ORTHODONTIC TOOTH MOVEMENT by A THESIS IN ANATOMY
Transcript of 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.
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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
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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.
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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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31
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11
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
Figure 10: Higher magnification of the above micrograph showing
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Figure 11: Twelve hours post-orthodontic stimulation at the interradic
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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Figure 15a: Seventy-two hours post-orthodontic stimulation at the
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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I
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•
St
11
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
^t
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tt=p < 0.001
X UJ a
I
4 -
2
I
J STIMULATED
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8
It
12 18 24 30
HOURS
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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