ORTHODONTIC TOOTH MOVEMENT by A THESIS IN ANATOMY

ALVEOLAR BONE RESORPTION IN RESPONSE TO

ORTHODONTIC TOOTH MOVEMENT

by

VERNON KAI-YING LIU, B.S.

A THESIS

IN

ANATOMY

Submitted to the Graduate Faculty of Texas Tech University School of Medicine at Lubbock

in partial Fulfillment of the requirements for

the Degree of

MASTER OF SCIENCE

Approved

Accepted

~n DeAiV of t^^ Graduate School v.-

May, 1979

?>^6

H^>X$ ACKNOWLEDGEMENTS

I would like to express my thanks and sincere appreciation to

Dr. John A. Yee for his continuous advice, encouragement and patience

during my graduate training. He has sacrificed his leisure time to

assist me whenever necessary.

Dr. Peter K. T. Pang, in addition to his continuous encouragement

and professional advice, has given freely of himself. Together Drs.

Yee and Pang have given me help to regain my confidence during diffi­

cult times.

I also wish to thank the other members of my committee, Drs.

James C. Hutson, Roger R. Markwald and William G. Seliger for their

time and critical evaluation of my thesis.

To my parents, I dedicate this work for all the sacrifices they

made for their children.

Special thanks to Ms. Yolanda Andrade for typing this manuscript

and Mrs. Cindy Frisbie for her patience in assisting me with the

illustrations.

11

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii

ABSTRACT iv

LIST OF FIGURES vi

I. INTRODUCTION 1

HISTORICAL BACKGROUND 5

II. MATERIALS AND METHODS 19

HISTOLOGIC PREPARATION AND CRITERIA 21

III. RESULTS 23

LIGHT MICROSCOPY OF PDL IN THE APICAL REGION 23

QUALITATIVE OBSERVATION OF PDL 23

QUANTITATIVE ANALYSIS OF PDL 24

LIGHT MICROSCOPY OF PDL IN THE INTERRADICULAR REGION 25

QUALITATIVE OBSERVATION OF PDL 25

QUANTITATIVE ANALYSIS OF PDL 26

MITOTIC ACTIVITY OF PDL CELLS IN THE INTERRADICULAR REGION . . 28

IV. DISCUSSION 57

LITERATURE CITED 64

111

ABSTRACT

In apical and interradicular regions of the periodontium in rats,

the generation of osteoclasts in response to mechanical pressure

created by orhtodontic tooth movement has been studied quantitatively

with light microscopy. Young male Sprague-Dawley rats were sacrificed

at 12, 18, 24, 30, 36, 44, 48, 54 and 72 hours post-orthodontic stimu­

lation. The proliferative activity of PDL cells in the interradicular

region was assessed by determining the mitotic index of PDL cells

following the administration of vinblastin sulfate. The rats for this

study were sacrificed at 12, 18, 24, 30 and 36 hours post-orthodontic

stimulation.

Following orthodontic tooth movement, maximal pathologic tissue

changes such as hyalinization of PDL and vascular disruption in the

apical region were observed by 30 hours post-stimulation. The repair

of periodontal tissues in this region was apparent by 54 hours. A

significant increase in the number of osteoclasts was observed at all

experimental periods. The total number of osteoclasts was maximal by

44 hours post-stimulation. In the interradicular region, significant

pathologic tissue disruption was not observed. A significant increase

in the number of osteoclasts was observed at all experimental periods.

These cells were generated at a rate of 0.32 osteoclast per hour. A

significant increase of alveolar bone loss was observed by 44 hours

post-stimulation. The mitotic activity of the PDL cells was maximal

by 24 hours post-stimulation. The data obtained from this study indi­

cate that mechanical pressure generated by orthodontic tooth movement

can stimulate osteoclastic alveolar bone resorption. This resorption

IV

activity is required to accommodate the displacement of the mesial

root of the first maxillary molar within its bone socket. The histo­

genesis of osteoclasts in interradicular region occurred in the

absence of inflammation. The quantitative data obtained from this

study can serve as the basis for future studies of osteoclast genera­

tion in the periodontium.

Figure

Figure

Figure

Figure

Figure

Figure

1.

2.

3.

Figure 4.

Figure 5.

6.

Figure 7.

8.

9.

Figure 10.

Figure 11.

LIST OF FIGURES

Text figure of the periodontium in rat.

Qualitative observations of non-orthodontically stimulated (controls) periodontal tissue in the apical region.

Morphologic changes of periodontal tissues by 12 hours post-stimulation in the apical region.

Morphologic changes of periodontal tissues by 54 hours post-stimulation in the apical region.

Morphologic changes of periodontal tissues by 54 hours post-stimulation in the apical region.

Quantitative analysis of osteoclasts generation in the apical region.

Quantitative analysis of osteoclast/mm bone surface in the apical region.

Quantitative analysis of nuclei/osteoclasts in the apical region.

Qualitative observations of non-orthodontically stimulated (controls) periodontal tissues in the interradicular region.

Qualitative observations of non-orthodontically-stimulated (controls) periodontal tissue in the interradicular region.

Qualitative observations of the periodontal tissue in the interradicular region by 12 hours post-stimulation.

Figure 12. Qualitative observations of the periodontal tissue in the interradicular region 36 hours post-stimulation.

Figure 13. Qualitative observations of the periodontal tissue in the interradicular region 44 hours post-stimulation.

Figure 14. Qualitative observations of the periodontal tissue in the interradicular region 72 hours post-stimulation.

Figure 15 a. Qualitative observations of the periodontal tissue in the interradicular region 72 hours post-stimulation.

Figure 15 b. Qualitative observations of the periodontal tissue in the interradicular region 72 hours post-stimulation.

vi

Figure 15 c. Qualitative observations of the periodontal tissue in the interradicular region 72 hours post-stimulation.

Figure 16. Quantitative analysis of osteoclasts generation in the interradicular region.

Figure 17. Quantitative analysis of osteoclast/mm bone surface in the interradicular region.

Figure 18. Quantitative analysis of nuclei/osteoclast in the inter­radicular region.

Figure 19. Quantitative analysis of percent alveolar bone in the interradicular region.

Figure 20. Mitotic activity of PDL Fibroblasts in the interradicular region of non-orthodontically stimulated (controls) rats.

Figure 21. Mitotic activity of PDL Fibroblasts in the interradicular region by 24 hours post-stimulation.

Figure 22. Mitotic index of PDL Fibroblasts in the interradicular region.

Vll

INTRODUCTION

The periodontium is a connective tissue organ which is composed

of cementum, periodontal ligament (PDL), alveolar bone and the lamina

propia of the gingiva. Embryologically, the periodontium is derived

from cells of dental follicle whose origin is from ectomesenchyme (Ten

Cate^lt al., 1971, 1972).

The periodontium is constructed in such a way that the PDL is

attached to the cementum at one end and the alveolar bone at the other.

Based on its anatomical relationships, the PDL serves the following

functions: 1) anchors the teeth in the alveolar bone socket; 2)

acts as a shock absorber to ease the pressure generated during masti­

cation; and 3) provides repair and wound healing capabilities follow­

ing orthodontic tooth movement and trauma (Melcher, 19 76b). Recent

studies on the migration of PDL fibroblasts suggest that PDL may also

play a role in tooth eruption (Beertsen et^ al., 1974; Beertsen, 1975).

The PDL is a dense regular collagenous connective tissue. When

examined by electron microscopy, the extracellular matrix can be seen

to be primarily composed of well organized banded collagen microfi­

brils. This fibrous component is embedded into an amorphous compo­

nent representing ground substance macromolecules such as acid muco­

polysaccharides, glycoproteins and proteoglycan (Melcher, 1976a).

While these extracellular matrical components have been demonstrated

histochemically, their nature is still somewhat unknown. Elastic

fibers are also present in the PDL but they are restricted to the

blood vessel walls only. Oxytalan fibers which appear to be immature

elastic fibers have been demonstrated in the PDL (Fullmer, 1967).

The function of these oxytalan fibers is unknown, but it has been sug-

gested that they may play a role in supporting the blood vessels in

the PDL (Fullmer, 1967).

There are various types of connective tissue cells residing with­

in the PDL, with fibroblasts being the most abundant. The main func­

tion of the PDL fibroblast is the synthesis of collagen and other

extracellular matrix macromolecules (Melcher 1976a). In addition,

phagocytosis of extracellular collagen microfibrils has been recently

described ultrastructurally in various animal species (Ten Gate, 1972;

Ten Cate^al., 1974, 1976; Listgarten, 1973; Beertsen al., 1974;

Garant, 1976). Other cell types such as mast cells, macrophages and

mononuclear leukocytes can also be found. In addition, there is

evidence which suggests that numerous populations of local progenitor

cells are also present (Melcher and Eastoe, 1969; Gould al_., 1977).

However, the nature of these progenitor cells has remained unclear.

A major problem in their identification being that progenitor cells

and functional fibroblasts are histologically indistinguishable by

light microscopy (Melcher and Eastoe, 1969) and electron microscopy

(Yee, 1979).

In order to maintain the structural and functional integrity of

the skeleton, bone matrix is constantly being remodeled. During the

remodeling process, bone resorption and formation are coupled. Bone

remodeling activity occurs in response to both biomechanical and

physiologic demands placed upon the skeleton (Frost, 1964). Similar

phenomena also occur within the periodontium. Constant turnover of

alveolar bone and PDL matrices are necessary in order to maintain

continuous tooth attachment in the alveolar bone socket. Throughout

life there is normally a constant state of physiologic tooth drift

(Stein and Weinmann, 1925). The direction of this drifting is depend­

ent on the animal species and varies between the maxillary and mandi­

bular molars. In humans, maxillary molars drift in a mesial direction

whereas a distal drift occurs in rat maxillary molar (Bjork, 1964;

Reitan and Kvam, 1971). During physiologic drift of maxillary molars

in rats, the tension which is generated in the PDL distal to the

mesial root stimulates alveolar bone formation while pressure on the

mesial surface leads to bone resorption (Oppenheim, 1911; Macapanpan

£^ al., 1954; Waldo and Rothblatt, 1954; Reitan, 1962). Bone resorp­

tion at the alveolar bone surface mesial to the PDL is due to: 1)

distal tipping of the tooth in response to abrasion in the contact

area between the first and second maxillary molars and 2) active

eruption in response to occlusal abrasion (Roberts, 1975).

Following the application of experimental orthodontic tooth

movement in rats, the alveolar bone surface distal to the mesial root

of the first maxillary molar, which is normally a site of bone for­

mation becomes a site of bone resorption (Oppenheim, 1911; Macapanpan

^ al., 1954; Waldo and Rothblatt, 1954; Reitan, 1962). Morphological

evidence obtained from previous studies suggest that mechanical pres­

sure resulting from orthodontic tooth movement can stimulate alveolar

bone resorption (Sandet, 1904; Schwarz, 1932; Reitan, 1952, 1962;

Kvam, 1972; Rygh, 1973a). The generation of osteoclasts can be

observed associated with this resorptive response.

Uncontrolled osteoclastic resorption of alveolar bone as occurs

in periodontal disease may lead to tooth detachment and eventual loss

of dentition. Although the severity of periodontal disease varies,

the most common pathologic feature is the extensive loss of alveolar

bone (Page and Schroeder, 1976). All the factors which contribute to

the extensive bone loss in periodontal disease have yet to be illucl-

dated. A more critical examination of factors which cause the gener­

ation of osteoclast in the periodontium is necessary in order to

understand the mechanisms involved in the loss of alveolar bone.

Therefore, the purpose of this study was to provide a quantitative

examination of the temporal nature of osteoclast histogenesis in the

rat periodontium following orthodontic tooth movement. The signifi­

cance of the data obtained from this study is that it will provide:

1) morphologic and histomorphometric information concerning the

biological events associated with alveolar bone resorption which

occurs as a result of orthodontic tooth movement and 2) basic data

needed for designing future studies on osteoclast histogenesis in the

periodontium.

HISTORICAL BACKGROUND

The periodontal ligament is a dense regular collagenous connec­

tive tissue. The collagen in the ligament is unique in that it under­

goes rapid turnover. Autoradiographic studies by various investiga­

tors showed that the PDL has a relatively higher rate of collagen

turnover as compared to other connective tissues such as skin and

tendon in various animal species (Stallard, 1963; Crumley, 1964;

Carneiro and Favo de Moraes, 1965; Carneiro, 1966; Anderson, 1969;

Koumas andMatthewS 1969; Skougaard et al., 1969; Baumrind and Buck,

1970; Rippin, 1976; Diaz, 1978). The alveolar bone adjacent to the

PDL was also shown to have more rapid collagen turnover than the tibia

(Crumley, 1964). Recent electron microscopic studies by Ten Gate and

co-workers (1972, 1974, 1976) have provided morphological evidence

supporting the rapid remodeling of the PDL as suggested by the pre­

vious autoradiographic studies. Other investigators demonstrated that

PDL fibroblasts are capable of degradating the extracellular collagen

in additional to their role in the synthesis of extracellular matrix

macromolecules (Ten Gate, 1972; Ten Gate £l., 1974, 1976; Listgarten

1973; Beertsen et al., 1974; Garant, 1976a).

During physiologic tooth drift, the PDL cells undergo constant

proliferation in response to functional demands by the periodontal

tissues (Messier and Leblond, 1960; Jensen and Toto, 1968; Weiss et al.,

3 1968). Autoradiographic examination of tritiated thymidine ( HTdR)

incorporation showed that these proliferating PDL cells can differen­

tiate into each functional ligament cells (fibroblasts, osteoblasts,

cementoblasts) (Toto and Magon, 1966). These functional PDL cells are

responsible for the synthesis of their corresponding extracellular

matrix macromolecules which are essential for the maintenance of the

periodontium. In order to understand how the functional and struc­

tural integrity of the periodontium is maintained, the cellular activ­

ity of PDL cells which is responsible for the extracellular matrix

turnover must be considered. Unfortunately, these PDL cells represent

a morphologically mixed population of functional fibroblasts and fibro-

blast-like progenitor cells (Melcher and Eastoe, 1969).

The proliferating progenitor cell population in the PDL undergoes

cell division and differentiation into cementoblasts, fibroblasts and

osteoblasts when under proper stimuli such as orthodontic tooth move­

ment (Baumrind and Buck, 1970; Kvam, 1972; Roberts and Jee, 1974;

Roberts, 1975; Yee £l.« > 1976) and surgical wound repair (Melcher,

1976b; Gould SLI., 1977). Following orthodontic tooth movement

achieved by inserting an elastic band between the first and second

3 maxillary molars of rats, an increased HTdR labeling index of the PDL

cells was observed at the tension side mesial to the first mesial

maxillary root (Baumrind and Buck, 1970; Roberts and Jee, 1974; Yee

3 ££ al., 1976). With multiple injection of HTdR following orthodontic

stimulation of bone formation at the tension side of the PDL, Yee

et al. (1976) demonstrated that osteoblasts were derived from the

proliferating PDL fibroblasts.

Despite numerous studies concerning the cell kinetics of the PDL

cells at the tension side of the ligament, there has been limited

study of cellular proliferation in the pressure side of the PDL.

Following the insertion of an orthodontic appliance between the first

3 and second maxillary molars in rats, Kvam (1972) reported a HTdR

labeling index of PDL cells which appeared as a bimodel shape resulted

at the pressure side of the PDL distal to the first mesial root. A

bimodel pattern in the labeling index response was due to invasion of

new PDL cells into the hyalinized area of the ligament. Migration of

cells into this region was then followed by a subsequent proliferation

of the PDL cells. The presence of labeled cementoblasts, fibroblasts,

osteoblasts and osteoclasts supports the multipotentiality of PDL

fibroblasts having cementogenic, fibrogenic and osteogenic capability

(Melcher and Eastoe, 1969). All of the above cell kinetic studies

suggest that: 1) orthodontic tooth movement can stimulate the pro­

liferation of PDL cells and 2) the presence of PDL progenitor cells

which have cementogenic, fibrogenic and osteogenic capability. Unfor­

tunately, the nature of the progenitor cells remains unknown. A

major problem being that the progenitor cells and functional fibro­

blasts are histologically indistinguishable with light microscopy and

electron microscopy (Melcher and Eastoe, 1969; Gould £t al.., 1977;

Yee, 1979). Whether a single progenitor stem cell population gives

rise to all functional PDL cells such as fibroblasts, cementoblasts

and osteoblasts; or whether each functional PDL cell is a direct

descendent of a specific population of progenitor cells still remains

unknown (Melcher, 1976a). Recent evidence obtained from studies con­

cerning surgical wound repair of the periodontium in mice (Gould et al_.,

1977) suggested that separate populations of vinblastin sulfate-

arrested mitotic cells were paravascularly located at three different

regions in the PDL of maxillary molars. Although these experimental

8

findings led Gould ejt al. (1977) to suggest that a separate population

of progenitor cells is present for each functional cell of the perio­

dontium, unequivocable evidence for this hypothesis is still lacking.

Despite the uncertain identity of the PDL progenitor cells, this

model offers several advantages over other models (fractured long bone

and endochondral osteogenesis) which have been employed to study the

skeletal tissue dynamics. Advantages of the PDL model are: 1) PDL

cells represent a population of relatively pure connective tissue

cells without the contamination of bone marrow cells; 2) the cellu­

lar response generated by tooth movement represents the normal func­

tional potential of PDL cells; and 3) a large population of osteo­

clasts can be generated at the alveolar bone surface in areas of

pressure in the PDL.

The histogenesis of osteoclasts associated with the alveolar bone

resorption has been studied in three models: 1) Mono-infection of

gnotobiotic animals with bacteria (Jordon ail., 1972; Irving et al.,

1974; Garant, 1976); 2) administration of parathyroid extract (PTE)

to generate osteoclastic resorption of alveolar bone (Toto and Magon,

1966; Roberts, 1975; Baron et al., 1977); and 3) Mechanical pressure

resulted from the orthodontic tooth movement (Kvam, 1972; Rygh, 1973a).

In gnotobiotic rats, alveolar bone resorption resulted following

mono-infection with the bacterium Actinomyces naesludii (Jordan jet al.,

1972; Irving et al., 1974; Garant, 1976b). Extensive alveolar bone

resorption similar to that manifested in periodontal disease is fre­

quently associated with constant plaque accumulation and constant

inflammatory challenge in monoinfected animals, Jordon et al_. (1972)

showed that osteoclastic bone resorption in the maxillary molar

usually occurred in the vicinity of plaque accumulation in Actinomyces

naesludii monoinfected hamster and rats. Following similar experimen-

3

tal procedure, Irving e;t al. (1974) employed H-proline autoradio­

graphy to study alveolar bone resorption at the distal root surface

which normally was the site of bone formation. Since no detectable

3 increase of H-proline labeling of the alveolar bone matrix adjacent

to the distal root of the first maxillary molar was observed and

alveolar bone resorption was not accompanied by the presence of osteo­

clasts, they concluded that alveolar bone loss was due to gradual

cessation of the bone formation rather than bone resporption. In

disagreement with Irving's interpretation of the mechanism of alveolar

bone loss, Garant (1976b), using both light and electron microscopy,

showed that alveolar bone destruction in rats monoinfected with

Actinomyces naesludii was due to osteoclastic activity. With electron

microscopy, multinucleated osteoclast-like cells were also observed

within the PDL away from the alveolar bone surface. No ruffled

borders or clear zones were developed in these osteoclast-like cells.

Garant suggests that these multinucleated cells might provide an

additional source of osteoclasts under the influence of local stimuli

such as osteoclastic activating factors (Horton et £l., 1972). When

fetal alveolar bone were explanted in. vitro and endotoxin containing

lipopolysaccharide was added into the culture medium, Hausman et al.

45 (1972, 1974) showed and increased Ca release from the alveolar bone

explant into the culture medium. He also correlated the increased

isotope release with the decrease of bone size and with an increased

10

number of osteoclasts. However, lipopolysaccharide lost the ability

to stimulate alveolar bone resorption when its lipid component was

removed. Therefore, they concluded endotoxin stimulated alveolar

bone resorption. Unfortunately, inflammatory response which occurred

in all monoinfected animals complicates the understanding of osteo­

clast generation associated with alveolar bone loss in the periodon­

tium.

Alveolar bone resorption following the administration of para­

thyroid hormone (PTH) has been studied in the periodontium. Eighteen

hours after the intraperitoneal administration of PTH, Toto and Magon

3 (1966) observed that osteoclasts containing both HTdR labeled and

unlabeled nuclei were found at the alveolar bone surface in rat man­

dibles. They suggested that osteoclasts were derived from the local

proliferating precursor cells in the PDL and the alveolar bone marrow.

Nine hours following the administration of parathyroid extract (PTE),

Roberts (1975) showed a net increase of PDL cells and osteoclasts in

zone I (adjacent to the alveolar bone surface) of the ligament. He

concluded that the net increase in PDL fibroblasts in zone I plus

this increased number of osteoclast could not be accounted for by

local mitotic activity. The additional osteoclasts were generated

from elsewhere and migrated into the ligament possibly via vascular

channels. However, whether the influx of macrophages and monocytes

into the ligament through the vascular channels had any contribution

for the histogenesis of osteoclasts was not known. In parathyroid-

ectomized rats. Baron et al. (1977) demonstrated that there was a 70%

decrease in the osteoclast population by eight days. Administration

11

of PTH into parathyroidectomized rats restored the osteoclast popula­

tion to normal size for 24 hours only. All of the above experimental

data demonstrate that PTH can stimulate osteoclastic resorption of

alveolar bone. However, they do not provide a clear indication of the

source of these osteoclasts.

During physiological drift of teeth in rats, the mesial side of

the PDL where pressure is exerted is normally a site of bone resorp­

tion (Roberts, 1975). The distal side of the PDL where tension is

generated is the site of bone formation (Oppenheim, 1911; Macapanpan

£t a]^., 1954; Waldo and Rothblatt, 1954; Reitan, 1975). Following

orthodontic tooth movement, the normal biological events are reversed.

Alveolar bone formation and resorption occur at the mesial and distal

PDL surfaces respectively (Oppenheim, 1911; Macapanpan et al., 1954;

Waldo and Rothblatt, 1954; Reitan, 1975). Although orthodontic stimu­

lation of alveolar bone formation at the tension side of the PDL has

been intensively studied (Crumley, 1964; Baumrind and Buck, 1970;

Roberts and Jee, 1974; Yee et al., 1976), few studies have been con­

cerned with the osteoclastic alveolar bone resorption at the pressure

side of the PDL.

Morphological changes which occur in pressure regions within the

PDL following orthodontic tooth movement have been examined with light

and electron microscopy (Sansted, 1904; Schwarz, 1932; Oppenheim, 1911;

Reitan 1951; Waldo and Rothblatt, 1954; Kvam 1972; Rygh 1972, 1972a,

1972b, 1973a, 1973b, 1974). The most striking morphological features

occurring in these areas were: 1) hyalinization of the PDL (the

hyalinized region of the PDL was a cell-free region containing necrotic

12

connective tissue which had a glassy appearance (Sandsted, 1904;

Schwarz, 1932; Reitan, 1951; Kvam, 1972; Rygh, 1972a, 1973a, 1974);

2) disruption of vascular channels due to the breakdown of the endo­

thelium of vessel walls which resulted from compression of the PDL

(Rygh, 1976); 3) disorganization of PDL collagen microfibrils (1973a,

1973b); and 4) eventual removal of hyalinized tissue by macrophages

(Kvam, 1972; Rygh, 1974) followed by the repopulation of new PDL cells

at the later stage of the tooth movement (Kvam, 1972). These studies

describe the qualitative tissue changes which occur in the ligament.

However, they provided no data concerning changes in osteogenic cells,

particularly osteoclasts.

Despite numerous studies concerning the osteoclastic bone resorp­

tion in various experimental models in the last few decades, the

origin of osteoclasts still remains as a controversial subject. Two

theories have been proposed: 1) some investigators suggest that

osteoclast histogenesis involves a progressive maturation of local

proliferating osteoprogenitor cells along specific pathways of cyto-

differentiation (Bloom and Bloom, 1941; Heller et al., 1950; Kember,

1960; Young, 1962, 1963; Scott, 1967, 1969; Bingham et al., 1969;

Thyberg, 1970); 2)other investigators suggest that osteoclasts may be

derived from cells of hematogenous origin such as monocytes and macro­

phages CFischman and Hay 1962; Jee and Nolan, 1963; Gothlin and

Ericcson, 1973; Walker, 1972, 1975a, 1975b).

The single osteoprogenitor cell theory was initially proposed

based on the studies of 1) medullary bone formation and resorption

in the reproductive cycle of female pigeons (Bloom and Bloom, 1941);

13

and 2) the cellular transformation in mammalian bones induced by PTE

(Heller et al., 1950). These investigators concluded that osteoblasts

and osteoclasts are modulation of one single osteoprogenitor cell.

The single osteoprogenitor cell theory was modified based on auto­

radiographic cell kinetic studies (Kember, 1960; Young, 1962, 1963).

3

By using HTdR autoradiography, Kember (1960) studied the cell pro­

liferation of the epiphyseal cartilage and the metaphyseal regions

in tibias of rats. Young (1962, 1963) studied the proliferation and

specialization of bone cells during endochondral osteogenesis in ribs

and tibias of young rats. Their results showed that labeling inten­

sity occurred in the osteoprogenitor cell population. Labeled osteo­

blasts and osteoclasts arose from the labeled proliferating osteo­

progenitor cells. Maximal labeling of osteoclasts was observed

between 40-48 hours post-injection. Based on their experimental

3 results, they concluded that incorporation of HTdR was restricted

to the cells in the osteoprogenitor population. After cell division,

3

HTdR labeled osteoprogenitor cells would either remain in the pro­

genitor pool, differentiate/modulate into osteoblasts or be incor­

porated into osteoclasts. Since all cell types in the osteoprogeni­

tor cell population are histologically indistinguishable at light 3

microscopic level, and all dividing cells can incorporate HTdR, it is likely that labeled osteoblasts and osteoclasts could have migrated

into the local area from extraskeletal sites via vascular channels.

3

Therefore, these HTdR autoradiographic studies do not provide con­

clusive evidence concerning the nature of osteoclast progenitor cells.

Evidence from other experiments (Scott, 1967, 1969; Bingham

14

et al., 1969; Thyberg et al., 1970) suggested that osteoblasts and

osteoclasts are derived from proliferating bipotential, osteopro­

genitor cells capable of specializing along separate pathways of

differentiation. By HTdR electron microscopic autoradiography in the

metaphysis of fetal rats, Scott (1967, 1969) demonstrated two morpho­

logically distinct cell types in the osteoprogenitor cell pool. One

of the cell types had morphological characteristics associated with

bone matrix production. Scott referred to these cells as preosteo-

blasts. The other cell type, referred to as preosteoclasts, resembled

developing neutrophilic leukocytes. Scott suggested the theory of

separate pathways for osteoblast and osteoclast cytodifferentiation

from a single bipotential osteoprogenitor cell.

3 3 3

Using various tritiated metabolites ( H-RNA, H-Leucine, H-glu-

cosamine) to measure RNA and protein synthesis, Bingham et al. (1969)

studied the effect of PTE on the osteogenic cellular activity at the

periosteal and endosteal surfaces in young rabbit femurs. Bone resorp­

tion and formation were examined at the endosteal and periosteal sur­

faces respectively. Parathyroid extract caused an increase in RNA

synthesis in the preosteoclast/osteoclast population at the endosteal

surface. Concurrently, there was a decreased incorporation of isotopes

in the preosteoblast/osteoblast population at the periosteal surface.

From these results, Bingham concluded that two distinct precursor cell

populations exist within the osteogenic cell system. Her experimental

data supported the theory of separate pathway of cytodifferentiation

of osteoblasts and osteoclasts.

Based on the electron microscopic studies concerning the osteogenic

15

cellular lysosomal enzyme activity at the metaphyseal bone in guinea

pigs, Thyberg et al. (1970) identified two distinct cell types located

perivascularly. Morphological evidence suggested that one cell type

resembled the preosteoblast/osteoblast while the other resembled pre­

osteoclast/osteoclast with macrophage-like morphology. The study

demonstrated that these two perivascular cells have different capabil­

ities for phagocytosis of extracellular matrix markers such as ferritin

and thorium dioxide. Active phagocytic activity of these extracellu­

lar matrix markers was observed by the presumptive preosteoclast/osteo­

clast cells, while limited phagocytic activity was demonstrated by

preosteoblast/osteoblast cells. Despite morphological evidence regard­

ing the possible existence of two different cell types within the

osteoprogenitor cell population, no concrete evidence has been provided

concerning the origin of osteoclast precursor cells.

Besides the theory of separate pathways of cytodifferentiation of

local osteoprogenitor cells, it has also been suggested that osteo­

clasts might be derived from cells of hematogenous origin such as

monocytes and macrophages (Fischman and Hay, 1962; Jee and Nolan, 1963;

Gothlin and Ericcson, 1972, 1973; Walker, 1972, 1975a, 1975b).

Tritiated thymidine autoradiographic study on limb regeneration of

newts by Fischman and Hay (1962) showed that osteoclasts were observed

at resorptive bone surface of the regenerating site. They suggested

that osteoclasts were derived from proliferating precursors of blood

forming tissue, which then migrated into the regenerating limb via

blood vessels. The histogenesis of multinucleated labeled osteoclasts

by fusion of mononuclear leukocytes was suggested.

16

By exmaining areas of ischemia induced by the administration of

carbon particles into the nutrient artery of rabbit femurs, Jee and

Nolan (1963) observed that initially carbon particles were present

only in macrophages (1-4 days). Subsequently (15-18 days) carbon

labeled osteoclasts were observed at the resorptive bone surface.

These observations led them to suggest that osteoclasts were formed

by the fusion of macrophages. At no time were carbon labeled osteo­

blasts found.

Electron micorscopic studies on fracture healing of femurs in

parabiotic rats was employed by Gothlin and Ericcson (1972, 1973) in

an attempt to determine the origin of the osteogenic cells. In the

parabiotic system one rat (A) was shielded during radiation while

the other rat (B) was exposed to a dose of cobalt radiation sufficient

enough to kill all the hematopoietic tissues. On the day of fractur­

ing the right femurs of both rats, the cross-circulation between the

rats was arrested for twenty minutes. During this time, rat A received

3 HTdR while rat B received a large dose of cold thymidine. Tritiated

labeled mono-and binuclear macrophages were observed at the fracture

site 21-28 days after fracture in rat B. In another experiment,

thorotrast-labeled periotoneal macrophages was injected into the

sibling rats which had their femur fractured, Gothlin and Ericcson

(1973) observed thorotrast-labeled osteoclasts at the fracture sites

21-28 days later. Their results suggested that precursors of osteo­

clasts were cells of hematogenous origin. Although the above experi­

ments provided morphological evidence suggesting that osteoclasts are

formed by the fusion of phagocytic cells of monocytic-macrophagic

17

origin. The inflammatory condition, which was generated at all the

skeletal sites examined in these studies, complicates the understand­

ing of osteoclast histogenesis under normal conditions.

In a series of experiments using parabiosis. Walker (1972, 1975a,

1975b) showed that when a congenital osteopetrotic mouse and a normal

littermate were connected parabiotically, a permanent cure of osteo­

petrotic disease resulted. Cell infusion of normal myloid tissue such

as spleen and bone marrow into the irradiated osteopetrotic mice could

also restore the normal bone resorbing capability in osteopetrotic mice.

On the contrary, the osteopetrotic condition could be induced in

irradiated normal mice following cell infusion of myloid tissues from

the congenital osteopetrotic mice. Based on his experimental results.

Walker concluded that migratory cells from myloid tissues play a role

in bone resorption. Whether these myloid tissue cells provide a

source of osteoclasts is unclear. Sequential ultrastructural exam­

ination during the transformation of myloid cells to osteoclasts

generated in osteopetrotic mice would provide additional information

regarding the histogenesis of osteoclasts.

An adequate knowledge of the origin of the osteoclast at the bone

surface is important for the understanding of diseases involving bone

loss such as periodontal disease. Although a large volume of informa­

tion concerning the histogenesis of osteoclasts is available, the

source of these cells in the periodontium is still unknown. Thus, a

more critical examination of osteoclast generation in the periodontium

is necessary in order to onderstand the mechanisms of alveolar bone

resorption involved in both tooth movement (physiologic and orthodontic)

18

and periodontal disease. The prerequisite in such understanding is

to characterize the nature of osteoclast generation. Therefore, the

purpose of this study was to examine the temporal sequence of osteo­

clast generation in the orthodontically stimulated rat periodontium

and to provide basic quantitative histomorphometric data concerning

this response.

MATERIALS AND METHODS

Male Sprague-Dawley rats, weighing 150 - 12 grams, were housed in

plastic animal cages in groups of five rats per cage. Water and food

(Purina Lab Chow) were available £d libitum. Prior to the start and

during the experiment, the rats were kept in an animal room which was

maintained on a 12 light-dark cycle.

While the rats were under light ether anesthesia, a piece of

orthodontic elastic band ( 2 m m X l m m X 0 . 2 mm, trimmed from No. J-104,

Rocky Mountain Co., Denver, Colorado) was inserted between the first

and second left maxillary molars to achieve orthodontic tooth movement.

The dimensions of the elastic band were chosen to avoid any inter­

ference with mastication. The corresponding area on the right maxil­

lary molars served as the control. The elastic band insertion proce­

dure was carried out between 10:00 a.m. and 12:00 p.m. A number of

rats were sacrificed at the following time intervals after orthodontic

tooth movement: 12 (3), 18 (3), 24 (8), 30 (3), 44 (3), 48 (8), 54 (3)

and 72 (7) hours. The number of rats sacrificed at each interval is

given in parentheses.

Cellular proliferation in the PDL in response to orthodontic tooth

movement was studied in a second group of rats. The general experimen­

tal procedure in this study was the same as described above. However,

six hours before sacrifice, each rat received an intraperitoneal

injection of vinblastin sulfate (0.1 ml of 1 mg/ml solution, Sigma

Co.). Two rats were sacrificed at each of the following time inter­

vals: 12, 18, 24, 30, 36 hours.

While under pentobarbital anesthesia (0.1 ml of 50 mg/ml of Sodium

19

20

Nembutal, Abbott Lab.), these rats were sacrificed and the maxillae

were initially fixed by intracardiac perfusion with 4% glutaraldehyde

in 0.1 M phosphate buffer at PH 7.2. The dissected maxillae were

subsequently fixed by immersion for two days in the same fixative.

Following fixation, tissues were washed in 0.1 M phosphate buffer and

the maxillae were decalcified in 4% EDTA (ethylene dinitrilo tetra­

acetate, American Drug and Chemical Co.) in 0.1 M phosphate buffer for

two weeks. Upon completion of decalcification, the maxillae were

dehydrated through a series of graded ethanol and acetone changes and

were embedded in methyl methacrylate embedding medium (Kimmel and Jee,

1975). Three micron-thick parasagittal sections from the mesial root

of the first maxillary molars were prepared on the Jung Model-K micro­

tome and affixed to slides coated with 1% gelatin. Each parasagittal

section of the periodontium included the entire length of the pulp

cavity of the first mesial root. The plastic on the histological sec­

tion was removed through a series of three ten-minute acetone changes.

Histological sections were stained with Mayer's hematoxylin and counter-

stained with eosin and coverslips were affixed with Permount (Fisher

Scientific Co.) .

The areas examined were the PDL and alveolar bone surfaces located

distal to the mesial root of the first maxillary molar at both the

apical and interradicular regions. Beginning at the pulp canal of the

first mesial root, three areas (0.4 mm X 0.4 mm each) were identified

in the apical region (fig. 1). Four areas were also identified at the

interradicular region (fig. 1). At the light microscopic level, the

following morphological features in the ligament at the apical and the

21

interradicular regions were examined: 1) the presence of hyaliniza­

tion distal to the mesial root at the apical region; hyalinization is

defined as a cell-free, glassy-looking regions of necrotic connective

tissues; 2) removal of hyalinization and repopulation by PDL cells;

3) the nature of PDL vascularity in response to pressure; and 4)

changes on the width of the ligament. Quantitative study included:

1) the temporal nature of osteoclast generation following orthodontic

tooth movement; 2) the number of osteoclast per bone surface peri­

meter (OCL/BSP); 3) the number of nuclei per osteoclast (nuclei/OCL);

4) the alveolar bone loss expressed as the percent percentage of

alveolar bone per unit area at the interradicular region only the per­

cent (%) of bone was determined by the point-hit method (Henning, 1958);

5) mitotic activity of the PDL cells was examined by determining the

mitotic index at the interradicular region in vinblastin sulfate-

injected rats.

The following criteria were employed for the identification of

PDL cells at light microscopic level: 1) osteoclast were generally

multinucleated cells, lying within the Howships' lacunae in the

alveolar bone surface, with eosinophilic cytoplasm and numerous

cytoplasmic vacuoles; 2) Fibrobilasts were cells observed in the PDL

which had little basophilic cytoplasm. Ligament fibroblasts are histo­

logically indistinguishable from local progenitor cells within the

ligament (Melcher and Eastoe, 1969; Yee, 1979); and 3) Mitotic cells,

arrested in metaphase of the cell cycle by vinblastin sulfate, were

characterized by their large amount of basophilic cytoplasm and their

condensed chromatin arranged at the center of the cells.

22

For quantitative analyses, all slides were coded with random

numbers before counting in order to avoid observer bias. Data obtained

from this study were tested for significance by Student's t test and/or

linear regression analysis (Dixon and Massey, 1969). Differences

between data from control and stimulated tissues were considered sig­

nificant if the p-value was less than 0.05.

RESULTS

!• Light microscopy of the PDL in the apical region.

A. Control animals.

In the apical region of the mesial root, the histological

appearance of the PDL and adjacent tissues (cementum and alveolar

bone) in non-rothodontic stimulated (control) rats were similar at

all time periods studied. Figure 2 illustrates the morphologic

appearance of this region. The PDL in this area was densely populated

with fibroblast-like cells. Organized collagen fibers of the ligament

extended from the cellular cementum to alveolar bone in an oblique

plane. Blood vessels were primarily confined to the alveolar bone

side of the ligament. The alveolar bone surface was covered by a

layer of spindle to cuboidal shaped osteoblasts. Few, if any, multi­

nucleated osteoclasts were observed along the alveolar bone surface

in the apical region control maxillae.

B. Experimental animals.

1, Qualitative observations in the paical region of the

pressure side of PDL following orthodontic tooth movement.

After 12 hours of orthodontic stimulation, osteoclasts were

often present along the alveolar bone surface. Otherwise, little mor­

phologic change was observed in the periodontal tissues. However, dis­

tal tipping of the mesial root was apparent in histologic sections as

evidenced by a decreased width of the PDL (Fig. 3).

By 30 hours post-orthodontic stimulation, progressive compres­

sion of the PDL followed by the formation of hyalinized areas in the

ligament were observed (Fig. 4). Vascular occlusion was maximal.

23

24

An inflammatory response characterized by invasion of neutrophilic

leukocytes in this hyalinized area was occasionally observed. Osteo­

clastic bone resorption at the alveolar bone surface adjacent to the

hyalinized PDL was suggested by an obvious increase in the number of

osteoclasts (Fig. 4).

By 54 hours post-orthodontic stimulation, both a gradual

cellular repopulation of the hyalinized PDL and vascular invasion had

begun (Fig. 5). Widening of the periodontal space, possibly due to

resorption of alveolar bone, had occurred to accommodate the new

position of the mesial root within the tooth socket.

By 72 hours post-orthodontic stimulation, progressive changes

in the PDL suggestive of repair continued. However, a complete return

of the periodontal tissue to a normal morphologic appearance did not

occur within the time frame of this study.

2. Quantitative analyses in the apical region of the pressure

side of PDL following orthodontic tooth movement.

The numerical data for quantitative analyses of changes

occurring in the apical region are given in figures 6-8. In the apical

region, the total number of osteoclasts and osteoclast per millimeter

of bone surface perimeter (OCL/BSP) were significantly increased in

most experimental periods (Fig. 6, 7). By 12 hours post-orthodontic

stimulation, the total number of osteoclasts observed at the alveolar

bone surface was 3.6 ± 0.6. This was a significant increase over the

number of osteoclasts presnet in control rats (1.4 t 0.5). The genera­

tion of osteoclasts in the apical region reached a peak of 12.6 t 1.4

after 44 hours post-orthodontic stimulation. This was followed by a

25

gradual decrease to 7.5 ± 1.5 and 3.5 ± 0.5 by 48 and 72 hours post-

stimulation respectively (Fig. 6). Changes in the OCL/BSP followed a

similar pattern (Fig. 7). There was 0.9 ± 0.1 OCL/BSP by 12 hours

post-stimulation. By 44 hours, the OCL/BSP reached a peak of 2.72 t

0.3 and followed by a gradual decline to 0.81 ± 0.1 OCL/BSP by 72

hours post-orthodontic stimulation. In the apical region, the average

number of nuclei per osteoclast was not significantly changed at any

time period studied (Fig. 8).

II. Light microscopic observation of the PDL in the interradicular

region.

A. Control animals. In the interradicular region distal to the

mesial root of the first maxillary molar, the collagen fibers of the

PDL were regularly arranged (Fig. 9, 10). Blood vessels were generally

located in the alveolar bone side of the ligament. Cuboidal shaped,

basophilic osteoblasts covered the alveolar bone surfaces (Fig. 9, 10).

Few osteoclasts were observed lying within the Howships' lacunae of the

alveolar bone surface in this region.

B. Experimental animals.

1. Qualitative observations in the interradicular region of

the pressure side of PDL following orthodontic tooth move­

ment.

In the interradicular region, contamination of the inflammatory

cells was never observed at any experimental period. During the first

36 hours following orthodontic stimulation, no significant morphologic

change was observed in the ligament as compared to the control rats.

The organization of the PDL fibers and the vascularity in the ligament

26

remained unchanged (Fig. 11, 12). Osteoclastic bone resorption was

initially observed at alveolar bone surfaces by 12 hours post-stimula­

tion (Fig. 11).

By 44 hours post-orthodontic stimulation, the width of the PDL

appeared to be slightly decreased. However, unlike the situation in

the apical region, vascularity of the PDL was maintained (Fig. 12).

Maximal changes in the morphology of periodontal tissues in the

interradicular region were observed after 72 hours orthodontic stimu­

lation (Fig. 14, 15a, 15b, 15c). Although considerable decrease in

width of the PDL and disorganization of the collagen fibers at the

interradicular crest were apparent (Fig. 15a, 15b), hyalinization of

the ligament similar to that in the apical region was not observed. A

decrease in the PDL vascularity was also observed. Maximal osteo­

clastic alveolar resorption was now occurring as demonstrated by the

presence of numerous osteoclasts lying on the surfaces of bony

spicules (Fig. 14, 15a, 15b, 15c).

2. Quantitative analyses in the interradicular region of the

pressure side of PDL following orhtodontic tooth movement.

Data obtained by histomorphometric analyses in the interradicular

region are given in figures 16-22. The total number of osteoclasts

located at the alveolar bone surface was significantly increased from

18 to 72 hours post-stimulation (Fig. 16). The total number of osteo­

clasts at 12 hours post-stimulation was 7.5 ± 0.5. The number of

osteoclasts rose steadily such that there was an average of 22.0 _ 2.4

total osteoclasts by 72 hours. The rate of increase in the number of

osteoclasts during the period from 12 to 72 hours was 0.32 osteoclast

27

per hour. A similar pattern of increase was displayed when the change

in osteoclast population was normalized to the bone surface perimeter.

The OCL/BSP over the duration of this study is shown in figure 17.

A significant increase in this parameter was found at all experimental

periods except the first 12 hours. This value increased at a rate of

approximately 0.035 OCL/BSP/hour.

Data for the number of nuclei per osteoclast in the interradicular

region are shown in figure 18. Although the data was quite variable,

there was a small but consistent increase in the number of nuclei per

osteoclast in orthodontic-stimulated versus non-stimulated maxillae.

This difference was most apparent at the later time periods (54 and 72

hours post-stimulation).

Figure 19 shows the regression lines generated from the measure­

ments of the percent alveolar bone in the interradicular region. In

control rats, alveolar bone occupied approximately 55.1 _ 5.6 percent

of the standard area measured. In contrast, there was a significant

loss of alveolar bone in the interradicular region of the orthodon­

tically stimulated rats was initially observed by 44 hours post-

stimulation. By 72 hours post-stimulation only 47 2.3 percent of

the standard area measured was occupied by alveolar bone.

3. Mitotic activity of PDL cells in the interradicular region.

Following orthodontic stimulation, the proliferation of PDL

cells in the interradicular region was assessed by determining the

mitotic index. Vinblastin sulfate arrested mitotic cells were randomly

distributed throughout the ligament of control and orthodontic stimu­

lated maxillae (Fig. 20, 21). Mitotic cells were occasionally observed

28

lying on or very near the laveolar bone surfaces (Fig. 20, 21). The

mitotic index was significantly increased as compared to the control

animals at 24 and 30 hours post-stimulation (Fig. 22).

29

n • I j> n

Figure 1: A diagramatic representation of a sagittal section o£ the

first maxillary molar and portion of the second molar. The

regions occupied by dashed lines represent the PDL. In this

study, areas under examination are the apical and inter­

radicular regions denoted by S, and S« respectively. These

regions are located along the PDL surfaces distal to the

mesial root. Alveolar bone (AB); cementum (Ce); dentin (D) ;

enamel (E); orthodontic elastic in contact areas between the

first and second maxillary molar (El); gingiva (G); pulp

cavity (P); periodontal ligament (PDL).

FIG.1

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Figure 2: Non-orthodontic stimulated (control) maxilla in the apical

region of the first maxillary molar. Regularly organized

PDL fibers extend from the Cementum (Ce) to the alveolar

bone (AB). The majority of PDL cells are fibroblasts.

Blood vessels (*) are closely located to the alveolar bone

side of the PDL. X 192

Figure 3: Twelve hours post-orthodontic stimulation in the apical

region. Initial distal tipping of the mesial root of the

first maxillary molar is seen. PDL fibers remain well

organized. Blood vessels (*) are located closely to the

alveolar bone surface. Dentin (D) X 156

Figure 4: Thirty hours post-orthodontic, stimulation in the apical

region. Hyalinization of PDL (H) is formed. Multinucleated

osteoclasts (arrow) are located within the Howships'

lacuna of the alveolar bone surface. No blood vessel is

observed in the area of hyalinization. X 192

Figure 5: Fifty-four hours post-orthodontic stimulation in the apical

region. Gradual repopulation of new PDL cells and vacular

invasion (*) into the hyalinized PDL have begun. Multi­

nucleated osteoclast (arrow) is present. X 192

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radicular region. PDL fibers are arranged in parallel

array. The majority of the PDL cells are fibroblasts.

Blood vessels (*) are closely located on the alveolar bone

side of the PDL. Numerous osteoblasts (arrow) cover the

alveolar bone surfaces. Dentin (D) X 230

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ular region of the first maxillary molar. Hyalinization of

PDL never occurs at any experimental period. Little mor­

phological change is observed as compared to the control

rats. Multinucleated osteoclast (arrow) lies within the

Howships' lacuna of the alveolar bone surface. Dentin (D)

X 152

Figure 12: Thirty-six hours post-orthodontic stimulation at the inter­

radicular region. Blood vessels (*) with intact structural

integrity are located adjacent to the alveolar bone. An

increase in the number of osteolcasts (arrows) is present.

X 192

Figure 13: Forty-four hours post-orthodontic stimulation at the inter­

radicular region. More osteoclasts (arrows) are observed.

A significant increase in alveolar bone loss is initially

observed. X 192

Figure 14: Seventy-two hours post-orthodontic stimulation at the inter­

radicular region. A slight decrease of vascularity in the

PDL adjacent to the alveolar bone is observed. The genera­

tion of osteoclasts Carrows) and the alveolar bone loss are

maximal. X 192

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interradicular region. Disorganized PDL fibers are

observed. Numerous osteoclasts (arrows) are located

in the Howships' lacunae along the alveolar bone

surface. Dentin (D) X 230

Figure 15b: Higher magnification of the above micrograph showing

multinucleated osteoclasts (arrows) located within the

Howships' lacunae of the alveolar bone surface. X 460

Figure 15c: Seventy-two hours post-orthodontic stimulation at the

interradicular region. Higher magnification of multi­

nucleated osteoclasts (arrows) are shown. More nuclei

per osteoclast are observed. X 460

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Figure 20: Non-orthodontic stimulated (control) maxilla at the inter­

radicular region. Vinblastin sulfate-arrested PDL mitotic

cells (M) are observed. These mitotic cells are character­

ized by the condensed chromatin and large basophilic cyto­

plasm. X 460

Figure 21: Twenty-four hours post-orthodontic stimulation. PDL

mitotic cells (M) are maximal at this time period.

Mitotic cells observed on the alveolar bone surface. X 460

^ .- * * ^ _ • * - PDL* ^ ^ - . - ^ * ^

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Figure 22: The mitotic index of PDL fibroblasts in the interradicu­

lar region was significantly increased by 24 and 30 hours

post-stimulation, t = p < 0.05 ft = p < 0.001

56

FIG. 22

MITOTIC INDEX AT INTERRADICULAR REGION

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It

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36

DISCUSSION

In the current study, the temporal response of osteoclast histo­

genesis and alveolar bone resorption following orthodontic tooth

movement has been described. An understanding of the cellular events

associated with alveolar bone destruction is important since uncon­

trolled bone loss is a major clinical problem in cases of chronic

periodontal disease. In order to understand the extensive alveolar

bone loss which results during the latter stage of this disease, a

more critical examination of osteoclastic resorption of alveolar bone

in a nonpathologic, normal biologic system is necessary. In the areas

examined in this study, simultaneous alveolar bone resorption and

formation in response to physiologic tooth drift represent normal

biological events. Orthodontic stimulation, as employed in this study,

was used to expedite and/or potentiate the occurrence of these events

in the rat periodontium with minimal pathologic tissue disruption.

In an attempt to establish base-line information concerning alveolar

bone resorption, this study was designed to provide quantitative and

qualitative data concerning the temporal nature of osteoclast genera­

tion following orthodontic tooth movement.

In this study, the morphologic changes in the apical region was

examined to compare our qualitative observations with other studies

(Kvam 1972, 1973; Rygh 1972, 1974). The interradicular region was

examined because: 1) minimal pathologic tissue changes occurs; and

2) the morphologic changes which result in response to orthodontic

tooth movement have never been examined in this area. In the apical

region, orthodontic tooth movement caused a significant displacement

57

I

58

of the mesial root in a distal direction. As a result, pathologic

tissue changes such as hyalinization of the PDL and vascular disrupton

occur in areas where pressure is exerted. A similar tissue response

to experimental tooth movement has been reported by other investigators

(Sandsted 1904, Schwarz 1932, Reitan 1951, Kvam 1972, Rygh, 1972, 1974).

Hyalinization of the ligament was well defined histologically by 30

hours post-orthodontic stimulation. Inflammatory cells such as neutro­

philic lentocytes were present adjacent to the hyalinized areas.

Repair of the periodontal tissue appears to have begun by 54 hours but

was still incomplete by 72 hours. Studies by Kvam (1972) and Rygh

1972a, 1974) indicate that complete repair of the hyalinized area

requires at least 7-8 days after the application of force. The fact

that complete repair did not occur in this area might be due to the

shorter duration of our experiment period.

Osteoclastic resorption of alveolar bone in response to pressure

following orthodontic tooth movement has been documented (Gianelly

and Goldman, 1971; Graber, 1972; Reitan, 1975). However, only quali- j I

tative descriptions concerning the histogenesis of osteoclasts have

been reported. In the current study, the temporal response of osteo­

clast generation associated with alveolar bone resorption has been

characterized quantitatively. Following displacement of the mesial

root in the tooth socket, a maximal increase in the total number of

osteoclasts in the apical region was observed by 44 hours post-ortho­

dontic stimulation. Progressive resorption of alveolar bone in the

apical region must take place in order to restore an adequate width

of the periodontal space such that: 1) new positional changes of the

It

11

59

mesial root within its socket can be accommodated; and 2) subsequent

repair of the PDL can be initiated. The results of this study pro­

vide quantitative verification that orthodontic tooth movement stimu­

lates osteoclast generation in the apical region where hyalinization

existed. Unfortunately, the presence of pathologic tissue disruption

and the inflammatory response make it difficult to interpret the

normal generation of osteoclasts in this area. The presence of

inflammatory cells in the hyalinized area might generate stimuli

necessary to initiate the generation of osteoclasts. Recent evidence

indicates that sensitized mononuclear leukocytes such as lymphocytes

might release an osteoclastic activating factor (OAF) when challenged

constantly with antigen (Horton et £l• > 1972).

Although osteoclastic resorption of alveolar bone has been

intensively studied in the periodontium, quantitative analyses on

the osteoclasts histogenesis in response to orthodontic force has

not been previously examined in the interradicular region. In con­

trast to the apical region, this area lacks the extensive pathologic

tissue damage such as hyalinization of the PDL and vascular disruption

following orthodontic stimulation. Although minimal compression of

the ligament and vascular changes were observed by 72 hours post-

orthodontic stimulation, no inflammatory response was observed.

Therefore, it is likely that the interradicular region might possibly

represent a normal biological system for the study of histogenesis of

osteoclasts in the future.

In the interradicular region, an initial significant increase in

the total number of osteoclasts was observed on the alveolar bone

60

surface by 18 hours post-stimulation. The maximal increase in osteo­

clasts was observed by 72 hours. A similar pattern of increase

results when the change of osteoclasts was normalized to the bone

surface perimeter. The similarity which exists between these

quantitative data indicates that the increase in total number of

osteoclasts is independent of the amount of bone surface available.

Since maximal alveolar bone loss was also observed in this region

by 72 hours post-stimulation, it is likely that active resorption

of alveolar bone takes place to facilitate the tipping of the mesial

root in its bony socket. Although these quantitative data indicate

that a significant increase in the total number of osteoclasts was

associated with increased alveolar bone resorption, the origin of

these osteogenic cells was not apparent.

So far, the origin of osteoclasts has remained as a mystery not

only in the periodontium but in the skeleton in general. In long

bones, ^HTdR studies by Tonna (1963) and Young (1962, 1963) showed

that HTdR labeled osteoclasts occurred between 6-28 hours in tibial

metaphyses of rats. These investigators suggest that osteoclasts

are derived from local proliferating osteoprogenitor cells along a

specific pathway of cytodifferentiation. Other studies suggest that

osteoclasts arise from mononuclear hematogenous cells (Fischman and

Hay, 1962; Jee and Nolan, 1963; Gothlin and Ericcson, 1972, 1973;

Kahn and Simmons, 1975; Walker, 1972, 1975a, 1975b).

In the periodontium, the histogenesis of a osteoclasts has been

studied by various investigators. In the non-orthodontically

3 stimulated periodontium, HTdR labeled osteoclasts were initially

•I *

61

observed in rat mandibles by 18 hours following the administration of

PTH (Toto and Magon, 1966). These investigators suggested that local

osteoprogenitor cells divide and differentiate into osteoclasts during

this time period. In parathyroidectomized rats, administration of

PTH restored the osteoclast population to normal size within 24 hours

(Baron et al., 1977). After the removal of orthodontic force, Kvam

3 (1972) observed HTdR labeled osteoclasts in area of pressure by 24

hours. However, the generation of osteoclast in the periodontium of

gnotobiotic rats requires 10-65 days following the mono-infection

of Actinomyces naesludii (Garant, 1976b). The above experimental

results have not provided quantitative data concerning the temporal

nature of osteoclast histogenesis. The only quantitative data con­

cerning the osteoclast histogenesis has been reported by Roberts

(1975a). He observed an increase in osteoclast in non-orthodontically

stimulated maxillae of rats in 2-9 hours following the administration

of PTE. Based on experimental data obtained from previous studies, i

the origin of osteoclasts in the periodontium is still uncertain. I* •I

They may arise from: 1) local proliferation of osteoprogenitor cells }

within the ligament, and/or 2) from progressive maturation of hemato­

genous cells such as monocytes and macrophages which migrate into the

periodontium via the blood vascular system in the PDL.

The presence of local proliferating progenitors in response to

tooth movement (physiologic and orthodontic) and wound repair have

been reported by numerous investigators (Melcher, 1972; Roberts, and

Jee, 1974; Gould al., 1976; Yee al., 1976; Yee, 1979). In an

attempt to examine the nature of local proliferation of progenitor

62

cells in our experimental system, a preliminary study on the mitotic

activity of PDL cells in the interradicular region was undertaken. A

significant increase in mitotic activity was noted following ortho­

dontic tooth movement. The proliferative activity of PDL cells was

maximal by 24 hours post-orthodontic stimulation as evidenced by

numerous vinblastin sulfate-arrested mitotic cells. However, the role

of these progenitor cells with respect to osteoclast histogenesis is

still unknown. It is possible that this proliferative activity has

nothing to do with osteoclast generation. On the other hand, osteo­

clast histogenesis and PDL cell proliferation may be directly related.

In the interradicular region, there was a time lapse between the

maximum proliferation of PDL cells and the subsequent maxima]

increase in total number of osteoclasts. Based on these observations,

it is tempting to suggest that osteoclasts are derived from the .

maturation of local proliferating progenitors within the ligament.

For this hypothesis to be correct, several questions would need to I .

answered. For example: 1) Does the PDL proliferating population |i

comprise a single progenitor stem cell pool capable of giving rise to il

all PDL functional ce-ls (fibroblasts, cementoblasts, osteoblasts

and osteoclasts)? or 2) Is each functional PDL cell derived from

eparate and specific progenitor cells? Before these questions can be

3

answered, a more sensitive cell kinetic study using HTdR autoradio­

graphy must be employed.

In summary, the temporal nature of osteoclast generation in the

apical and interradicular regions distal to the mesial root of the

first maxillary molar in rats have been studied following orthodontic

63

tooth movement. Alveolar bone resorption in this study occurs to

facilitate the displacement of the mesial root within its bony socket.

A significant increase in osteoclast generation with time occurs in

the interradicular region. This is preceeded by an increase of

cellular proliferation of local PDL progenitor cells. Although the

relationship between these events has not yet been resolved, the

data provided by this study should serve as a basis for future studies

concerning the mechanism of osteoclast histogenesis and alveolar bone

destruction.

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