Scanning Microscopy Vol. 12, No. 2, 1998 (Pages 317-327) 0891-7035/98$5.00+.25 Scanning Microscopy International, Chicago (AMFSynthesis O’Hare), of IL Sharpey’s 60666 USA fibers

SYNTHESIS OF SHARPEY’S FIBER PROTEINS WITHIN RODENT ALVEOLAR

R.B. Johnson1* and R.H. Martinez2

1Dept. of Diagnostic Sciences, Univ. of Mississippi School of Dentistry, 2500 North State Street, Jackson, Mississippi 39216-4505; 2Departmento de Ortodoncia, Escuela Colombiana de Medicina, Bogotá, Colombia

(Received for publication April 29, 1996 and in revised form September 24, 1996)

Abstract Introduction: Review of the Literature

Physiologic drift of teeth requires remodeling alve- Sharpey’s fibers attach tendons and to olar bone at the socket wall. Remodeling must be bone. They consist of a bundle of collagen fibers and are regulated so that collagenous attachments are maintained usually partially mineralized (Selvig, 1965; Boyde and Jones, as new tooth positions are established. Thus, alveolar 1968; Shackleford, 1973; Jones and Boyde, 1974; Johnson, remodeling must occur coincident to remodeling of the 1983). The fibers are surrounded by a mineralized, Sharpey’s fibers which are embedded within that bone. collagenous sheath (Johnson, 1983). Studies suggest that these fibers are severed at resorptive For many years, it was presumed that Sharpey’s sites on the alveolar wall and continuity with periodontal fibers were relatively inert. However, recent studies suggest fibers is reestablished by splicing, de novo that they readily adapt to stress/strain forces coincident to synthesis, or adhesion to the base of the Howship’s lacuna functional movements of adjacent teeth. The site of at the alveolar wall; however, little information is available adaptation is at the periosteal surface of bone, where the concerning the mechanisms of these events. collagenous fibers of the tendon or ligament enter the This article reviews types of periodontal Sharpey’s osteoid, becoming a Sharpey’s fiber. fibers and quantifies and compares protein deposition into A convenient place to study the effects of functional these fibers with that of adjacent bone. Effects of ortho- forces on the metabolism of Sharpey’s fibers is within the dontic tooth movement on the protein incorporation into periodontal ligament of the rodent teeth. This ligament the fibers were also studied. Coincident to orthodontic supports the tooth root and is composed of a large number tooth movement, sites of bone matrix deposition (without of unmineralized collagen fiber bundles, which are embedded previous resorption) and resorption/reversal were evident. in root and alveolar bone as Sharpey’s fibers. In At sites of deposition, periodontal ligament fibers were addition, the turnover rate of matrix components of alveolar passively entrapped as Sharpey’s fibers by the new bone bone is approximately 10 times more rapid in alveolar bone matrix. Incorporation of 3H-proline-labeled proteins into than at other sites (Vignery and Baron, 1980); thus, changes bone matrix adjacent to Sharpey’s fibers was significantly in the rate of metabolism of the Sharpey’s fiber and its increased; however, radiolabeled protein incorporation into surrounding bone matrix are more readily evident in alveolar the Sharpey’s fiber was not affected. At resorption/reversal bone than at other skeletal sites. sites, Sharpey’s fibers had been severed and periodontal Rodent molar teeth drift in a distal direction, requir- ligament fiber attachments to the alveolar wall were ing continuous remodeling of the periodontal ligament and becoming reestablished. Incorporation of radiolabeled alveolar bone to assure adequate tooth support. Bone proteins into both Sharpey’s fibers and adjacent matrix was deposition occurs on the distal surface of the interdental significantly increased at these sites. Thus, movement of septum of alveolar bone, which maintains a positive bone adjacent teeth affect the relative metabolism of these fibers, balance (Stallard, 1963; Crumley, 1964; Baumhammers et al., assuring adequate support of the tooth during its relocation. 1965; Carneiro and Fava de Moraes, 1965; Anderson, 1967; Kraw and Enlow, 1967; Kenney and Ramfjord, 1969; Vignery Key Words: Sharpey’s fibers, periodontal ligament, proteins, and Baron, 1980; Dreyer and Sampson, 1984). The mesial, alveolar bone. or remodeling, side of the interdental septum has focal regions of bone resorption and reversal, and has a negative *Address for correspondence: bone balance (Baron, 1973; Vignery and Baron, 1980). How- Roger B. Johnson, address as above. ever, there is always some synthetic activity evident on Telephone number: (601) 984-6010 these surfaces (Johnson, 1987). For tooth movement to FAX number: (601) 984-6014 occur, remodeling of Sharpey’s fibers at the bone interface E-mail: [email protected] is necessary (Garant, 1976; Beertsen et al., 1978; Deporter

317 R.B. Johnson and R.H. Martinez and Ten Cate, 1980; Rygh, 1982). To effect remodeling, microscopic radioautographic studies, suggest that unmineralized periodontal ligament fibers are detached from Sharpey’s fibers and alveolar bone may be simultaneously the bone-embedded Sharpey’s fiber (Deporter and Ten Cate, formed on these surfaces. 1980) and reattached in several ways. They are classified Effects of normal and altered functional forces on with respect to their position relative to old bone (bone Sharpey’s fibers of the periodontal ligament present before resorption), the reversal line, and new bone Metabolism of Sharpey’s fibers is affected by the (bone deposited subsequent to reversal) (Johnson, 1987). function of adjacent teeth. Functional forces determine the Resorptive surfaces mineralization patterns, diameters, and density (fibers/unit On resorptive surfaces, bone removal at the alveolar area) of Sharpey’s fibers at the alveolar wall (Martinez and wall severs the attachment of periodontal ligament and Johnson, 1987; Short and Johnson, 1990) and cementum Sharpey’s fiber (Johnson, 1987). In many instances, (Akiyoshi and Inoue, 1963), which assures adequate support reattachment never occurs and severed Sharpey’s fibers for teeth coincident to changes in their function. There is remain evident within old bone, deep to the reversal lines. no evidence that functional forces determine the type of There are three mechanisms for remodeling of peri- Sharpey’s fiber present at the alveolar wall. odontal ligament attachments to bone on the resorptive Physiologic forces There is no evidence to indicate surface: (1) de novo synthesis of Sharpey’s fibers and new that the compression and tensile forces coincident to bone, (2) formation of adhesive attachments, and (3) splicing physiologic tooth movements have significant effects on of periodontal fibers to existing Sharpey’s fibers. Sharpey fiber diameters or densities (Short and Johnson, Adhesive attachments Kurihara and Enlow (1980a,b) 1990). However, unmineralized cores of Sharpey’s fibers and Johnson (1986) proposed “adhesive” attachments, a are larger at depository than at resorptive/reversal surfaces fibrous meshwork composed of proteoglycans and (Boyde and Jones, 1968; Shackleford, 1973; Johnson, 1983). randomly arranged unit collagen fibrils which reattach Hypofunction and partial function Short-term periodontal ligament fibers onto resorbed surfaces of nonfunction of adjacent teeth results in significant increases alveolar bone. At these sites, the deep portions of the in woven bone deposition at the alveolar crest of the rodent Sharpey’s fibers appear to attach to the reversal line, and interdental septum (Glickman, 1945; Stallard, 1964; Levy and the fiber then extends into the adjacent periodontal ligament. Mailland, 1980; Tran Van and Mailland, 1981; Johnson, 1990). This is the least common method for periodontal remodeling In a recent study (Short and Johnson, 1990), non- and (Johnson, 1987). The bone matrix collagen subjacent to the hypofunctional mandibular molar teeth were created by adhesive attachment is often very dense. selective extraction of rat maxillary molar teeth (Cohn, 1966), De novo synthesis Sharpey’s fibers are often formed which placed teeth in the contralateral quadrant in coincident to deposition of bone matrix onto a reversal hyperfuntion. Sharpey’s fibers supporting non- or surface. These fibers are embedded within new bone, hypofunctional teeth had larger unmineralized cores than superficial to the reversal line. There is no evidence that controls. Mean Sharpey fiber diameters were significantly they are attached to the reversal line, as are adhesive greater and their mean density significantly less in the attachments (Johnson, 1987). supporting non- and in hypo- or Continuous attachments These are presumed to be hyperfunctional teeth. Establishment of partial function to splices between collagen of the periodontal ligament fibers nonfunctional teeth ameliorated the atrophic effects of and the Sharpey’s fiber bundles and are the most common nonfunction on mean diameter and density, but had little method for periodontal remodeling (Johnson, 1987). There effect on the mineralization of Sharpey’s fibers, suggesting is morphological evidence suggesting that when that their mineralization may be controlled by factors other periodontal ligament and Sharpey fiber continuity had been than occlusal forces on adjacent teeth (Short and Johnson, severed during the resorption phase, the fibers were then 1990). rejoined by an unbanded protein (Johnson, 1987). This Orthodontic forces A recent study has demon- protein probably serves as a scaffold for collagen deposition strated changes on the diameter of Sharpey’s fibers at the and could be either masked or replaced by mature collagen. alveolar wall coincident to orthodontic forces. The study These fiber bundles cross the reversal line (Johnson, 1987). presumed that these fibers experienced tension when Depository surfaces adjacent teeth were separated by an orthodontic appliance. These Sharpey’s fibers had significantly greater diameters There is morphological evidence suggesting that than untreated controls (Martinez and Johnson, 1987). How- periodontal ligament fibers are passively entrapped by the ever, their morphology was not significantly altered by the advancing front of bone at depository surfaces (Kraw and forces. It is possible that increased Sharpey’s fiber diameters Enlow, 1967; Kurihara and Enlow, 1980a,b; Johnson, 1984). could be a biological attempt to prevent tearing of the fiber Garant and Cho (1979) and Johnson (1986), in light bundle and separation of the bundle from alveolar bone.

318 Synthesis of Sharpey’s fibers

Although there is morphological evidence to suggest for two hours (37°C) and with aqueous lead citrate for one that synthesis of proteins of Sharpey’s fibers and adjacent hour (20°C), carbon-coated, and viewed in a JEOL-1000B bone matrix is affected by movement of adjacent teeth, there high-voltage electron microscope at an accelerating voltage is little functional data supporting these observations. In of 1000 kV (JEOL, Tokyo, Japan). addition, there is little information concerning the effects of tooth movement on the relative synthesis of bone matrix Radioautography, light microscope and Sharpey’s fiber proteins. To this end, we have Physiologic tooth movement Male Sprague-Dawley conducted a radioautographic study to assess and compare rats, aged 12 weeks, were weighed and given an intra- the effects of normal physiologic and orthodontic tooth peritoneal injection of 3H-proline (2 µCi/g; L-[5-3H] proline; movement on the deposition of these matrix components. specific activity, 40 Ci/mmol; Amersham). Four animals were killed 48 hours after injections. Materials and Methods Orthodontic tooth movement Six-week-old female Sprague-Dawley rats were weighed and anesthetized; sep- Radioautography, high-voltage electron microscope arating springs were placed between the right maxillary left Male Swiss mice, aged 45 days, were weighed and first and second molar teeth (Hadj-Salem, 1971). Then the given an intravenous injection of an appropriate dosage of rats were given an intraperitoneal injection of 3H-proline (5 3H-proline (2 µCi/g; L-[5-3H] proline; specific activity, 40 Ci/ µCi/g; L-[2,3-3H] proline; specific activity, 33 Ci/mmol; mmol, Amersham, Arlington Heights, IL). Animals had been Amersham). Four animals were killed 1-5 days later. The entrained to a cycle of 12 hours light/ 12 hours darkness, right maxillary quadrant was untreated and served as an which was maintained throughout the experiment. internal control. In addition, eight rats received sham- Twenty-four hours after injections, animals were operations and radioisotope injections, and their right anesthetized with a 1:4 solution of ketamine/ xylazine (0.23 maxillary arch served as an external control. ml/100 g body weight) and perfused through the left ventricle Radioautographic procedures Maxillae were re- with Ringer lactate solution containing 4.5 mg of “cold” moved by blunt dissection and immediately fixed in Bouin’s proline/ml to compete with the free 3H-proline remaining in fixative to minimize the loss of radioactive label (Beertsen the tissue (Marchi and Leblond, 1983). Perfusion continued and Tonino, 1975). Specimens were demineralized in 4.13% with 5% formaldehyde in 0.1 M cacodylate buffer (pH 7.3) EDTA at pH 7.0 (Warshawsky and Moore, 1967), dehydrated followed by Karnovsky’s fixative (pH 7.3) (Karnovsky, 1965). in dioxane, and embedded in paraffin wax. Serial sections, 6 The preceding fixation regimen has been suggested by µm thick, were cut in a sagittal plane through the roots of Marchi and Leblond (1983) to reduce binding of free-labeled the teeth, mounted on slides, hydrated, dipped in NTB-2 proline to cells and extracellular substances as a result of emulsion (Kodak), and exposed in a refrigerator for 14 days. reactions between glutaraldehyde containing fixatives, free Slides were developed in D-19 developer (Kodak) and amino acids, and tissue proteins. stained by the Van Gieson method (Luna, 1968). Mandibles were removed by blunt dissection, rinsed Analysis Grain counts were made using an ocular in 0.1 M cacodylate buffer (pH 7.3) and demineralized in grid demarcating 100 µm2. Counts were made on every 4.13% EDTA (ethylenediaminetetraacetic acid; pH 7.0) µ (Warshawsky and Moore, 1967). Interdental septae were sixth section (36 m between each area of analysis) over removed and post-fixed in 2% cacodylate-buffered osmium either a Sharpey’s fiber bundle or adjacent bone matrix. tetroxide, rinsed in 0.1 M cacodylate buffer, dehydrated in Counts were also made over the emulsion for detection of ethanols, and embedded in Epon-Araldite. background and adjusted mean counts (total count minus Radioautography procedures were performed essen- background) calculated for each area of each section. Mean tially by the techniques of Pylypas et al. (1990). Serial counts were calculated for each animal and were compared by factorial analysis of variance and Duncan’s New Multiple sections, 0.5 µm in thickness, were made in coronal and Range test. sagittal planes, collected on glass slides coated with 1% celloidin, carbon-coated, and prepared for radioautography. Results Slides were dipped into diluted Ilford L4 emulsion (1:3; Ilford, U.K.), drained, placed into light-tight boxes and exposed Sites of Sharpey fiber protein synthesis during for 17 weeks at 4°C. Sections were developed (at 20°C) in physiologic drift either Microdol-X (to yield fine grains) or diluted D-19 (1:10) 3 (to yield filamentous grains), fixed in 25% sodium thiosulfate The distribution of H-proline-labeled protein was (at 20°C), floated off slides, and collected on Formvar coated limited to periodontal ligament cells and fibers and new bone matrix (matrix synthesized subsequent to injection of the grids. Grids were stained with 5% uranyl acetate in methanol radioisotope). Cells and fibers of old bone (matrix present

319 R.B. Johnson and R.H. Martinez before injection of radioisotope) were not labeled (Fig. 1). (Figures1-4 on facing page) Twenty-four hours after injection of 3H-proline, Figure 1. Central region of alveolar bone 24 hours after numerous silver grains were evident at the distal surface of injection of 3H-proline, D-19 developer. There are no silver the interdental septum, suggesting bone matrix deposition grains evident over either Sharpey’s fibers (SF) or there. Silver grains were located over the osteoid, but not surrounding bone (B); Bar = 1 µm. over intrabony portions of principal fibers of the periodontal ligament (Sharpey’s fibers). In this situation, periodontal Figure 2. Depository surface of alveolar bone 24 hours ligament fibers were likely being passively entrapped by after injection of 3H-proline, D-19 developer. In some areas, the advancing front of new bone (Fig. 2). silver grains are located over both principal fibers (PF) of The mesial surface of the interdental septum had the periodontal ligament and newly deposited osteoid (NB). focal areas of bone resorption and deposition and numer- There are no silver grains over either bone matrix present ous reversal lines. At sites of reversal, silver grains were prior to injection of the radioisotope (OB) or Sharpey’s over both principal fibers and new bone matrix (Figs. 3 and fibers; Bar = 1 µm. 4), suggesting that Sharpey’s fibers and new bone matrix were being simultaneously formed. At some sites, Sharpey’s Figure 3. Reversal surface of alveolar bone 24 hours after fibers did not deeply penetrate alveolar bone and did not injection of 3H-proline, D-19 developer. Silver grains are cross the reversal line. Their terminal ends blended with over recently deposited bone matrix (NB) and principal fibers the bone matrix collagen (Fig. 3). These fibers were likely (PF) of the periodontal ligament. These fibers are an example formed de novo. of Sharpey’s fibers formed de novo, as they are being At sites of matrix resorption prior to reversal and synthesized coincident to osteoid and are not becoming deposition of new bone, Howship’s lacunae were being filled linked to a severed Sharpey’s fiber within old bone (OB). by an anastomosing network of unit collagen fibrils, forming There are no silver grains over bone matrix prior to the a structure resembling an adhesive attachment (Fig. 5). injection of radioisotope (OB); Bar = 1 µm. Silver grains were over unit collagen fibrils in these areas. Fibrils appeared to attach directly onto the surface of the alveolar wall (Fig. 5). The collagenous bone matrix there Figure 4. Alveolar bone near a reversal surface 24 hours 3 appeared to be slightly denser than subsurface bone matrix following injection of H-proline, Microdol-X developer. Silver grains are over both new bone matrix (NB) and (Fig. 5). µ In many areas, Sharpey’s fibers crossed reversal lines Sharpey’s fibers; Bar = 1 m. and became continuous with principal fibers of the periodontal ligament. At the alveolar wall, silver grains were over the junction of Sharpey’s and principal fibers (Fig. 6). Unit collagen fibrils of the two fiber bundles were joined by fine, unstriated fibrils. Silver grains were over these fibrils over bone at depository surfaces than at resorptive/reversal (Fig. 7). Splicing of fiber bundles was evident in areas of surfaces. Orthodontic tooth movement produced a new bone deposition (Fig. 6), but also occurred in areas significant increase in silver grains over the bone matrix at where new bone was not being formed (Fig. 8). depository surfaces, but had no effect on numbers of silver grains over the adjacent Sharpey’s fibers as compared to Effects of orthodontic tooth movement maxillary bone matrix of sham-operated external controls Placement of a separating spring between the first (Table 1). The number of grains over bone formed coinci- and second molar teeth tipped the first molar in a mesial dent to orthodontic forces was significantly greater than in direction, creating a region of tension at the alveolar crest sham-operated external controls. At resorptive/ reversal and a region of compression near the root apices (Fig. 9). surfaces, the numbers of silver grains over both matrix and Thus, numerous areas of alveolar bone deposition and Sharpey’s fibers was significantly increased by orthodontic resorption/reversal were evident on both mesial and distal forces compared to sham-operated external controls (Table surfaces of the alveolus. Histomorphometric analysis of 1). the area of the interdental septum demonstrated net interdental septal bone loss from 1-5 days following spring Discussion placement, probably resulting from tipping of the molar teeth and coincident compression of the interdental septum by The present study describes the pattern of 3H-pro- the adjacent tooth roots (Fig. 10). In specimens prepared line incorporation into bone and Sharpey’s fibers at de- for radioautography, sites of protein deposition were marked pository and resorptive/reversal surfaces of the interdental by silver grains over both Sharpey’s fibers and the adjacent septum of the rodent during both physiologic and ortho- bone matrix. In general, more silver grains were evident dontic tooth movement. Since the time after isotope

320 Synthesis of Sharpey’s fibers

321 R.B. Johnson and R.H. Martinez

Table 1. Mean silver grain counts (± standard error of mean) Figure 5. Alveolar bone near a reversal surface 24 hours 3 over 100 µm2 areas of Sharpey’s fibers and adjacent alveolar following injection of H-proline, D-19 developer. Within bone matrix from animals experiencing orthodontic tooth Howship’s lacunae, an anastomosing network of unit movement for 2 days and sham-operated external controls. collagen fibrils adheres to the alveolar wall (AW). Cellular N = 4 for each group. All counts from reversal/resorption membranes (RB) are evident within the meshwork. The sites were significantly different from the corresponding alveolar wall (AW) has denser packing of matrix collagen deposition site, p < 0.001. than the underlying bone (OB). Silver grains are over the anastomosing network of unit collagen fibrils. The alveolar wall and subjacent bone are unlabeled; Bar = 1 µm. Site Tissue Treatment Count Figure 6. Alveolar bone near a reversal surface 24 hours Deposition(D) Bone Experi- 36.69 ± 3.58*,tt following injection of 3H-proline, D-19 developer. In some Matrix(M) mental (E) areas, Sharpey’s fibers (SF) maintain continuity with principal fibers (PF) of the periodontal ligament by fibril splicing at the bone surface. Silver grains are over recently D M Control© 19.00 ± 0.88tt deposited osteoid (NB) and the junction of principal and D Sharpey’s E 24.78 ± 0.98 Sharpey’s fibers (arrow). The remainder of the bone (OB) is fibers(F) µ D F C 24.04 ± 0.93 unlabeled. O indicates osteoblast; Bar = 1 m. Resorption M E 11.23 ± 1.14** /Reversal (R) Figure 7. Reversal of a resorptive surface of the alveolar R M C 6.52 ± 0.74 wall 24 hours following injection of 3H-proline, D-19 R F E 12.22 ± 1.79** developer. Higher magnification of junction between R F C 7.35 ± 0.92 principal fiber (PF) and Sharpey’s fiber (SF) at the bone surface. Silver grains are over non-crossbanded fibrils Significantly different from controls (C): connecting unit collagen fibrils of the two fiber bundles; at *p < 0.001; **p < 0.01 12,000x. Bar = 1 µm. Significantly different from Sharpey’s fibers (F): t tt p < 0.001; p < 0.01 Figure 8. Alveolar bone near a depository surface 24 hours after injection of 3H-proline, Microdol-X developer. Silver grains are over Sharpey’s fibers (SF). There are no silver grains over the remaining bone (OB); at 8,000x. Bar = 1 µm. injection into rodents in our experiments was standard, we can assume that differences in quantity of silver grains within a tissue represented the net rate of incorporation of proline into those proteins. To maintain attachment of teeth to bone on remodeling surfaces of the alveolar wall, on morphological evidence. The present work represents periodontal ligament fibers must be severed, and the fibers the only electron microscopic radioautographic study of then reattached to the alveolar wall bone matrix or to severed these mechanisms and the only light microscopic Sharpey’s fibers. To assess this phenomenon, semi-thin radioautographic study of the effect of orthodontic tooth tissue sections were studied using high-voltage electron movement on Sharpey fiber protein metabolism. This data microscopic techniques. These techniques allowed study demonstrates (1) the development and maintenance of the of relatively thick sections and provided more confident various types of Sharpey’s fibers, and (2) the effects of the assessment of periodontal/Sharpey fiber continuity. In tissue stress/strain microenvironment on patterns and addition, we could more readily determine whether fibers quantities of Sharpey fiber and bone matrix proteins. This had been severed or had passed from the plane of section, study extends other experiments by reporting changes in producing severance artifacts. patterns of protein synthesis in both Sharpey’s fibers and Patterns of deposition of 3H-proline-labeled proteins their adjacent alveolar bone matrix coincident to movement herein suggest three types of periodontal ligament attach- of adjacent teeth and suggests that attachment of ments to resorptive bone surfaces: (1) de novo synthesis of periodontal ligament fibers to bone requires continuous and Sharpey’s fibers and new bone, (2) formation of adhesive coordinated synthesis of collagenous fibers of both alveolar attachments, and (3) splicing of periodontal fibers to existing bone matrix and Sharpey’s fibers. Separation of teeth by Sharpey’s fibers, and confirmed and extended observations orthodontic force altered the stress/strain microenvironment of Johnson (1987), who proposed these patterns based only of the supporting periodontium, which adapted to these

322 Synthesis of Sharpey’s fibers

323 R.B. Johnson and R.H. Martinez

changes by alteration of the pattern and rate of synthesis of matrix proteins. Our study also suggests how separation of adjacent teeth might alter Sharpey fiber metabolism. Placement of an orthodontic spring created areas of tension (cervical third) and compression (middle and apical thirds) of the periodontium. In areas of tension, bone deposition was evident, and there was little evidence of resorption/ reversal. There was no effect on resultant protein incorporation into the Sharpey fiber bundle at those sites, but a nearly doubled protein incorporation into the adjacent matrix was evident. Thus, both the fiber bundle and adjacent matrix likely became more resistant to damage from the tensile force. These results also suggest passive entrapment of pre-existing periodontal ligament principal fibers by the new bone matrix to become a Sharpey fiber, as protein incorporation into the periodontal and Sharpey fiber bundles were similar. At sites Figure 9. Diagram of spring (S) placement between the first experiencing resorption/reversal, incorporation of proteins and second molar teeth of a rat. Arrows suggest the into bone matrix and Sharpey’s fibers subsequent to reversal movement of the teeth; separation of the crowns creating a was nearly doubled as compared to sham-operated external tensile force in the cervical third of the periodontal ligament controls; however, the quantity of proteins incorporated and approximation of the roots creating a compressive force into the matrix and Sharpey’s fibers was equivalent, in the apical third of the periodontal ligament (Martinez and suggesting simultaneous deposition of protein into the two Johnson, 1987). tissues (and not entrapment). Thus, the attachment of periodontal ligament to new bone was likely strengthened to resist damage from adjacent tooth movements. Several studies have suggested that Sharpey fiber diameters were correlated to functional forces on 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 protein incorporated into alveolar bone matrix and adjacent Sharpey’s fibers coincident to orthodontic tooth movement only at areas of resorption/ reversal, suggesting that Sharpey fiber diameter may be more dependent on the extent of adjacent tooth movement than on the type of functional force on the adjacent teeth. Thus, the movements of adjacent teeth affect both the quantity and ratios of protein incorporation into Sharpey’s fibers and adjacent alveolar bone, which is dependent on the characteristics of the stress/strain microenvironment produced by these movements.

References

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324 Synthesis of Sharpey’s fibers

Baron R (1973) Remaniement de l’os alveolaire et Anat Rec 216: 339-348. des fibres desmodontales au cours de la migration Johnson RB (1987) A classification of Sharpey’s physiologique (Remodeling of alveolar bone and periodon- fibers within the alveolar bone of the mouse: A high-voltage tal fibers undergoing physiological movement). J Biol electron microscope study. Anat Rec 217: 339-347. Buccale 1: 151-170. Johnson RB (1990) Effect of altered occlusal function Baumhammers A, Stallard RE, Zander HA (1965) on transseptal ligament and new bone thicknesses in the Remodeling of alveolar bone. J Periodontol 36: 439-442. periodontium of the rat. Am J Anat 187: 91-97. Beertsen W, Tonino GJM (1975) Effects of fixation Jones SJ, Boyde A (1974) The organization and gross and demineralization on the intensity of autoradiographic mineralization patterns of the collagen fibres in Sharpey labeling over the periodontal ligament of the mouse after fiber bone. 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Shackleford JM (1973) Ultrastructural and microradio- Johnson RB (1986) The distribution of 3H-proline in graphic characteristics of Sharpey’s fibers in dog alveolar alveolar bone of the mouse as seen by radioautography. bone. Ala J Med Sci 10: 11-20.

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Short E, Johnson RB (1990) Effects of tooth func- the resultant fiber diameters. Thus, in our model of tion on adjacent alveolar bone and Sharpey’s fibers of the orthodontic tooth movement, the magnitude of the forces rat periodontium. Anat Rec 227: 391-396. to the periodontium produced by tipping the adjacent teeth Stallard RE (1963) The utilization of 3H-proline by would be equivalent in areas of tension and compression, the elements of the periodontium. Perio- producing increases in Sharpey’s fiber diameter at both dontics 1: 185-188. tension and compression zones. Stallard RE (1964) The effect of occlusal alterations on collagen formation within the periodontium. Periodon- S.B. Jones: Which cells do you think are contributing (pre- tics 2: 49-52. cursors of) collagen to the intrinsic fibers and the Sharpey’s Tran Van P, Mailland ML (1981) Short-term effects (extrinsic) fibers? Are they always the same, or are they of occlusal hypofunction following agonist tooth extrac- variable? tion upon periodontal tissues in the rat. J Biol Buccale 9: Authors: Since 3H-proline is incorporated into collagen 385-400. produced by both fibroblasts and osteoblasts, the source Vignery A, Baron R (1980) Dynamic histomor- of the collagen to the intrinsic and Sharpey’s fibers could phometry of alveolar bone remodeling in the adult rat. Anat not be determined. Another biomarker would be required Rec 196: 191-200. to study this process. Warshawsky H, Moore G (1967) A technique for the fixation and decalcification of rat incisors for electron S.B. Jones: At a reversal line, have you seen any evidence microscopy. J Histochem Cytochem 15: 542-549. in your electron microscopy studies that demineralized collagen of Sharpey’s fibers, as well as collagen that has Discussion with Reviewers never mineralized, is available for splicing? Or does any demineralized collagen self-destruct or be removed before A.R. Ten Cate: It would be helpful if the authors could new collagen assembles? explain why they chose not to examine bone surfaces with Authors: We reported indirect evidence of unmineralized high voltage electron microscopy (HVEM) following cores in Sharpey’s fibers at reversal lines in HVEM and a “orthodontic” tooth movement in addition to preparing suggested incomplete severance of Sharpey’s at the alveolar conventional paraffin embedded sectioned for counting wall (Johnson, 1987). Although incomplete severance is purposes. While it is likely that changes on these surfaces difficult to prove, it would provide a template for slicing of following orthodontic movement reflect enhancement of new collagen to existing Sharpey’s fibers to form continuous physiological adaptations, this assumption cannot be fairly fibers. made. Authors: It was very difficult to quantify the radiography S.B. Jones: Where a Sharpey’s fiber crosses a reversal line, in HVEM due to low density of the marker. Since we could does part of the fiber in the old bone have an unmineralized distinguish the type of Sharpey’s fibers and the surrounding core always, sometimes or never? tissues using a light microscope, we chose this method. Authors: We believe that the fiber within old bone always Light microscopy also allowed more extensive evaluation has an unmineralized core (Martinez and Johnson, 1987; of the pattern and quantity of protein deposition into those Johnson, 1987). However, the cores become reduced in size tissues. as the fiber becomes more deeply embedded in alveolar bone (Martinez and Johnson, 1987). C.A. McCulloch: How can one explain increase of Sharp- ey’s fiber diameter on both compression and tension zones H. Warshawsky: The radiographic data show sites of pro- with the same mechanism? tein synthesis and matrix deposition. How does this data Authors: We do not believe that tensile or compressive confirm the morphological evidence that suggests three forces alone determine the diameter of the Sharpey’s fiber. types of periodontal attachment to resorptive bone surfaces? The magnitude of the force seems to be more important. Authors: Our morphological data did not confirm forma- Our present data, in addition to that in a study of the tion of three types of periodontal attachments, but sug- hypofunctional periodontium by Short and Johnson (1990), gested that there may be three types of patterns of suggests that the magnitude of the load exerted by teeth on detachment of periodontal ligament fibers from alveolar the periodontium is the most important determinant of the bone. Radiographic data proved that these attachments diameter of the periodontal ligament and Sharpey’s fibers. are under constant renewal. Whether the tooth movement enhances tensile forces (as in reactivated eruption) or compressive forces (as in H. Warshawsky: How do you explain splicing of collagen orthodontic tooth movement) seems to have little effect on filaments to existing cut ends of Sharpey’s fibers in terms of

326 Synthesis of Sharpey’s fibers the quarter-stagger mechanism of collagen fibrillogenesis? Authors: We cannot explain this from our data. There must be some coordination between collagen removal and deposition within these fiber bundles. We could only speculate on that mechanism.

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