sections were then deplasticized by using acetone and xylene and stained with a modified Masson trichrome stain protocol optimized for methylmethacrylate. Each distobuccal root was observed and photographed under a microscope with 75 times magnification. A standardized grid (about 35 3 35 mm) was superimposed on each figure by using ImageJ (public

domain NIH image-editing software). The compressive side of the PDL of the distobuccal root was then divided into 3 equal regions on the basis of the FE data: high stress (near furcation), medium stress, and low stress (near apex). The number of squares in a region that contained resorption cavities adjacent to the root was divided by the total number of squares (about 10) in that region to obtain the final resorption index. Similarly, the hyalinization score was obtained by dividing the total number of squares that contained necrotic tissue by the total number of squares in the compressive side of the PDL. These methods are higher-resolution modifications of a previously developed protocol.34,35 Cell types were identified with the assistance of an experienced pathologist. The PDL-bone interface of the distopalatal root was chosen for bone formation analysis; interlabel distance in the socket of the distopalatal root was quantified by averaging the distances between the centers of the calcein green and alizarin complexone labels at 3 locations. The sampling regions were determined by orienting the long axis of the tooth vertically, tracing parallel lines at the average cervical bone level and the apical contour of bone, and then dividing the interval into 4 equal segments (Fig 7). The 3 FE calculated nodal stresses (n1, n2, and n3) and the 3 measured bone interlabel distances (d1, d2, and d3) shown in the figure were averaged to obtain stress (mean nodal stress in each region) and mean bone interlabel distance), respectively. This was performed with ImageJ by calibrating 250-mm bars according to the pixel size of the photograph. All data from KO and WT specimens, in each region, were averaged for each type. The values were recorded for statistical regression.

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Fig 7. Method to relate FE-computed maximum PDL tensile stresses (left) to histologically measured interlabel bone distances (right). In-plane maximum tensile stresses, acting horizontally, decreased from regions 8 to 5. The pattern of PDL deformation was correctly predicted by the FEA. In bone measurements of the picture, d3 would be discarded because it is in a remodeling cavity, and only d1 and d2 would be averaged. The scale shows the maximum tensile stress values in pascals.

Statistical analysis

The Mann-Whitney nonparametric test was used to compare single measurements (tooth movement, hyalinization score, tooth volume, and ERR score) between the 2 mouse types. Kruskal-Wallis analysis of variance (ANOVA) and Tamhane post-hoc tests were used to compare ERR in different regions of the distobuccal root in each mouse type. More conservative nonparametric tests (and interquartile range as a measurement of dispersion) were chosen because the assumption of a Gaussian distribution of data was questionable (n\20 in each group). In some cases, the data clearly did not follow a Gaussian distribution (eg, ERR control section scores were skewed toward 0). This justifies the use of medians as the measurement of central tendency. Linear regression was conducted to assess the relationship of bone formation with stress after a natural logarithmic transformation. Analysis of covariance (ANCOVA) was used to compare slopes of bone formation vs stress lines in KO and WT mice. The significance level for all analyses was set at 5%. Reliability analysis was performed by repeated measurements of 10 randomly chosen specimens or data points for each method, at least a week later, and calculating the Pearson correlation. The reliability coefficients of the measurement methods of root volume, bone morphometry, bone interlabel distances, and hyalinization or root resorption scores were 97%, 94%, 92%, and 89%, respectively.

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Fig 8. Comparison of tooth hyalinization scores in treated KO and WT mice (P \ 0.001). RESULTS

Ten days after the force was applied, the KO mice had about 8 times more (P\0.001) hyalinized (necrotic PDL) tissue than did the WT mice (Fig 8). There was no difference in the incidence of ERR between the regions for all control animals across both strains. For the WT force group, ERR was 1.8 times higher (P 5 0.002) and 3.7 times higher (P \ 0.001) in the area of high stress compared with medium and low stress, respectively. A similar pattern was noted for the KO force group: respective scores were 4.7 times (P 5 0.001) and 3.5 times (P 5 0.002) higher (Fig 9). When comparing ERR between KO and WT mice in different regions, the Kruskal-Wallis test determined that there was a difference, and that ERR was elevated in the force groups. The post-hoc test showed that the significant difference was in the region of the highest PDL compressive stress, with 27% more (P \ 0.02) root resorption craters in the KO mice (Fig 10). There were no significant differences between the WT and KO controls in any region, or in region 2 or 3 in the treated mice. ERR was rarely observed in the PDL tension areas, and there was no detectable hyalinized tissue associated with these few lesions. The few observed lesions were shallow compared with those in PDL compressive zones. To confirm that the 2D root resorption data were consistent with the 3D assessments, tooth volumes between strains in the control and treated animals after the 10-day experimental period were compared. Figure 11 illustrates the approximately10-mm scan resolution of the micro-CT scan and how it can detect resorption lesions. The test showed no difference in tooth volume between the WT and KO control animals after the experimental period. However, tooth volume was 8%

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Fig 9. Comparison of ERR proportion score by region (Fig 11) in each strain of treated mice. Significant differences in the WT and KO mice were found between regions 1 and 2 (P 5 0.002 and P 5 0.001), and 1 and 3 (P \ 0.001 and P 5 0.002).

Fig 10. Comparison of ERR proportion score by type in the region of high stress (P \ 0.02).

Fig 11. Micro-CT root resorption detection: A, histologic section of the distobuccal root of a KO force group mouse; B, micro-CT reconstructed section of the same region; C, its segmentation. Such segmentation of all sections allows 3D rendering of the structures and volume estimation. D, Visualization of resorption cavities (c) and the area of hyalinized PDL tissue (Hz).

larger (P 5 0.002) in the treated WT mice compared with the treated KO mice; this is consistent with the elevated ERR in the KO animals (Fig 12). Histologic sections showed differences between the KO and WT mice. Typically, many neutrophils, characterized by lobular-shaped nuclei and small cytoplasm, were found accompanying a mass of macrophages that accumulated at the border of the hyalinized zone in the WT mice (Fig 13). In many WT sections, the small neutrophils, identified by their characteristic nuclear shape, were active enough to penetrate the narrow hyalinization zone to resorb necrotic tissue. The PDL space in the KO mice was characterized by cells of larger cytoplasmic area and large distances between regular-shaped nuclei,

suggesting that the predominant cells were macrophages. Neutrophils were rare in the KO mice. The macrophages did not penetrate the compressed hyalinized tissue, although they accumulated at its periphery and failed to remove the hyalinized tissue as effectively as the cells in the WT mice. Severe ERR and undermining bone resorption occurred in the regions adjacent to the cellular mass. The within-mouse-type comparison of alveolar bone morphometric characteristics of control and treated animals showed that orthodontic force causes significant bone resorption in the alveolar process and at surfaces far from the PDL/bone interface (Table I). This is

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Fig 12. Tooth volume by type in control and force groups. A significant difference was found only after force application, confirming that KO mice had more ERR (P 5 0.002).

a manifestation of the regional acceleratory phenomenon. It is especially evidenced by statistically significant decreases in bone mineral density and bone volume fraction, and an increase in bone surface-volume ratio in the WT and KO mice. Table I shows that the observed regional acceleratory phenomenon caused dramatic catabolic changes in bone morphology and density far from the PDL interface in both types of mice. Distopalatal socket interlabel bone distances were used to evaluate the relationship between bone formation in each mouse type and the associated FEcalculated PDL maximum tensile stress. Regions 5 through 8 were used for analysis because they had no evidence of bone formation (growth) in the controls (Fig 14) and because their maximum tensile stress directions were aligned with the plane of the histologic section. A typical histologic section with corresponding stress calculation is shown in Figure 7. Although the original curve was nonlinear, a natural logarithmic transformation of stress values allowed linear regression with bone formation measurements. All coefficients were statistically different (P \ 0.006) from 0, showing that a logarithmic function was statistically adequate to represent the relationship between bone formation and PDL stress (Fig 15). This showed that, in both mouse types, the bone-formation rate initially increased with stress but, after excessive tension, started to decrease and stopped completely. No statistically significant difference (P . 0.2) was found between bone formation in the 2 mouse types. A previous study found no differences in the bone morphometry of untreated P2X7R KO and WT control

Fig 13. Example sections of A and C, WT mouse, and B and D, KO mouse histology. C and D are magnified highstress regions of A and B, respectively. Note the increased presence of neutrophils (lobular-shaped nuclei) in the WT mouse section.

mice.7 However, with force application, our results indicated a significant difference in the structure model index of the 2 mouse types (Table II). The WT mice had a slightly higher structure model index (P \ 0.05) with a minor tendency toward a rod-like trabecular structure. The data showed that KO mice had slightly less resorption activity in trabecular bone distant from the PDL interface, suggesting that the regional acceleratory phenomenon was slightly less pronounced when P2X7R was absent. After 10 days of force application, no statistically significant differences (P . 0.9) were observed in the amounts of tooth movement of each mouse type. DISCUSSION

The difference in the extent of hyalinized tissue after 10 days indicated that KO mice are less efficient at removing necrotic tissue. These data can be explained

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Comparison of bone morphometry parameters of alveolar trabecular bone before and after application of orthodontic forces in WT and KO mice

Table I.

Control Measurement BMD (g/cm3) WT KO BV/TV (%) WT KO BS/BV (/mm) WT KO Tr.Th (mm) WT KO Tr.Sp (mm) WT KO Tr.N (/mm) WT KO Tr.Pf (/mm) WT KO SMI WT KO DA WT KO FD WT KO

Force

Median

Range

Median

Range

P value

0.78 0.91

0.15 0.20

0.54 0.57

0.14 0.14

\0.001 \0.001

45.0 45.0

10.0 7.0

26.0 25.0

10.0 12.0

\0.001 \0.01

50.0 49.0

15.0 16.0

64.0 74.0

20.0 16.0

0.001 0.008

0.08 0.09

0.02 0.03

0.07 0.06

0.02 0.01

0.001 0.01

0.13 0.15

0.02 0.05

0.16 0.16

0.02 0.09

0.008 0.87

5.6 5.1

1.1 1.4

3.8 4.1

1.6 1.2

\0.001 0.19

3.0 1.0

10.0 14.0

14.0 10.0

12.0 10.0

\0.001 0.17

1.0 1.8

0.8 0.5

2.0 1.8

0.5 0.4

\0.001 0.22

2.2 2.1

1.3 1.1

2.9 2.1

2.3 1.1

0.57 0.29

2.24 2.12

0.07 0.10

2.08 2.11

0.08 0.11

\0.01 0.11

BMD, Bone mineral density; BV/TV, bone volume fraction; BS/BV, bone surface-volume ratio; Tr.Th, trabecular thickness; Tr.Sp, trabecular separation; Tr.N, trabecular number; Tr.Pf, trabecular pattern factor; SMI, structure model index; DA, degree of anisotropy; FD, fractal dimension.

by the lack of ATP-driven stimulation of IL-1 secretion via the P2X7 receptor, leading to a poor initial neutrophil response to remove necrotic tissue and chemically mediate its metabolism. This finding agrees with previous studies13-21 on the role of this receptor in necrotic tissue metabolism. Both KO and WT mice had some residual hyalinized tissue after 10 days of force application. This must be considered when interpreting our results, especially the lack of a difference between WT and KO mice in the amount of tooth movement. It was possible to extrapolate the mechanical threshold for clinical hyalinization from the FEA coordinated histology. Assuming that little necrotic tissue resorption took place in the KO mice (Fig 8) and that hyalinization occurs equally in areas of maximum PDL compression

Fig 14. Example section of a labeled control WT mouse distopalatal root. Note normal bone apposition around blood vessels. New bone is also observed on the palatal periosteal surface and near the apex, suggesting slight eruption during the experimental period. Because of this interference, the apical regions (2 apical region 5 points and region 6) were eliminated from linear regression analysis in the treated mice.

in man and mouse, then FEA shows that a compressive stress higher than about 10 KPa produces PDL hyalinization. In the human maxillary canine, the computed minimum bracket load magnitudes to cause this stress level are approximately 0.4 N for tipping and 1.2 N (plus the necessary moment) for translation. Figure 16 illustrates the compressive stress-hyalinization relationship. These data are relatively consistent with values reported by Reitan,22,36 who observed hyalinization in maxillary incisors (smaller teeth), with tipping forces as low as 0.3 N. However, these results demonstrated that PDL hyalinization occurred at stress levels significantly lower than vascular blood pressure. The association between vascular blood pressure in the PDL and hyalinization is a popular theory in orthodontics. However, our results demonstrate that this theory fails to consider the mechanotransduction signaling that leads to cell apoptosis and necrosis, which can begin by mechanical stimulation of the cells when blood supply is still

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Table II. Comparison alveolar trabecular bone morphometry after force application in WT and KO mice WT

Fig 15. Linear regression plots of the natural logarithm of maximum principal stress [Ln (stress)] vs bone interlabel distance in the distopalatal root of WT and KO mice. The outer lines in each regression are the limits of the 95% confidence interval.

present. These preliminary data indicate that coordinated histology and FEA is a promising strategy to establish scientific evidence for optimal orthodontic load magnitudes in future experiments in which the time course of tooth movement will be studied in more detail. Histology showed that severe ERR occurred mainly in areas with dense accumulations of leukocytes adjacent to high compression zones. This suggests that, in the PDL, dense inflammatory cell accumulation leads to tissue destruction, as seen in general inflammatory processes. In the WT mice, despite considerable resorption of the hyalinized tissue in areas of medium stress, less ERR was found. The statistically significant differences, up to about 4 times, in the incidence of ERR in the higher stress regions in WT mice provide strong evidence that localized elevation in PDL stress leads to a dramatic increase in ERR. This is an important finding because this is the first study to control genetics and the mechanical stress in the histologic sectioning plane. According to this study, the variable with the largest impact on ERR was the PDL compressive stress magnitude (all principal stresses were compressive in the PDL ERR zone). Areas of high stress had 4 times or more ERR compared with areas of low stress in both mouse types. In the areas of higher stress, the lack of P2X7R (and consequently IL-1b) caused about a 20% increase in ERR in the KO mice. The difference in ERR between mouse types occurred at the hyalinized areas where the stress was highest after force application, and where most cell accumulation occurred. Near the furcation region, there is an interface between a high compressive stress region at the lateral surface of the alveolar crest and the relatively constraint-free region immediately occlusal to

KO

Measurement

Median

Range

Median

Range

P value

BMD (g/cm3) BV/TV (%) BS/BV (/mm) Tr.Th (mm) Tr.Sp (mm) Tr.N (/mm) Tr.Pf (/mm) SMI DA FD

0.54 26.0 64.0 0.07 0.16 3.8 14.0 2.0 2.9 2.08

0.14 10.0 20.0 0.02 0.02 1.6 12.0 0.5 2.3 0.08

0.57 25.0 74.0 0.06 0.16 4.1 10.0 1.8 2.1 2.11

0.14 12.0 16.0 0.01 0.09 1.2 10.0 0.4 1.1 0.11

0.58 0.61 0.27 0.31 0.94 0.19 0.14 \0.05 0.41 0.23

BMD, Bone mineral density; BV/TV, bone volume fraction; BS/BV, bone surface-volume ratio; Tr.Th, trabecular thickness; Tr.Sp, trabecular separation; Tr.N, trabecular number; Tr.Pf, trabecular pattern factor; SMI, structure model index; DA, degree of anisotropy; FD, fractal dimension.

the crest. This makes leukocyte accumulation viable. This constraint-free region probably facilitates diapedesis from blood vessels to the area of high compression, resulting in resorption of root, bone, and hyalinized tissue. From a clinical perspective, this observation explains why ERR occurs primarily at the apex of singlerooted teeth: the apex is relatively free of constraints and adjacent to an area of high compressive stress (the lateral compressed zone near the apex). Compared with the equilibrated macrophage and neutrophil infiltration for orthodontically stimulated WT animals, KO mice had a massive macrophage response. This suggests that the PDL reaction in KO mice is analogous to an inefficient acute-phase response associated with inadequate levels of P2X7 and IL-1b. An inefficient acute-phase response leads to exacerbated macrophage infiltration, with slow resolution of necrotic hyalinization and excessive tissue damage, characterized by ERR. The association of macrophages with ERR was reported previously, but the role of neutrophils has been overlooked in most studies, which typically analyze later stages of tooth movement.25,37-40 An interesting contrast in the orthodontic catabolic process in the KO mouse is that more ERR was observed without increased bone resorption away from the PDLbone interface. Lack of IL-1b contributes to less bone resorption away from the PDL-bone interface, consistent with the role of this cytokine in bone physiology. However, it also contributes to more catabolic activity (tissue damage) in the periphery of the necrotic areas of the PDL, consistent with the role of P2X7 and IL-1b in necrotic tissue metabolism. This explains why the lack of this cytokine can result in less bone resorption (away

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Fig 16. A, Histologic section of a KO mouse. Large area of hyalinized tissue remains on the compression side of the distobuccal root. B, Corresponding section of the FE model. Note that the FEA correctly predicted the deformation of the PDL. Hyalinization occurs at PDL stress levels . about 10 KPa; this can be smaller than vascular blood pressure.

from PDL interface) but more bone and root resorption in areas adjacent to necrotic tissue. The decreased bone resorption away from the necrotic areas (undermining resorption) in the KO mouse, associated with a lack of IL-1b, contributes to a vicious circle, leading to denser accumulation of macrophages and more ERR. Although many investigators have suggested associations between genetic factors and ERR, the mechanical environments were inadequately controlled or assumed to be trivial after application of a known force.29,30,35,41-43 This study is the first to appropriately control the mechanical environment and select histologic sectioning planes based on FEA stress computations. Thus, it was demonstrated that the influence of genetics on ERR can be independent of mechanical and anatomic factors. In clinical terms, hypothetically, patients with the same root morphology, treated with identical orthodontic mechanics, might have dissimilar ERR responses as a direct consequence of genetic differences. The lack of statistical difference in ERR between KO and WT mice in areas of low (\10 KPa) PDL compression suggests that keeping orthodontic loads at the lowest effective level could be a viable strategy for minimizing ERR in all patients, particularly those being treated with the new anti-inflammatory drugs that block the P2X7 receptor or with polymorphisms that cause reduced P2X7 function. Although the concept of light therapeutic loads is appealing, it might not always be practical because orthodontic loads are relatively light mechanics superimposed on heavy functional and postural loads, which might increase after tooth displacement because of altered occlusion.24,29 In addition, commonly used appliances often produce uncontrollable statically indeterminate force systems. Avoiding ex-

cessive occlusal trauma and using statically determinate force systems when possible are probably as important as using low load mechanics. An important follow-up to this study would be to study the rate of sustained tooth movement in WT and KO mice. Cytokines could affect the initiation of and sustained tooth movements differently. For instance, patients homozygous for allele 1 of IL-1b (diminished levels of IL-b) had an accentuated ERR response attributed to slow initiation of tooth movement.29 However, in a recent clinical study, patients homozygous for allele 1 of IL-1b had increased rates of sustained tooth movement compared with other patients.44 This inconsistency might be biologically valid and related to the timing of the study (the initial presence of necrotic tissue), or it might reflect an uncontrolled mechanical environment in the clinical setting. Relative to current scientific literature, our data suggest a differential role for cytokines in the initiation and sustained tooth-movement phases; this is possibly associated with the period of necrotic tissue clearance. The controversy about the role of cytokines in initiating and sustaining tooth movement could be resolved by varying the timing of force application, mechanical environment, and sampling sites in this mouse orthodontic model. For fluorescent-label bone-formation analysis, the distopalatal root was used because it had lower (\14 KPa) PDL maximum tensile stress magnitudes, and a limited nonlinear relationship was shown between stress magnitudes and bone apposition. Excessive tension did not result in bone apposition (Figs 6 and 14). Little or no osteoid was observed on the relatively high-tension side of the distobuccal root, the smaller root originally chosen for ERR analysis, because of

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small cupping deformation of the buccal alveolar plate. This is difficult to observe histologically but can be exaggerated for clear observation in the FE model. This heretofore unreported phenomenon could be observed by matching the histologic sections with the FEA results. It demonstrates that orthodontic tooth movement can lead to bone modeling far from the PDL-bone interface. Finally, the remnants of hyalinized tissue after 10 days probably affected the comparisons of bone formation and tooth movement because their limiting factors are hyalinized tissue and bone resorption. P2X7 receptor activation is a possible pathway for the release of PGE2; however, no statistically significant effects in bone formation and tooth movement were observed in this study. It would not be possible to initially predict the timing of hyalinization clearance because tooth and PDL morphology are different in humans and mice, and hyalinized tissue metabolism can (as demonstrated) vary with genetics. Thus, the lack of demonstrated statistical differences in bone formation and tooth movement does not mean that significant effects would not become apparent with longer treatment times. Fig 17. Structures around the high-tension PDL of the distobuccal root. Note the stretched fibroblasts and the absence of bone formation and inflammatory cells in the socket. Bone formation, however, is prominent on the periosteum of the buccal surface. 1, Root; 2, PDL with stretched fibroblasts; 3, buccal alveolar wall; 4, osteoid; 5, osteoblasts; 6, trapped osteoblast that would become an osteocyte; 7, cervical area where once there was resorption and now there is formation.

CONCLUSIONS

1.

2.

3. higher stress levels. Instead, bone cavities with few active osteoclasts were observed (Fig 17). This suggests that the resorption phase occurred before 10 days. The lack of initial bone formation in high PDL tensile stresses levels can be clinically related to the tooth mobility associated with high-force appliances such as headgear. Of course, in such cases, bone formation can occur to restore the original PDL width after the mechanical stimulus is halted. Alternatively, a resorption/ formation cycle, a result of a delayed regional acceleratory phenomenon, could be maintained to achieve a sustained response. Future studies could examine the dynamics of this process by observing bone formation at various time points. Interestingly, bone apposition was observed on the buccal alveolar periosteum of all treated mice (Fig 17) but not in the control mice. This unexpected response is most likely related to PDL tension associated with

4.

5. 6.

7.

8.

There is a direct relationship between PDL compressive stress magnitude and the incidence of ERR. Severe ERR is associated with necrotic (hyalinized) PDL tissue, which occurs mainly because of high compressive stresses. The lack of the P2X7R gene (in the KO mice) contributed to slower removal of the hyalinized tissue. These results complement previous evidence, suggesting that ERR and hyalinized tissue metabolism are mediated by the ATP-P2X7- IL-1b mechanotransduction pathway. Absence of the P2X7R gene caused increased ERR in the mouse model. Bone resorptive activity is slightly diminished away from the PDL/bone interface in the P2X7R KO mice. There is a limited interval of nonlinear relationship between PDL tensile stress magnitude and bone formation. High tensile stresses do not initially lead to bone formation, although they can promote a delayed response. Stress analysis (eg, FEA) must be an integral component of experimental mechanotransduction studies to adequately control and assess the mechanical environment.

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41. Harris EF, Kineret SE, Tolley EA. A heritable component for external apical root resorption in patients treated orthodontically. Am J Orthod Dentofacial Orthop 1997;111:301-9. 42. Al-Qawasmi RA, Hartsfield JK Jr, Everett ET, Flury L, Liu L, Foroud TM, et al. Genetic predisposition to external apical root resorption in orthodontic patients: linkage of chromosome-18 marker. J Dent Res 2003;82:356-60. 43. Ngan DC, Kharbanda OP, Byloff FK, Darendeliler MA. The genetic contribution to orthodontic root resorption: a retrospective twin study. Aust Orthod J 2004;20:1-9. 44. Iwasaki LR, Gibson CS, Crouch LD, Marx DB, Pandey JP, Nickel JC. Speed of tooth movement is related to stress and IL1 gene polymorphisms. Am J Orthod Dentofacial Orthop 2006; 130:698.e1-9.