Synthesis of alveolar bone Sharpey's fibers during experimental tooth movement in the rat

Authors

  • Roger B. Johnson

    Corresponding author
    1. Department of Periodontics and Preventive Sciences, University of Mississippi Medical Center School of Dentistry, Jackson, Mississippi
    • Department of Periodontics and Preventive Sciences, School of Dentistry, University of Mississippi, 2500 North State Street, Jackson, MS 39216
    Search for more papers by this author
    • Fax: 601-984-6120


Abstract

There is little information concerning the effects of tooth movement on the relative synthesis of bone matrix and Sharpey's fiber collagenous proteins. The purpose of this study was to investigate this situation using radioautographic techniques. The maxillary first molar tooth in rats was tipped toward the midline using an appliance and the animals were injected with 3H-proline after 3 days and sacrificed 24 hr later. Maxillae were sectioned and silver grain proportional areas (grain density/5,000 μm2) evaluated over Sharpey's fibers and adjacent alveolar bone matrix using computerized densitometry and histomorphometric techniques. These data were compared to a group of untreated animals by Fisher's exact test. At depository surfaces of experimental tissues, the silver grain proportional area over bone matrix was significantly greater than over Sharpey's fibers (P < 0.05) and control bone matrix (P < 0.01). The silver grain proportional area over Sharpey's fibers was not different between the groups. At resorptive surfaces, the silver grain proportional area over both bone matrix and Sharpey's fibers was significantly greater in experimental tissues compared to controls (P < 0.01). Thus, movements of adjacent teeth affect both the quantity and ratios of collagenous protein incorporation into Sharpey's fibers and adjacent alveolar bone, which is dependent on the intensity and characteristics of the force. © 2005 Wiley-Liss, Inc.

Tooth movement occurs by alveolus translocation, which is a unique type of remodeling, featuring simultaneous bone formation and resorption on opposite sides of the alveolus. Taken together, these processes result in drift of the entire alveolus in parallel to tooth drift to maintain tooth support (Roberts et al., 1981) and require remodeling of the principal fibers of the periodontal ligament (PDL), alveolar bone matrix, and the embedded Sharpey's fibers.

Tooth movement produces stress/strain forces within the PDL, which are transferred to the alveolus (Tanne et al., 1987; Katona et al., 1995; Middleton et al., 1996; Puente et al., 1996; Tamatsu et al., 1996) and become transduced into a cellular response within the periodontium. In response to these forces, bone is deposited on the alveolar wall in regions of tension, and bone resorption occurs at sites experiencing pressure forces (Macapanpan et al., 1954; Waldo and Rothblatt, 1954; Zaki and Van Huysen, 1963; Azuma, 1970; Lopez Otero et al., 1973; Heller and Nanda, 1979; Yamasaki et al., 1980; Lilya et al., 1984; Martinez and Johnson, 1987; Chao et al., 1988; Lee, 1990; King et al., 1991a, b; King and Keeling, 1995; Ashizawa and Sahara, 1998). Rat molar teeth drift in a distal direction under physiologic conditions because the alveolus maintains net bone deposition on its mesial surface and net bone resorption on its distal surface (Sicher and Weinmann, 1944).

The principal PDL fibers are attached to alveolar bone by Sharpey's fibers, a bundle of partially mineralized collagen fibers embedded within the alveolar bone matrix (Selvig, 1965; Boyde and Jones, 1968; Shackleford, 1973; Jones and Boyde, 1974; Johnson, 1983). These fiber bundles are surrounded by a partially mineralized collagenous sheath, which is a component of the alveolar bone matrix (Johnson, 1983).

For many years, it was assumed that Sharpey's fibers were relatively inert; however, recent studies suggest that they readily adapt to stress/strain forces produced by functional movements of the adjacent teeth. The site of this adaptation is at the interface between alveolar bone and PDL. Recent studies suggest that stress/strain forces from adjacent teeth determine the mineralization patterns, diameters, and density (fibers/unit area) of Sharpey's fibers at the alveolar wall (Martinez and Johnson, 1987; Short and Johnson, 1990). For any tooth movement to occur, remodeling of Sharpey's fibers at the PDL interface with the alveolus is required (Garant, 1976; Beertsen et al., 1978; Deporter and Ten Cate, 1980; Rygh, 1982; Fukui et al., 2003). Morphological studies report that some PDL fibers become detached from their bone-embedded Sharpey's fiber components during remodeling of the alveolus (Deporter and Ten Cate, 1980), while some remain attached (Johnson, 1987). Many detached PDL fibers become reattached to either the bone surface or to nonresorbed Sharpey's fibers during the remodeling process (Johnson, 1987).

A convenient place to study the effects of tooth movements on the remodeling of Sharpey's fibers is within the molar periodontium of the rat. The turnover rate of matrix components of alveolar bone has been reported to be approximately 10 times more rapid than within bone at other sites (Vignery and Baron, 1980). Thus, changes in the rate of remodeling of the Sharpey's fibers and the surrounding bone matrix should also occur more rapidly within alveolar bone than at other skeletal sites. In addition, the turnover rate of collagenous proteins is more rapid within the PDL than within other connective tissues, as the half-life of collagen in the rat PDL has been reported to be less than 3 days (Sodek, 1977; Imberman et al., 1986; Sodek and Ferrier, 1988).

Experimental tooth movement by application of light forces to the rat molar teeth (< 10 g) occurs in two phases: an initial shift of the tooth (occurring within the initial 56 hr) followed by a smoother, gradual rate of movement in the direction of the applied force (Kohno et al., 2002). Heavier forces (> 20 g) produce another sequence of events: an initial strain producing distraction of the alveolar bone, followed by a lag phase featuring hyalinization of the PDL, and finally a progressive tooth movement into sites of undermining bone resorption in the direction of the force (Reitan, 1967; Storey, 1973). Experimental tooth movement using high forces produces tissue damage, such as disruption of collagen fibers (Zaki and Van Huysen, 1963; Reitan, 1967; Azuma, 1970), areas of compression and expansion of the PDL (Reitan, 1967; Hong et al., 1992), and resorption of the bone and tooth root (Zaki and Van Huysen, 1963; Reitan, 1964, 1967; Tanaka et al., 1990).

Although there is morphological evidence to suggest that synthesis of bone matrix collagenous proteins of Sharpey's fibers and adjacent alveolar bone is affected by movement of adjacent teeth, there is little functional data supporting these observations. In addition, there is little information concerning the effects of tooth movement on the relative synthesis of bone matrix and Sharpey's fiber collagenous proteins. To this end, we have conducted a radioautographic study to assess the effects of experimental tooth movements using a moderate force level on the pattern of deposition of collagenous proteins into these components of the periodontium. These data should yield additional information about the biologic mechanism for maintenance of tooth attachment during alveolus translocation.

MATERIALS AND METHODS

Animals

This study was approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center. Seventeen female Sprague-Dawley rats (190 ± 15 g) were studied. The animals were weighed and then anesthetized with a 4:1 solution of ketamine/xylazine (ketamine HCl, 50 mg/ml, 20 mg/ml) at a dosage of 0.15 ml/100 g body weight. A stainless steel appliance constructed of orthodontic wire was attached to the right first maxillary molar (M1), moving it in an anterior direction (Fig. 1). The left side was untreated and served as an internal control. In addition, 17 age- and weight-matched rats were sham-operated and served as external controls. Rat chow and water were available to all animals ad libitum. Rat chow was ground into powder prior to feeding all animals to minimize potential damage to the appliance in the experimental quadrants.

Figure 1.

The appliance for movement of the right maxillary first molar tooth (M1) of the rat. The appliance is attached to the maxillary incisor teeth (I) with a band and to M1 by a stainless steel fine mesh anchored by cyanoacrylate cement. The teeth were joined by a 0.022 stainless steel closing coil (S) supported by a 0.020 stainless steel wire. When activated, the closing coil produced a 15 ± 1 g anterior force to M1.

Appliance Insertion and Activation

The appliance for tooth movement used in this study has been used by us in previous studies (Row and Johnson, 1990; Murrell et al., 1996). It was assembled on a plastic model of the rat dentition and was then bonded with cyanoacrylate cement to occlusal surface of the right M1 and to a band surrounding the central incisor teeth. Following activation of the spring component, the appliance moved M1 in an anterior direction with a 15 ± 1 g force (Fig. 1). Mandibular right M1 and central incisor teeth were ground with a diamond stone to eliminate occlusal interference with the appliance. All animals received an intraperitoneal injection of 3H-proline (5 μCi/g; L-{2,3-3H} proline; specific activity, 33 Ci/mmol; Amersham) after 3 days and were euthanized 24 hr later, as previous studies have indicated rapid bone formation within 4 days of the initiation of experimental tooth movement (Pavlin and Gluhak-Heinrich, 2001).

Radioautographic Procedures

Following anesthesia, maxillae were removed by blunt dissection and immediately fixed in Bouin's fixative to minimize the loss of radioactive label (Beertsen and Tonino, 1975). Specimens were demineralized in 4.13% EDTA at pH 7.0 (Warshawsky and Moore, 1967), dehydrated in ethanols, and embedded in paraffin wax. To establish periodontal baseline measurements, a 6 μm thick tissue section was made in a sagittal plane through the central developmental groove of the tooth crown in one experimental internal control and external control quadrant to provide a longitudinal profile of the entire tooth and periodontium (Figs. 2 and 3). In 16 remaining rats, serial sections 6 μm thick were made in a coronal plane, providing a cross-sectional profile of the periodontium. Tissue sections were mounted on slides, hydrated, dipped in NTB-2 emulsion (Kodak), and exposed for 14 days at 4°C in a light-tight box containing CaSO4 drying agent. Slides were developed in D-19 developer (21°C for 5 min; Kodak) and fixed in 25% sodium thiosulfate (21°C for 5 min; Kodak) (Rogers, 1979). Following development, slides were stained through the emulsion by the Van Gieson method (Luna, 1968).

Figure 2.

Silver grain distribution at depository (D; adjacent to the mesial periodontal ligament, or MPDL) and resorptive (R; adjacent to the distal periodontal ligament, or DPDL) surfaces of the interdental septum (B) between the maxillary right first molar (M1) and second molar (M2) teeth of the rat coincident to normal physiologic drift. The direction of physiologic tooth drift is indicated by an arrow. Van Gieson stain, 100×.

Figure 3.

Silver grain distribution at depository (D; adjacent to the DPDL) and resorptive (R; adjacent to the MPDL) surfaces of the interdental septum (B) between the first molar (M1) and second molar (M2) teeth of the rat coincident to experimental tooth movement. The experimental movement of M1 (arrow) has reversed the direction of normal physiologic drift of the teeth. Several areas of root resorption (asterisks) are evident on the root of M2. Van Gieson stain, 100×.

Regions for Analysis

Images (300×) were captured from every sixth section (36 μm between each area of analysis) of the cross-sectional profiles using a photomicroscope with a video camera (JVC Professional Products, Wayne, NJ) interfaced through an image capture card (Photometrics V, Munich, Germany) with a Dell computer. Images were grouped based on their location within the periodontium [apical, middle, and cervical thirds, with superior borders 0.45 mm (cervical group), 0.90 mm (middle group), and 1.35 mm (apical group) distance from the apex of the crest of the furcation between the mesiolingual and distolingual roots (Ashizawa and Sahara, 1998)]. The alveolar wall and root surface of the mesiobuccal root of the second molar (M2) and the distobuccal root of M1 were identified on the captured images. Quadrants for analysis were defined by radii drawn from the center of each root at 45°, 135°, 225°, and 315° to the midline of the tooth, subdividing the adjacent periodontium into mesial, buccal, palatal, and distal quadrants (Murrell et al., 1996). The mesial quadrant of the PDL adjacent to M2 and the distal quadrant of the PDL of M1 were evaluated. Sharpey's fibers were chosen at random in 300× images for further analysis at 1,000×.

Silver Grain Analysis

Images of the Sharpey's fibers and adjacent alveolar bone within the appropriate quadrants were captured at 1,000× and 3H-proline silver grains were identified using computer densitometric software (SigmaScan Pro; SPSS, Chicago, IL; Fig. 3). Silver grain density (proportional area of 5,000 μm2 field) over either a Sharpey's fiber bundle or adjacent bone matrix (within the collagenous proteins of the alveolar wall deposited subsequent to 3H-proline administration) was calculated by the software. Dimensions of the analysis area could be adjusted so that grain counts were only over either the Sharpey's fiber or the adjacent bone matrix; however, the total area of analysis remained constant (5,000 μm2). Evaluation of the density of silver grains was also made within 5,000 μm2 regions over the enamel space for detection of background, and adjusted mean proportional areas (total proportional area minus background) were calculated for each section.

Statistical Analysis

Mean adjusted proportional silver grain areas within Sharpey's fibers or adjacent bone matrix were calculated for each animal and treatment group and compared by Fisher's exact test using SPSS v12 (SPSS) software. Mean differences were considered to be significant when P < 0.05.

RESULTS

The appliance moved M1 in an anterior direction, reversing the direction of normal physiologic drift. This abrupt change in the direction of drift produced changes in the pattern of deposition and resorption on the walls of the interdental septum separating M1 from M2 (Figs. 2 and 3). Internal and external control data were not significantly different, so we used only the external control data (C) for comparison to the experimental data (E).

Movement of M1 in an anterior direction created a region of bone deposition at the alveolar crest and on the distal surface of the alveolar wall adjacent to M1 and resorption/reversal on the medial alveolar wall adjacent to M2 (Fig. 3), which was opposite to that pattern during normal physiologic drift (Fig. 2).

Sites of collagenous protein deposition were marked by silver grains over both PDL and Sharpey's fibers and the adjacent bone matrix (Fig. 4) on both depository and resorption/reversal surfaces (Figs. 4 and 5). On those surfaces, PDL fibers were heavily labeled, but Sharpey's fibers had a lower density of the label (Fig. 5). There was no effect of experimental tooth movement on 3H-proline incorporation into Sharpey's fibers at depository sites, but a nearly threefold increase in the radiolabel into these fiber bundles at resorptive/reversal sites. Silver grain proportional areas were significantly greater over bone matrix at depository sites than at resorptive/reversal sites (P < 0.001). However, at the E depository sites, the silver grain proportional areas over bone matrix were significantly greater than over both Sharpey's fibers and C bone matrix (P < 0.05). Similarly, at the resorptive/reversal surfaces, there was nearly a twofold increase in silver grain proportional areas within bone matrix, but no difference between the groups in density of label within the Sharpey's fibers (Table 1). Experimental tooth movement produced no significant differences in silver grain proportional areas over Sharpey's fibers as compared to controls at both depository and resorptive sites. However, at resorptive/reversal surfaces, the proportional silver grain areas over Sharpey's fibers and adjacent bone matrix were significantly greater in experimental compared to control rats (P < 0.001; Table 1).

Figure 4.

Silver grain distribution at a depository surface (D; adjacent to the DPDL) of the interdental septum (B) between the first molar (M1) and second molar (M2) teeth of the rat coincident to experimental tooth movement. Silver grains are located over both Sharpey's fibers (SF) and bone matrix (B). Van Gieson stain, 800×.

Figure 5.

Silver grain distribution at resorptive/reversal surfaces (adjacent to the MPDL) of the interdental septum (B) between the first molar (M1) and second molar (M2) teeth of the rat coincident to experimental tooth movement. Silver grains are located over both newly synthesized periodontal ligament fibers (PF) and new bone matrix (NB). No silver grains are located over bone present prior to the injection of 3H-proline (NB). Van Gieson stain, 800×.

Table 1. Mean silver grain proportional area ± SEM*
SurfaceTissueTreatment% area
  • *

    Adjusted for background within 5,000 μm2 regions of Sharpey's fibers (SF) and adjacent alveolar bone matrix (B) from animals experiencing experimental tooth movement for 4 days (E) and sham-operated controls (C). All proportional areas at depository surfaces are significantly greater than those at resorptive surfaces (P < 0.001). n = 16 for each treatment group.

  • a

    Significantly different from control: P < 0.01.

  • b

    Ditto. P < 0.05.

  • c

    Significantly different from Sharpey's fibers: P < 0.05.

DepositoryBE36.69 ± 3.58ac
 BC19.00 ± 1.88
 SFE24.78 ± 1.98
 SFC24.04 ± 1.93
Resorptive/reversalBE14.23 ± 1.14b
 BC6.52 ± 0.74
 SFE12.22 ± 1.79b
 SFC4.35 ± 0.52

DISCUSSION

It has long been recognized that rat molar teeth drift in a posterior direction during physiologic conditions, requiring that the walls of each tooth socket undergo net bone deposition on the medial and net bone resorption on the distal sides (Sicher and Weinmann, 1944). In our experiment, we moved the maxillary first molar tooth in an anterior direction (opposite to the direction of physiologic distal drift), which induced a bone remodeling pattern opposite to that observed during physiologic drift. This change probably occurs as a result of changes in the stress/strain forces on the periodontium produced by the experimental forces on the adjacent teeth (Macapanpan et al., 1954; Waldo and Rothblatt, 1954; Zaki and Van Huysen, 1963; Azuma, 1970; Lopez Otero et al., 1973; Heller and Nanda, 1979; Yamasaki et al., 1980; Lilya et al., 1984; Martinez and Johnson, 1987; Chao et al., 1988; Lee, 1990; King et al., 1991a, b; King and Keeling, 1995; Ashizawa and Sahara, 1998). This compensatory bone deposition results from enhanced differentiation of osteoblasts from precursor cells within the PDL and the alveolar bone coincident to the tensile forces during the first 4 days of tooth movement (Pavlin and Gluhak-Heinrich, 2001), the time period used in our study.

Our data describes and compares for the first time the collagenous protein deposition into Sharpey's fibers and the adjacent alveolar bone matrix during both physiologic and experimental tooth movement. These data extend others by reporting changes in both the patterns and quantities of collagenous protein deposition into both Sharpey's fibers and their adjacent alveolar bone matrix coincident to movement of adjacent teeth and suggest that attachment of PDL fibers to bone required continuous and coordinated synthesis of collagenous fibers of both alveolar bone matrix and adjacent Sharpey's fibers as a response to the experimental forces. Enhanced incorporation of 3H-proline into the PDL fibers adjacent to alveolar bone and a significant increase in label over Sharpey's fibers at the resorptive/reversal sites of the alveolar bone surface of E suggest that the PDL fibers are newly synthesized and could be forming an attachment to Sharpey's fibers coincident to the new bone deposition (as both 3H-proline incorporation into Sharpey's fibers and adjacent bone matrix were significantly greater in E than in C animals), supporting several previous studies (Deporter and Ten Cate, 1980; Johnson, 1987).

Previous data have suggested that Sharpey's fiber diameters at the alveolar wall were correlated to function of adjacent teeth and that fibers became larger when these forces were increased and smaller when the forces were decreased (Martinez and Johnson, 1987; Short and Johnson, 1990). Neither study suggested a mechanism for this change in diameter. Our study demonstrated an increased quantity of collagenous protein incorporation into alveolar bone matrix and adjacent Sharpey's fibers in E animals at all sites, suggesting that the magnitude of forces on the PDL produced significant changes in their attachment to bone. At depository sites in E animals, 3H-proline label was significantly greater within alveolar bone matrix than within Sharpey's fibers, which was a pattern also evident within C animals. At resorption/reversal sites, the labeling density within bone matrix of E was not significantly different from the Sharpey's fibers, a situation also evident in the C group. However, the E and C groups were not different. Taken together, our data suggest that Sharpey's fiber metabolism may also be dependent on the force characteristics, as resorptive/reversal and depository sides of the alveolus likely experienced predominately pressure and tension forces, respectively.

Our data propose that therapeutic tooth movements are possible because experimental forces increase the rate of collagenous protein deposition into both bone matrix and Sharpey's fibers in depository regions as compared to resorption/reversal regions. Thus, during experimental tooth movements, collagenous protein deposition into bone matrix and Sharpey's fibers increases at the sites of the tensile force (possibly increasing the Sharpey's fiber diameters) and is much lower in the pressure regions, ensuring net movement of the tooth socket in the direction of the force. Incorporation of collagenous proteins into Sharpey's fibers at both sites ensures adequate tooth support during alveolar remodeling.

Ancillary