Histochemistry of enzymes associated with tissue degradation incident to orthodontic tooth movement

Histochemistry of enzymes associated with tissue degradation incident to orthodontic tooth movement E. Lilja, S. Lindskog, Stockholm, Sweden

Dr. Lilja

and L. Hammarstrbm

Orthodontic tooth movement in rats was examined by histochemical techniques for some enzymes associated with bone resorption and tissue damage. The maxillary first molar was moved buccally by means of a fixed appliance with predetermined forces for periods of from 10 hours to 6 days. The activities of acid phosphatase and lactate dehydrogenase were higher in cells in the connective tissue of the periodontal membrane (PDM) than in the oral mucosa. A low orthodontic force resulted in an initial redistribution of acid phosphatase-containing cells in the PDM followed by an increased activity of acid phosphatase. The activity of lactate dehydrogenase in the PDM was not affected by low orthodontic forces. The changes in distribution and activity of acid phosphatase and lactate dehydrogenase incident to a high orthodontic force were similar to those seen incident to a low force. However, there was one definite difference. A zone which lacked acid phosphatase activity and lactate dehydrogenase activity developed in the most compressed areas of the PDM. Prostaglandin synthetase activity was found exclusively in the bone marrow and seemed not to be affected by the orthodontic forces. However, some prostaglandin synthetase activity was found in the oral mucosa corresponding to the site of the orthodontic appliance. The adjacent bone surface was covered with cells showing an intense acid phosphatase activity. In the present study the magnitude of the orthodontic force seemed to be a determining factor for the vitality of the PDM but not for the tissue-degradation activity.

Key words: Experimental tooth movement, rats, acid phosphatase,lactate dehydrogenase,prostaglandin synthetase

W

hen an orthodontic force is applied to a tooth, the periodontal membrane (PDM) and the alveolar bone are remodeled. The alveolar bone adjacent to the pressure zones is resorbed, while new bone is formed in the tension zones. The events leading to these reactions have been the subject of many investigations. i-C Forces applied with fixed orthodontic appliances in rats have been shown to increase the number of osteoclasts and resorption lacunae in the pressure zones after 2 days. ‘3 ’ In addition to osteoclasts, resorbing macrophages and fibroblasts participate in tissue degradation on the pressure side. 8-1o All these resorptive cells have a high activity of acid hydrolases.“-‘” Furthermore, osteoclasts have been shown to have a very high activity of oxidoreductases.‘d, I5 However, there have been very few studies of these enzymes in the PDM and of possible changes in their activity with time during orthodontic tooth movement. The only studies published so far were made by Deguchi, Mori, and Takimoto,16-18 From the Department of Oral Pathology, School of Dentistry, Karoline Institutet. This article was written in partial fulfillment of the requirements for the Ph.D. degree.

62

who studied the activity of some oxidative and hydrolytic enzymes in the PDM after the insertion of a rubber dam between the first and second upper molars in rats. An increased number of osteoclasts with a high activity of succinic dehydrogenase appeared in the pressure zone in the PDM after 24 hours, but there was no marked change in the distribution of acid phosphatase and lactate dehydrogenase (LDH) in the PDM. Orthodontic tooth movement is often accompanied by the development of hyaline zones in the compressed PDM. In light microscopic histologic sections these zones lack all cellular structures.‘, I92 *O At an ultrastructural level, however, a variety of cell fragments have been shown.4-fi Experimental studies on cell death have shown that dying cells loose their lactate dehydrogenase activity early in the degenerative process, while lysosomal enzymes are among the last to disappear.*l The purpose of the present investigation was to study the activity of acid phosphatase and LDH in the PDM and the alveolar bone as indicators of bone resorption and tissue damage during orthodontic tooth movement. In the present study a modified orthodontic appliance ,22 originally constructed by Kvam,’ was OOOZ-9416/83/010062+14$01.40/0

0

1983 The C.V. Mosby Co.

Volume 83 Number 1

En~mes associated with tissue degradation

63

Table I. Distribution and activity of acid phosphatase in most compressed areas of periodontal membrane of maxillary first molar in rat

Osteocytes

Buccal p*essure zone

7 t

-

r 0

i

I,

r

i

:

r

Bone Treatment

Force

time

10 hours 1 day

3 days 4 days 6 days

Low High Low High

force force force force

(50 mN) (330 mN) (60 mN) (300 mN)

Low High Low High Low High

force force force force force force

(50 mN) (250 mN) (50 mN) (250 mN) (60 mN) (360 mN)

WlA~MW

-

T 0 t 0 t 0

Lingual pressure zone

r 0 t 0 t ‘r’ 0

Noncompressed areas

i -

0 = No activity. T = Increased activity. 1 = Decreased activity. - = No change. Arrows show changes in activity in relation to the preceding treatment time. Size of arrows indicates magnitude of the change

used. With this appliance it is possible to apply welldefined forces to a tooth. This is essential for a high reproducibility of the tissue reactions in the PDM and the alveolar bone. In addition, we found it of interest to study the activity of prostaglandin synthetase since some prostaglandins have been shown to be important local activators of bone resorption.23-2fi MATERIAL AND METHODS General procedure The maxillary right first molar in ten SpragueDawley rats was moved buccally by means of a fixed appliance with predetermined forces ranging from 50 mN (5.0 gram force) to 360 mN (36.0 gram force) for periods of from 10 hours to 6 days. At the end of each treatment period the rats were killed. After sacrifice the upper jaw from each rat was embedded in carboxymethyl cellulose and frozen. Thin frozen sections were taken up on adhesive tape and immediately incubated for acid phosphatase, LDH, and prostaglandin synthetase. The glycol methacrylate technique was not used. This embedding procedure is very tedious. It does not allow histochemical detection of oxidative enzymes and prostaglandin synthetase as was part of the purpose of the study. Subsequently, it is possible to map the distribution of these enzymes throughout the entire alveolus.

was taken in alginate (Zelgan)* and a gypsum model was made. A modificatiot? of an orthodontic appliance originally constructed by Kvam’ was manufactured on the models. The orthodontic forces were adjusted on the models and the appliances were then cemented to the rat incisors. The reproduceability of a force measured on the model and in place in vivo was +- 10 mN.** The maxillary right first molar in each rat was moved in a buccal direction. The forces and treatment times are shown in Table I. The forces were selected on the basis of a previous study in which forces above 200 mN produced bleeding in the pressure zones.22 They were designated high forces in this study. Forces below 100 mN, which did not produce bleeding in the pressure zones, were defined as low forces. Five rats were treated with high forces and five rats with low forces. After 10 hours, 1, 3, 4, and 6 days one rat from each group was killed by ether inhalation and decapitated, followed by a final measurement of the orthodontic force.” At the same time, the clinical appearance of the gingiva around the orthodontic spring and the arch wire was examined through a dissecting microscope. Two nontreated rats of the same weight were included for control purposes. Histochemical

procedure

Ten Sprague-Dawley rats weighing 280 + 40 grams each were treated orthodontically. Each rat was anesthetized with Hypnorm vet* in doses of 1 ml. per kilogram of body weight. An impression of each upper jaw

The upper jaw from each rat was dissected free, embedded in a 4 percent aqueous solution of carboxymethyl cellulose, and frozen at -70 degrees in hexane cooled with solid carbon dioxide. In order to obtain sections in the same plane from all specimens, the jaws were reproducibly fixed during the embedding.“’ Frozen sections (10 pm) were taken up on adhesive tapej

*Leo PharmaceuticalCompany, Helsingborg,Sweden.

tNo. 190, MinnesotaMining and ManufacturingCompany..St. Paul. Mml.

Orthodontic treatment

*De Trey, London, England.

64

Lilja, Lindskog, and Hammarstrijm

fig. 1. Acid phosphatase activity in the palatal median bone suture of a nontreated rat. The enzyme activity was higher in the suture than in the oral mucosa(OM). E, Oral epithelium. PB, Palatal bone. (Magnification, x20.)

according to the method of Ullberg.*‘* 28The mesial roots of the two upper first molars were sectionedin a frontal aspect,extendingforty levels 20 pm apart in a distal direction (total distance, 800 pm). Every third section was immediately incubatedfor acid phosphatase (E.C. 3.1.3.2), LDH (E.C. 1.1.1.27), or prostaglandin synthetase(E.C. 1.14.99.1).* The simultaneousazocouplingmethodzgwas used to visualize acid phosphataseactivity. The incubation was performed at pH 5.0 for 10 m inutes at 37” C., using 4 m M Ix-naphthyl acid phosphatase(Sigma N-7000) as substrateand 3 m M hexazotizedpararosanilin as coupling agentaccordingto the methodof Barka and Andersson,30modified by Lojda, Veierek, and Pelichova.31For hexazotization, equal amounts of *Enzyme Commission’s classification of enzymes

Am. J. Orthod. Januarv 1983

Fig. 1A. Acid phosphatase activity in a frontal frozen section of nontreated tooth and alveolar bone. The activity was high in the oral epithelium (E), and in the periodontal membrane (PLIM) cells with a very high enzyme activity were randomly distributed along the bone surface of the alveolus. BC, Buccal alveolar crest. D, Dentin. (Magnification, x6.5.)

pararosanilin(Sigma 3X0), dissolved in hot 2N HCl (40 mg./ml.) and a 4 percent sodium nitrite solution, were m ixed fresh before each incubation; 0.5 m l. of this m ixture was addedto the incubationmedium. Incubationfor LDH was performedby the tetrazolium method32using 0.1 M DL-lactic acid (Sigma 1375) as substrateand 0.1 m M nitro blue tetrazolium for the electron capture(Sigma N-6876). The sections were incubatedat pH 7.2 for 20 m inutes at 37”C. Prostaglandinsynthetaseactivity was detectedby the benzidinetechnique33at pH 8.0, accordingto the method of Janzenand Nugkeren.34The medium contained 0.15 m M 8.11.14-eicosatrienoicacid (Sigma E-4504) as substrateand 0.5 m M 3.3-diaminobenzidine (Sigma D-5637) for the visualization of enzyme

Volume a3 Number 1

Enzymes associated with tissue degradation

65

Fig. 1 B. Detail showing acid phosphatase activity at the buccal alveolar crest (SC). The enzyme activity was higher on the bony side of the alveolus than on the dental side. Note the high activity in the supracrestal connective tissue close to the tooth. D, Dentin. E, Oral epithelium. PDM, Periodontal membrane. (Magnification, x20.)

Fig. 2. Acid phosphatase activity in the buccal pressure zone incident to a low orthodontic force. Enzyme activity increased, compared to the control, incident to the orthodontic force after 10 hours. SC, Buccal alveolar crest. C, Cementum. D, Dentin. PDM, Periodontal membrane. (Magnification, x20.)

activity. The incubation time was 30 minutes at 37” C. Control incubation with complete incubation media except substrate showed no staining of the sections. Following incubation, the sections were washed in distilled water in vacua to remove gas bubbles and mounted in glycerin-gelatin (Kebo 9242). All concentrations indicate final concentrations in the incubation media.

had been produced by the orthodontic forces, and thus two pressure zones (a buccal-cervical and a lingualapical zone) developed in the PDM. Step-serial sectioning made it possible to follow the distribution and activity of all three enzymes throughout the entire PDM and the alveolar bone surrounding the mesial roots of the first molars. Despite the fact that the specimens were not decalcified, the relationship between the PDM, the root, and the alveolar bone was well preserved in the frozen sections. In the nontreated rats the activities of acid phosphatase and LDH were higher in the cells in the connective tissue of the PDM and the bone sutures, such as the palatal median bone suture, than in the oral mucosa. In addition, cells with a very high acid phos-

RESULTS

The appliances were well tolerated by the experimental animals. Clinical inspection of the gingiva around the experimental teeth revealed that a mild inflammation had developed under the orthodontic springs and around the arch wires. A tipping movement

66

Lilja, Lindskog, and Hammarstriim

Am. J. Orthod. Junuaiy

Fig. 3. Acid phosphatase activity in the buccal pressure zone incident to a high orthodontic force after 1 day. The central part of the pressure zone (Ef) lacked enzyme activity. SC, Buccal alveolar crest. Ef, Enzyme-free zone. D, Dentin. PDhn, Periodontal membrane. (Magnification, x20.)

phatase activity were randomly distributed along the bone surface in the alveoli. Prostaglandin synthetase activity was found exclusively in the bone marrow, and no enzyme activity was demonstrable in the PDM. During the experimental period there was a gradual change in both activity and distribution of LDH and acid phosphatase. A low orthodontic force resulted in a rapid redistribution to the pressure zones of cells with a high acid phosphatase activity. In addition, there was a gradual increase in the activity of this enzyme in the pressure zones and the marrow spaces. A high acid phosphatase activity in these areas was accompanied by a high enzyme activity in the adjacent osteocytes. Low forces caused no change in the distribution and activity of LDH at any time during the treatment. A number of cells with prostaglandin synthetase activity appeared in

1983

Fig. 4. Acid phosphatase activity was very high in the lingual pressure zone incident to a high force after 3 days. The cementurn (C) and alveolar bone (AS) contain cells (arrows) with a high enzyme activity. Note that these intensely stained cells were present also adjacent to the enzyme-free area (IF). PDM, Periodontal membrane. (Original magnification, x80.) the gingival and palatal mucosa under the orthodontic appliance. No change in the activity of this enzyme was found in the bone marrow during the treatment. High forces induced changes in enzyme activity and distribution similar to those induced by low forces. In the most compressed parts of the PDM, however, a zone lacking both acid phosphatase and LDH developed. The changes in distribution and activity of the individual enzymes will be described below. Acid phosphatase A high activity of acid phosphatase was seen in the bone sutures, in the PDM beneath the crest, the gingival connective tissue close to the tooth, and a narrow band of connective tissue covering the alveolar crests in the nontreated rats (Fig. 1). The enzyme activity was

Volume 83 Number 1

Enzymes associated with tissue degradation

67

Fig. 5A. Frozen section from a rat treated with a high force for 4 days. Acid phosphatase activity was very high in the bone marrow spaces (B/V) and on the pressure sides. Note the enzyme-free area in the buccal pressure zone (arrow). PC, Palatal alveolar crest. (Magnification, x5.)

Fig. 58. Frozen section of a nontreated rat from the same area as shown in Fig. 5A. Note the difference in acid phosphatase activity compared to the orthodontically treated rat in Fig. 5A. PDM, Periodontal membrane. BM, Bone marrow. PC, Palatal alveolar crest. (Magnification, x5.)

demonstrable in most cells of the PDM. It was higher on the bony side of the alveoli than on the cemental side (Fig. 1B). In addition, there were some cells which showed a very high acid phosphatase activity and which seemed to be randomly distributed along the bone surfaces of the alveoli and in the marrow spaces (Fig. 1A). After 10 hours of orthodontic treatment with a low force, cells with a very high acid phosphatase activity were found almost exclusively in the pressure zones (Fig. 2, Table I). This distribution of cells with a high acid phosphatase activity was consistent for the remaining treatment periods. However, there was a gradual change in the activity of acid phosphatase in the PDM and alveolar bone with time. After 1 day a slightly increased enzyme activity was demonstrated in the cells

of the bone marrow close to the pressure zones (Table I). The enzyme activity of the bone marrow cells had increased markedly after 3 and 4 days of orthodontic treatment. By this time the buccal and lingual pressure zones exhibited an increased acid phosphatase activity (Table I). The increased acid phosphatase activity in the PDM and bone marrow was accompanied by an elevated enzyme activity in the adjacent osteocytes and cementocytes. After 6 days of orthodontic treatment with a low force the cells in the PDM, the alveolar bone, and the bone marrow exhibited a further increase in enzyme activity (Table I). It could not be determined whether the elevated enzyme activity was due to an increased enzyme activity of the osteoclasts or an increased number of osteoclasts, or whether the increased enzyme activity was confined to cells other than osteo-

66

Lilja, Lindskog, and Hammarstrhn

Fig. 6. Acid phosphatase activity was very high in the DUCCal pressure zone incident to a low orthodontic force after 6 days. Resorption lacunae (arrows) were found on the intraalveolar bone surface. BC, Buccal alveolar crest. PDM, Periodontal membrane. 0, Dentin. (Magnification, x20.)

clasts. Signs of bone resorption were now evident, and resorption lacunae filled with cells with a very high acid phosphatase activity bordered the entire bone surface of the pressure zones (Fig. 6). In addition, cells with a very high acid phosphatase activity were found in resorption lacunae on the palatal bone surface below the oral epithelium (Fig. 8.4). This was associated with an elevated activity of the adjacent osteocytes (Fig. 8B). The changes in activity and distribution of acid phosphatase incident to a high orthodontic force were similar to those seen incident to a low force. Thus, there was a gradual increase in acid phosphatase activity with time in the PDM and bone marrow of the orthodontically treated rats (Figs. 5, A and B). However, there was one definite difference-a zone devoid of enzyme activity in the pressure zones. This zone devoid of acid phosphatase activity had developed in

Fig. 7. Acid phosphatase activity in the buccal pressure zone incident to a high orthodontic force after 6 days. Enzyme-free zone (EF) surrounded by a very high activity of acid phosphatase. Resorption lacunae (arrowsj were found on the it-&a-alveolar bone surface and at the alveolar crest. BC, Buccal alveolar crest. PDM, Periodontal membrane. 0, Dentin. (Magnification, x20.)

the buccal pressure zone after 1 day (Fig. 3, Table I). The lingual pressure zones exhibited enzyme-free areas incident to a high orthodontic force after 3 days (Fig. 4, Table I). This pattern, seen after 3 days, was consistent after 4 and 6 days of orthodontic treatment with a high force. The enzyme-free areas in the PDM were surrounded by cells with a very high activity of acid phosphatase (Fig. 4). Resorption lacunae were found in the marrow spaces facing the zones devoid of acid phosphatase and on the buccal alveolar crest in the rat treated with a high force (Fig. 7). Lactate dehydrogenase

(LDH)

In the nontreated rats a high activity of LDH was seen in the intra-alveolar part of the PDM, the gingival connective tissue close to the tooth, the periosteum

Enzymes associated with tissue degradation

Volume 83 Number 1

69

Fig. 8A. Frozen section of the palatal bone (P6) surface of a rat after 6 days of orthodontic treatment with a low force. Cells (arrows) with an intense acid phosphatase activity outlined the bone surface below the location of the orthodontic appliance. D, Dentin. f, Epithelium. PDM, Periodontal membrane. (Magnification x20.)

Fig. 8B. Detail showing the palatal bone surface (PB) and the adjacent connective tissue (CT). Osteocytes (arrows) adjacent to an area with a very high acid phosphatase activity exhibited an intense enzyme activity. (Magnification, x 130.)

covering the alveolar crests, and the sutures of the scull (Fig. 9, A and B). LDH activity was demonstrable in all cells. The enzyme activity was rather uniform in the cells of the PDM, with the exception of some cells with a markedly higher enzyme activity close to the bone

surface of the alveoli. A low LDH activity was seen in ail of the osteocytes and in the cementocytes in the superficial layer of the cementurn. The cementocytes in the deeper parts of the cementum facing the dentin lacked demonstrable LDH activity. A low orthodontic

70

Lilja, Lindskog, and Hammarstriim

Fig. 9A. Lactate dehydrogenase activity in a frontal frozen section of nontreated tooth and alveolar bone. Enzyme activity was high in the intra-alveolar part of the periodontal membrane (PDM), the gingival connective tissue close to the tooth, and the oral epithelium (E). BC, Buccal alveolar crest. (Magnification, x20.) force did not affect the distribution and activity of LDH at any time (Fig. 10). After 1 day incident to a high orthodontic force a zone devoid of demonstrable LDH activity developed in the buccal pressure zone (Fig. 11). After 3 days both pressure zones lacked demonstrable LDH activity incident to a high orthodontic force. This pattern remained unchanged after 4 and 6 days. The osteocytes close to the enzyme-free areas had a normal LDH activity (Fig. 12). Prostaglandin synthetase Prostaglandin synthetase activity was found exclusively in the bone marrow in the nontreated rats and in rats treated for 10 hours with either low or high forces.

Am. J. Orthod. Januaty 1983

Fig. 98. Lactate dehydrogenase activity in the palatal median bone suture of a nontreated rat. The enzyme activity was higher in the suture than in the oral mucosa (OM). E, Oral epithelium. PB, Palatal bone. (Magnification, x20.)

The distribution of this enzyme activity was uneven in the marrow spaces and seemed to be confined to specific cells. After 1 day of treatment with a low or a high force, infiltrates of prostaglandin synthetase-containing cells were seen also below the palatal gingival pocket and in the oral mucosa corresponding to the site of the orthodontic appliance (Fig. 13). The activity and distribution of the enzyme remained unchanged throughout the experimental period. No prostaglandin synthetase activity was at any time histochemically demonstrable in the PDM by the present method. DISCUSSION The PDM is a specialized connective tissue which attaches the teeth to the jaw and, at the same time, separates the dental roots from the surrounding alveolar bone. The organization of the PDM has been compared

Volume a3 Number 1

Enzymes associated with tissue degradation

71

Fig. 10. Frozen section of the buccal pressure zone from a tooth moved in a buccal direction for 3 days with a low force. Lactate dehydrogenase was high, uniform, and not affected by the orthodontic treatment. E, Oral epithelium. SC, Buccal alveolar crest, PDM, Periodontal membrane. D, Dentin. (Magnification, x20.)

Fig. 11. Frozen section of the buccal pressure zone from a tooth moved in a buccal direction for 3 days with a high force. An area devoid of lactate dehydrogenase (LDH) activity was found in the buccal pressure zone. EF, Enzyme-free zone. E, Oral epithelium. BC, Buccal alveolar crest. PDM, Periodontal membrane. 0, Dentin. (Magnification, x20.)

with the sutures of the skull bones.35 It is therefore of interest to note that the activities of acid phosphatase36,37 and LDH in the PDM as well as in the palatal median bone suture were similar to and higher than in the connective tissue of the oral mucosa. The high activities of LDH and acid phosphatase in the PDM probably reflect a high metabolic activity and a rapid turnover of the connective tissue .3*40 The metabolic activity seemed to be higher near the bone surface than near the cemental surface in the alveoli. A more rapid turnover near the bone side as compared to the cemental side has been found by means of autoradiography.41a42Furthermore, acid phosphatase is a lysosomal enzyme which has a high activity in bone-resorbing cells, such as osteoclasts and macrophages.“, 12,43The large cells with a

very high activity of this enzyme in the present study were probably involved in bone resorption, while acid phosphatase activity in the PDM in general reflected remodeling of periodontal fibers.‘“, 44 During the first 10 hours of orthodontic treatment the distribution of acid phosphatase-containing cells changed from a nonspecific distribution in the alveolus to a local accumulation in the pressure zones. They may have been redistributed from other areas in the PDM, or they may be new osteoclasts from the bone marrow. The mechanisms which direct the osteoclastic resorptive activity to specific sites on the bone surfaces are not known. Piezoelectricity45-48 streaming potentials,4s and chemotaxis due to local tissue damagejO*51 have been proposed.

72

Lilja, Lindskog, and Hammarstrijm

Am. J. Onhod. Janualy 1983

Fig. 12. Frontal frozen section of the lingual pressure zone incident to a high orthodontic force after 3 days. The osteocytes and cementocytes (arrows) in the vicinity of the enzyme-free zone (EF) exhibited a moderate lactate dehydrogenase activity. C, Cementum. AB, Alveolar bone. (Magnification, x 130.)

It has been shown that osteoclasts originate from the bone marrow.52z 53The increased acid phosphatase activity in the bone marrow, which was noted as early as 1 day after the beginning of the orthodontic treatment, could be a sign of increased formation of osteoclasts. The fact that prostaglandin synthetase was limited to the marrow spaces lends further support to this idea. The formation of osteoclasts in the bone marrow might be stimulated by the orthodontic treatment and followed by a migration of these cells to the pressure zones in the PDM. Yamasaki and associatesS4have presented indirect evidence that prostaglandins are involved in bone resorption during orthodontic tooth movement. After administration of indomethacin, an inhibitor of prostaglandin synthetase activity, they found that the number of osteoclasts decreased during experimental tooth movement. Numerous cells with a demonstrable prostaglandin synthetase activity were found in the palatal mucosa under the orthodontic appliance. These were probably inflammatory cells.55 Prostaglandins of the E and F series have been reported to be powerful mediators of bone resorption.23, 24* 26 It is tempting to suggest a local relation between the prostaglandin synthetase activity in the oral mucosa and the resorption of the neighboring palatal bone. Compression of the PDM to a certain degree induces a hyalinization of the most compressed areas.j6

In the present study neither LDH nor acid phosphatase activity was demonstrable in the central parts of the pressure zones when the PDM was compressed by forces exceeding 200 mN. This lack of enzyme activity during experimental tooth movement has not previously been shown. LDH participates in the anaerobic glycolysis and is thus involved in the energy metabolism of the cell.j7 The activity of this enzyme has previously been used as a measure of cell vitality with an accuracy similar to that of the trypan blue exclusion test.j8, 5g Our findings of a nonvital tissue are thus in agreement with previous morphologic studies in which the hyaline zone has been described as an area of local aseptic necrosis. 4-6 The hyaline zone is resistant to degradation and persists for a long time in the pressure zone, depending on the magnitude of the force.j6 The lack of lysosomal enzymes in the hyaline zone, as shown by staining for acid phosphatase, might explain why the elimination of the hyaline zone is a slow process and why it proceeds from its periphery. The presence of necrotic tissue in the PDM could, however, be important for the direction of the resorptive activity. Necrotic tissue is chemotactic on macrophages.‘jOThese cells have been found surrounding the hyaline zone. s, lo In the present study this could be seen as a very high acid phosphatase activity apical and coronal to the hyaline zones, which seemed to be directed to remove this zone.

Volume83

Enzymes associated with tissue degradation

Number 1

73

Osteocytes are capable of both bone resorption and bone formation.61, 62In the present study resorption of a bone surface always seemed to be associated with a resorptive activity in the adjacent osteocytes. Resorptive osteocytes are rarely found; only about 1 percent of the total osteocytes in the skeleton are in the resorptive stage. 63There is morphologic evidence of a communication between bone-lining cells and osteocytes.37S64 The findings in this study support the concept of a functional relationship between osteocytes and resorbing cells on a bone surface.65 The question of whether or not the tissue reaction incident to high and low orthodontic forces differs has been the subject of many investigations. Evidence both favoring and contradicting this view has been presented.” 56*~3 67In the present study the magnitude of the force seemed to be a determining factor for the vitality of the PDM but not for the tissue-degradation activity. This investigation cal Research Council Dentistry, Karolinska SBllskapet

was supported by the Swedish Medi(Grant No. X06001-02A), School of Institutet and Svenska TandI&are-

REFERENCES 1. Kvam, E.: Tissue changes incident to movement of rat molars, Thesis, Universitetsforlaget, Oslo, 1967. 2. Lopez, 0. R., Parodi, R. J., Ubios, A. M., Carranza, F. A., Jr., and Cabrini, R. L.: Histologic and histometric study of bone resorption after tooth movement in rats, .l. Periodont. Res. 8:327-333, 1973. 3. Macapanpan, L. C., Weinmann, J. P., and Brodie, A. G.: Early tissue changes following tooth movement in rats, Angle Orthod. 24:79-95) 1974. 4. Rygh, P.: Ultrastructural cellular reactions in pressure zones of rat molar periodontium incident to orthodontic tooth movement, Acta Odontol. Stand. 30:575-593, 1972. 5. Rygh, P.: Ultrastructural vascular changes in pressure zones of rat molar periodontium incident to orthodontic movement, Stand. J. Dent. Res. 80:307-321, 1972. 6. Rygh, P.: Ultrastructural changes in pressure zones of human periodontium incident to orthodontic tooth movement, Acta Odontol. Stand. 31: 109-122, 1973. 7. Azuma, M.: Study on histologic changes of periodontal membrane incident to experimental tooth movement, Bull. Tokyo Med. Dent. Univ. 17:149-178, 1970. 8. Kurihara, S.: An electron microscopic observation on cells found in bone resorption area incident to experimental tooth movement, Bull. Tokyo Med. Dent. Univ. 24:103-123, 1977. 9. Kvam, E.: A study of the cell-free zone following experimental tooth movement in rat, Trans. Eur. Orthod. Sot., pp. 419-434, 1970. 10. Rygh, P.: Elimination of hyalinized periodontal tissues associated with orthodontic tooth movement, Stand. 3. Dent. Res. 82:57-73, 1974. 11. Burstone, M. S.: Histochemical demonstration of acid phosphatase activity in osteoclasts, J. Histochem. Cytochem. 7:3941, 19.59.

Fig. 13. Frontal frozen section of the palatal alveolar bone close to a tooth treated with a low orthodontic force for 1 day. Infiltrates of prostaglandin synthetase-containing cells were seen in the oral mucosa (Ok4), above the alveolar bone, corresponding to the site of the orthodontic appliance. 0, Dentin. PC, Palatal alveolar crest. PDM, Periodontal membrane. (Magnification, x 15.) 12. Cohn, Z. A., and Wiener, E.: The particulate hydrolases of macrophages. I. Comparative enzymology, isolation and properties, J. Exp. Med. 118:991-1008, 1963. 13. Deporter, D. A., and Ten Cate, A. R.: Fine structural localization of acid and alkaline phosphatase in collagen-containing vesicles of fibroblasts, J. Anat. 114~457-461, 1973. 14. Balogh, K., Dudley, H. R., and Cohen, R. B.: Oxidative enzyme activity in skeletal cartilage and bone; a histochemical study, Lab. Invest. l&839-845, 1961. 15. Burstone, M. S.: Histochemical demonstration of succinic dehydrogenase activity in osteoclasts, Nature 185886, 1960. 16. Deguchi, T., and Mori, M.: Histochemical observations on oxidative enzymes in periodontal tissue during experimental tooth movement in rat, Arch. Oral Biol. 13:49-59, 1968. 17. Takimoto, K., Deguchi, T., and Mori, M.: Histochemical detection of succinic dehydrogenase in osteoclasts following experimental tooth movement, J. Dent. Res. 45: 1473-1476, 1966.

74

Lilja,

Lindskog,

Am. J. Orthod. Januay 1983

and Hammarstrtim

18. Takimoto, K., Deguchi, T., and Mori, M.: Histochemical detection of acid and alkaline phosphatase in periodontal tissue following experimental tooth movement, J. Dent. Res. 47:340, 1968. 19. Sandstedt, C.: Einige heitr~ge zur theorie der zahnregulierung, Nord Tandlaeg. Tidskr. 5:236-256, 1904. 20. Sandstedt, C.: Einige beitrige zur theorie der zahnmgulienmg, Nord Tandlaeg. Tidskr. 6:1-25, 1905. 21. Trump, B. S., and Mergner, W. I.: Cell injury. In Zweifach, B. W., Garant, L., and McCluskey, R. T. (editors): The inflammatory process, New York 1974, Academic Press, Inc., chap. 3. 22. Lilja, E., Lindskog, S., and Hammarstriim, L.: Orthodontic forces and periodontal compression-A new method and its application, Acta Odontol. Stand. 39:367-378, 1981. 23. Elattar, T. M. A.: Prostaglandins: Physiology, biochemistry, pharmacology and clinical applications, J. Oral Pathol. 7:253282, 1978. 24. Holtrop, M. E., King, G. I., and Raisy, L. G.: Factors influencing osteoclast activity as measured by ultrastructural morphometry. In Coop, D. H., and Talmage, R. V. (editors): Proceedings of the Sixth Parathyroid Conference, 1977, Amsterdam, 1978, Excerpta Medica. 25. Klein, D. C., and Raisz, L. G.: Pmstaglandins: Stimulation of bone resorption in tissue culture, Endocrinology 86: 1436-1440, 1970. 26. Tashijan, A. H., Jr.: Pmstaglandins as local mediators of bone resorption. In Horton, .I. E., Tarpley, T. M., and Davis, W. F. (editors): Mechanisms of localized bone loss, Calcif. Tiss. Abstr. Suppl., pp. 173-179, 1978. 27. Ullberg, S.: Studies on the distribution and fate of S3”labelled benzylpenicillin in the body, Acta Radiol., Suppl. 118, 1954. 28. Ullberg, S.: Autoradiographic studies on the distribution of labelled drugs in the body, Second U.N. Int. Conf. Peaceful Uses Atomic Energy 24:248-254, 1958. 29. Burstone, M. S.: Histochemical comparison of naphthol ASphosphates for the demonstration of phosphatases, J. Natl. Cancer Inst. 20:601-616, 1958. 30. Barka, T., and Anderson, P. J.: Histochemical methods for acid phosphatase using hexazonium pararosanilin as coupler, J. His&hem. Cytochem. 10:741-753, 1962. 3 1. Lojda, Z., Vecerek, B _, and Pelichova, H.: Some remarks concerning the histochemical detection of acid phosphatase by azocoupling reactions, Histochemie 3:428-454, 1964. 32. Pears, A. G. E.: Histochemistry, theoretical and applied, London, 1960. J. & A. Churchill, Ltd., p. 536. 33. Graham, R. C.. and Kamowsky, M. J.: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem. 14:291-302, 1966. 34. Janzen, F. H. A., and Nugkemn, G. H.: His&chemical localization of prostaglandin synthetase, Histochemie 27: 159-164, 1971. 35. Evans, C. A.: Facial growth; mechanisms. In Shaw, J. H., Sweeney, E. A., Cappuccino, C. C., and Meller, S. M. (editors): Textbook of oral biology, Philadelphia, 1978, W. B. Saunders Company, p. 67. 36. Todo, H., and Mori, M.: Histochemical observations on hydrolytic and oxidative enzymes in tooth supporting tissues in mature rodents, Arch. Oral Biol. 2:921-930, 1966. 37. Vilmann, H.: Characteristics of growing bone surfaces, Stand. J. Dent. Res. 87:65-72, 1979. 38. Rippin, J. W.: Collagen turnover in the periodontal ligament

39.

40.

41. 42.

under normal and altered functional forces. I. Young rat molars, J. Periodont. Res. ll:lOl-107, 1976. Rippin, J. W.: Collagen turnover in the periodontal ligament under normal and altered functional forces. II. Adult rat molars, J. Periodont. Res. 13:149-154, 1978. Weiss, R., Stahl, S. S., and Tonna, E. A.: Functional demands on the cell proliferative activity of the rat periodontium studied autoradiographically, J. Dent. Res. 47:1153-l 157, 1968. CrumIey, P. I.: Collagen formation in the normal and stressed periodontium, Periodontics 2:53-61, 1964. Diaz, E.: Periodontal ligament collagen response to tooth movement: Histochemical and autoradiographic reactions, AM. J. ORTHOD. 73:443-458,

1978.

43. Parakkal, P. F.: Involvement of macrophages in collagen resorption, J. Cell Biol. 41:345-354, 1969. 44. Beertsen, W.: Remodelling of collagen fibers in the periodontal ligament and the supra-alveolar region, Angle Orthod. 49:218224, 1979. 45. Bassett, C. A. L.: Electromechanical factors regulating bone architecture. In Fleisch, H., Blachwcod, H. J. J., and Owen, H. (editors): Third European Symposium on Calcified Tissues, Berlin, 1966, Springer-Verlag, pp. 78-89. 46. Cochran, G. V. B., Pawluk, R. J., and Bassett, C. A. L.: Stress-generated electric potentials in the mandible and teeth, Arch. Oral Biol. 12:917-920, 1967. 47. Gillooly, C. J.. Hosley, R. T., Mathews, J. R., and Jewett, D. L.: Electric potentials recorded from mandibular alveolar bone as a result of forces applied to the tooth, AM. J. ORTHOD. 54:649-654, 1968. 48. Zengo, A. N., Bassett, C. A. L., and Prountzos, G: In vivo bioelectric potentials in the dentoalveolar complex, AM. J. ORTHOD 66: 130- 139, 1974. 49. Bassett, C. A. L.: Biophysical principles affecting bone structure. In Boume, G. H. (editor): The biochemistry and physiology of bone, New York, 1971, Academic Press, Inc. 50. Dexter, T. M., and Spooncer, E.: Resorbing bone is chemotactic for monocytes, Nature 275: 132-136, 1978. 5 1. Hurley, J. V., and Spector, W. G.: Endogenous factors responsible for leucocytic emigration in viva, J. Pathol. Bacterial. 82:403-420, 1961. 52. Fishman, D. A., and Hay, E. D.: Origin of osteoclasts from mononuclear leucocytes in regenerating new limbs, Anat. Rec. 143:329,

1962.

53. Leder, L-D.: Der Blutmonocyt., Berlin, 1967, Springer-Verlag, pp. 87-155. 54. Yamasaki, K., Miura, E., and Suda, T.: Prostaglandin as a mediator of bone resorption induced by experimental tooth movement in rats, J. Dent. Res. 59:1635-1642, 1980. 55. Elattar, T. M. A.: Prostaglandins: Physiology, biochemistry, pharmacology and clinical applications, J. Oral Patbol. 7: 175. 207, 1978. 56. Reitan, K.: Biomechanical principles and reactions. In Graber, T. M. (editor): Current orthodontic principles and techniques, vol. 1, Philadelphia, 1969, W. B. Saunders Company, pp. 56150. 57. Lehninger, A. L.: Biochemistry, ed. 2, New York, 1977, Worth Publishers, Inc., p. 431. 58. Berg, T., Boman, D., and Seglen, P. 0.: Induction of tryptophan oxygenase in primary rat liver suspensions by glucocorticoid hormone, Exp. Cell Res. 72:571-574, 1972. 59. Rubin, K., Kjellen, L., and (ibrink, B.: Intercellular adhesion between juvenile liver cells, Exp. Cell Res. 109:413-422, 1977. 60. Stashenko, P. P.: ImmunoIogy. In Shaw, J. H., Sweeney,

Volume 83 Number I

E. A., Cappuccino, C. C., and Meller, S. M. (editors). Textbook of oral biology. Philadelphia, 1978, W. B. Saunders Company, p. 171. 61. Jande, S. S.: Fine structural study of osteocytes and their surrounding bone matrix with respect of their age in young chicks, J. Ultrastmct. Res. 37:279-300, 1971. 62. Jande, S. S., and Belanger, L. F.: Electron microscopy of osteocytes and the pericellular matrix in rat trabecular bone, Calcif. Tissue Res. 6:280-289, 1971. 63. Simmons, D. J.: Fracture healing. In Urist, M. R. (editor): Fundamental and clinical bone physiology, Philadelphia, 1980, J. B. Lippincott Company, p. 289.

Enzymesassociatedwith tissue degradation 75 64. Whitson, S. W.: Tight junction formation in the osteon, Clin. Orthop. Rel. Res. 86:206-213, 1972. 65. Baylink, D. J., and Wergedal, J. E.: Bone formation and resorption by osteocytes. In Nichols, G., and Wasserman, R. H. (editors): Cellular mechanisms for calcium transfer and homeostasis, New York, 1971, Academic Press, Inc., pp. 257-286. 66. Botting, P. S., and Storey, E.: Bone changes associated with the application of very low forces to incisor teeth in the guinea pig, Aust. Dent. J. l&240-245, 1973. 67. Utley, R. K.: The activity of alveolar bone incident to orthodontic tooth movement as studied by oxytetracycline-induced fluorescence, AM. J. ORTHOD. 54:167-201, 1968.