Osteoblasts, Osteoclasts, and Osteocytes:

Unveiling Their Intimate-Associated

Responses to Applied Orthodontic Forces
Ulf H. Lerner

Bone is remodeled and modeled by the concerted activities of 3 cell types—

osteoblasts, osteocytes, and osteoclasts. Osteoblasts are the cells that pro-
duce bone extracellular matrix and are responsible for its mineralization.
Osteoblasts also have endocrine activity through secretion of osteocalcin,
which regulates fat and energy metabolism. These cells also control the
differentiation and activity of osteoclasts. Osteocytes are osteoblasts that
have been incorporated into bone matrix and are cells with extensive den-
dritic processes through which the cells communicate with other osteocytes
and with osteoblasts. Mechanical loading is sensitized by the dendritic
processes and transferred to biochemical responses involved in control of
osteoblast and osteoclast function. Osteocytes also have endocrine activity
by releasing fibroblast growth factor 23, which is involved in phosphate
secretion in kidneys. Differentiation of osteoclast mononuclear progenitors
to mature multinucleated osteoclasts is regulated by macrophage colony-
stimulating factor and receptor activator of NF-␬B ligand, expressed by
stromal cells in bone marrow or osteoblasts in bone, as well as by osteo-
cytes. The integrated endo- and paracrine control of osteoblasts, osteocytes,
and osteoclasts is important for maintaining bone mass and for control of
remodeling and modeling processes in bone, including during orthodontic-
induced tooth movement. (Semin Orthod 2012;18:237-248.) © 2012 Elsevier
Inc. All rights reserved.

Remodeling and Modeling of Bone up by osteons of a central Haversian canal

Tissue
Histologically, the skeleton comprises 2 types
of bone tissue: cortical (compact) and trabec-
ular (cancellous) bone. The cortical bone
makes up 80% of the volume in the adult
skeleton and the trabecular bone makes up
the remaining 20%. The cortical bone forms a
peripheral shell in all kinds of bone. It is built
Department of Molecular Periodontology, Umeå University,
Umeå, Sweden; and Center for Bone and Arthritis Research, Insti-
tute of Medicine, Sahlgrenska Academy, University of Gothenburg,
Gothenburg, Sweden.
Address correspondence to Ulf H. Lerner, DDS, PhD, Depart-
ment of Molecular Periodontology, University of Umeå, SE-901 87
Umeå, Sweden. E-mail: ulf.lerner@odont.umu.se.
© 2012 Elsevier Inc. All rights reserved.
1073-8746/12/1804-0$30.00/0
http://dx.doi.org/10.1053/j.sodo.2012.06.002
with blood vessels and nerve fibers surrounded
by bone tissue in concentric parallel layers.
The trabecular bone forms spicules (spongy
bone) in the interior of some parts of the
skeleton. The vertebrae are most rich in tra-
becular bone, but it also fills up large parts of
the flat bones in the skull, pelvis, and sacrum
and in the distal and proximal parts of long
bones. Both the cortical and trabecular bones
are important for bone strength. In the jaw
bones, the cortical bone surrounds the teeth
and alveoli and delimits the periodontal liga-
ment from interior trabecular bone. The inte-
rior of the bones contains bone marrow com-
prising the hematopoietic tissue, which in
elderly people becomes more and more adi-
pocytic. Because interesting interplay between
bone cells and hematopoietic cells has been

Seminars in Orthodontics, Vol 18, No 4 (December), 2012: pp 237-248 237

238
demonstrated during the past decade, the
new research field— osteoimmunology— has
emerged.1
Bone tissue is not static but is continuously
rebuilt to adapt to external requirements and
for renewal of the extracellular matrix. In the
adult skeleton, both cortical and trabecular
bones are rebuilt by 2 processes called remodel-
ing and modeling.2 Modeling of the bone is a
process that changes the size and shape of bone
either by bone resorption without subsequent
bone formation or bone formation without pre-
vious bone resorption. Remodeling of bone is
the process by which old bone is replaced by new
bone. It is initiated by bone resorption to re-
move damaged bone and is followed by new
bone formation in the area resorbed. Remodel-
ing does not change the size or shape of the
bones. Remodeling is more frequent in trabec-
ular bone than in cortical bone, one of the
reasons why a metabolic bone disease such as
postmenopausal osteoporosis affects primarily
bones with proportionally increased amounts of
trabecular bone, for example, vertebrae, distal
radius, and proximal femur. It has been sug-
gested that microcracks in bone and subsequent
apoptosis in osteocytes will activate lining cells in
the remodeling process to stimulate osteoclast
formation and bone resorption of the mi-
crodamaged area. During the resorption pro-
cess, growth factors in bone are released, which
attract and activate osteoblasts to form new bone
under a canopy of bone lining cells.3-5 When
equal amounts of bone are formed as that re-
sorbed, bone remodeling is in balance and bone
mass is kept constant. This equilibrium involves
bidirectional interactions between osteoclasts
and osteoblasts to fine tune the balance between
bone formation and bone resorption. It has
been suggested that one such bidirectional
mechanism might be the interaction between
ephrinB2 expressed by osteoclasts and EphB4
expressed by osteoblasts.6
How the bone tissue reaction induced by
orthodontic treatment should be classified in
relation to the well-described physiological pro-
cesses in the skeleton is not clear. Although it is
sometimes referred to as a process with resem-
blances to inflammation-induced remodeling, it
should probably be more relevant to classify the
process as a modeling process to reshape the
bone. The aim of the present review is to sum-
Lerner
marize the current knowledge on bone cell bi-
ology and to describe how the cells interact with
each other and respond to mechanical load.
Osteoblasts—Key Cells in Bone
Formation and Resorption
Osteoblasts, the cells that form bone, perform
other functions also along with this (Fig 1). Os-
teoblasts form one continuous cell layer that
covers all bone surfaces, both trabecular bone
and endosteal and periosteal surfaces of cortical
bone. These cells also cover the alveolar bone
surfaces in the periodontium. Thus, all bone
tissues will be surrounded by this syncytium of
osteoblasts, which implies that when osteoclasts
will resorb bone, the osteoblastic cell layer must
be opened up, which is one of the starting points
in the bone resorption process (see later in the
text).
Osteoblast Differentiation
Osteoblasts are of mesenchymal origin and
share stem cells with adipocytes, chondrocytes,
myoblasts, tenocytes, and stromal cells in bone
marrow and endothelial cells. The fate of stem
cells is determined by the concerted action of
intracellular proteins called transcription fac-
tors. When activated by appropriate signals,
these factors translocate to the nucleus and bind
to specific response elements in the DNA called
promoters. Transcription factors form com-
plexes with several other activating and suppress-
ing proteins, and it is this complex that either
activates or inhibits the promoters and, thereby,
determines whether a gene should be turned on
or shut off.
Several transcription factors have been shown
to be important for osteoblast differentiation,7
the most crucial ones being core-binding factor
␣-1 (cbfa1 or Runx2) and osterix. Mice that lack
either of these proteins can embryonically form
a cartilaginous skeleton, but are unable to form
a skeleton consisting of bone tissue and die at
birth. Key transcription factors for chondrocytes
and adipocytes are Sox9 and PPAR␥, respec-
tively.
Although osteoblast biology is generally viewed
on as similar in different parts of the skeleton, this
is not necessarily true. It seems as if bone extracel-
lular matrix produced by osteoblasts is similar in
 Osteoblasts, Osteoclasts, and Osteocytes 239

Figure 1. Osteoblasts form a 1 cell layer covering all bone surfaces. One function is to synthesize an extracellular
matrix, consisting of type I collagen fibers and several other proteins, and to subsequently mineralize this matrix
into bone. Osteocalcin, one of the proteins in the matrix, is released also into the circulation and acts as a
hormone to influence fat and energy metabolism by interacting with adipocytes and ␤-cells in the pancreas. In
addition, osteoblasts can be regulated to stimulate osteoclast formation and bone resorption by increasing
formation of RANKL and decreasing that of OPG and, thereby, paracrinally induce osteoclast progenitor cell
differentiation. Some osteoblasts become incorporated into bone matrix and differentiate to dendritic osteo-
cytes, which can sensitize mechanical loading and through decreased sclerostin expression increase Wnt/␤-
catenin signaling in osteoblasts to enhance bone formation. The osteocytes are also important in bone
remodeling, as microcracks damaging old bone causes osteocytic apoptosis and increased RANKL production
and, thereby, increased osteoclast formation to resorb the old damaged bone. The resorption process releases
coupling factors from the matrix (eg, TGF␤, BMPs), which enhance osteoblastic bone formation and subsequent
new bone formation to substitute the resorbed bone, a process denoted bone remodeling. In addition, osteocytes
secrete FGF23, a systemic hormone acting on kidneys to regulate phosphate excretion. Inset: Wnt is a large
family of different proteins with many different functions. In osteoblasts, Wnt acts by binding to the nonsignaling
transmembrane protein Lrp5 that leads to dimerization with the signal, 7 transmembrane signaling molecule
Frizzled. Downstream events include several steps and several cytosolic proteins, eventually causing activation of
the transcription factor ␤-catenin. Through interaction with responsive elements in different promoters,
␤-catenin will enhance osteoblast proliferation and activity, and a new bone is formed. Wnt/␤-catenin signaling
can be inhibited by sclerostin and Dkk1, which are 2 proteins that through binding to Lrp5 inhibits its
interaction with Wnt.

different parts of the skeleton, but osteoblasts- data also indicate that systemic hormones regu-
forming cortical bone is probably different from late osteoblast function differently in different
osteoblasts-producing trabecular bone with dif- parts of the skeleton, as exemplified by the ob-
ferent morphology and turnover rate. Recent servations that estrogen, as well as parathyroid
240
hormone (PTH), mainly affects periosteal osteo-
blasts.8,9 Another important issue is whether the
osteoblasts in bone formed embryonically
through intramembranous bone formation are
identical to those present in bones formed by
endochondral bone formation. Thus, it seems
more likely that osteoblasts in different bone
areas are more heterogeneous than previously
thought.10-12 Few investigations on osteoblasts
include studies on effects on osteoblasts in jaw
bones, and it should be kept in mind that text-
book descriptions of osteoblast biology may not
necessarily be transferred in all aspects to the
biology of these cells in jaw bones.
Osteoblast Functions
Osteoblasts form bone by synthesizing an extra-
cellular matrix consisting of a wide variety of
proteins, the most abundant being type I colla-
gen fibers. Many proteins are also found in other
extracellular matrices. So far, only 2 proteins
have been found to be specific for bone, namely
osteocalcin and bone sialoprotein. Interestingly,
bone matrix also contains several growth factors
including transforming growth factor ␤ (TGF␤),
insulin-like growth factors, bone morphogenetic
proteins (BMPs), platelet-derived growth factor,
and fibroblast growth factors (FGFs). It is hy-
pothesized that these molecules are stored in
the matrix to be used during the remodeling
process to stimulate osteoblast recruitment.13
Most unexpectedly, it was found during the
late 1970s that receptors in bone for the well-
known bone resorbing hormones PTH and
1,25(OH)2-vitamin D3 were not present in oste-
oclasts but in osteoblasts.14 In the classical article
by Rodan and Martin,14 it was suggested that
osteoblasts play crucial roles in the control of os-
teoclast formation, although the molecular link
between the receptors for PTH and 1,25(OH)2-
vitamin D3 in osteoblasts and their paracrine con-
trol of osteoclasts was unknown. Later, during the
1980s, Takahashi et al showed that osteoclast for-
mation in bone marrow cultures stimulated by
either PTH or 1,25(OH)2-vitamin D3 was ob-
served only in areas with islands of bone marrow
stromal cells or cocultured osteoblasts, and that
these cells and osteoclast progenitor cells had to
make cell-to-cell contact for osteoclasts to be
formed.15 In late 1990s, 2 groups independently
showed that the molecules involved are receptor
Lerner
activator of NF-␬B ligand (RANKL), expressed
by stromal cells or osteoblasts, which stimulate
the receptor RANK on the osteoclast progenitor
cells (Fig 1; see later in the text).16,17 Thus,
osteoblasts are the cells in bone that regulate
both the amount of bone formed and the
amount of bone-resorbing osteoclasts.
In addition to their important roles in ana-
bolic and catabolic events in bone, it has re-
cently been reported that osteoblasts might have
endocrine activities (Fig 1). Mice lacking osteo-
calcin exhibit a limited bone phenotype with
slightly increased bone mass (instead of de-
creased as expected), but become fat. A detailed
phenotyping showed that the mice had in-
creased amount of visceral adipocytes, increased
serum levels of glucose, hypoinsulinemia, and
enhanced amount of triglycerides in blood.18
This phenotype has several similarities to meta-
bolic syndrome in humans, and, in fact, patients
with metabolic syndrome turned out to have low
levels of circulating osteocalcin. Secretion of sys-
temically active molecules seems not to be re-
stricted to osteoblasts in bone because osteocytes
also seem to have endocrine functions (see later
in the text).
Regulation of Osteoblast Activities
There is a great need to increase the knowledge
of mechanisms controlling osteoblast activities
and how they can discriminate between being a
bone-forming cell and a cell which also can ac-
tivate bone resorption. This is particularly true
for the development of drugs stimulating new
bone formation in patients with decreased bone
mass, such as in osteoporosis, but also in patients
with decreased alveolar bone mass because of
marginal periodontitis or peri-implantitis.
Endocrine Regulation
Osteoblasts express receptors for PTH that seems
to be linked to both increased bone resorption
and bone formation. If these cells are exposed to
chronically increased amounts of PTH, like in hy-
perparathyroidism, osteoblasts will enhance the
expression of RANKL and, thereby, stimulate os-
teoclast formation and bone resorption. However,
if the cells only are transiently exposed to the
hormone, osteoblasts will increase their bone-
forming activity. Subcutaneous daily injection with
low dose of either recombinant full-length (amino
 Osteoblasts, Osteoclasts, and Osteocytes
acids 1-84) human PTH, or a synthetic peptide
consisting of the first 34 amino acids, results in
increased bone mass and decreased fractures in
patients with postmenopausal osteoporosis.19 The
molecular mechanisms behind the chronic and
intermittent stimulation of the PTH receptor in
osteoblasts leading to divergent effects are not fully
known. Interestingly, preliminary data indicate
that intermittent PTH injections can increase alve-
olar bone level in patients with periodontitis.20
Vitamin D3 is widely used, in combination
with calcium as a treatment to enhance bone
mass in patients with osteoporosis, although the
efficacy of this treatment is debated.21 However,
there are no data to support that 1,25(OH)2-
vitamin D3 receptors in osteoblasts are directly
coupled to enhanced bone formation, and the
positive role of 1,25(OH)2-vitamin D3 in skeletal
metabolism is more likely to be indirect by in-
creased intestinal uptake of calcium.22
Paracrine Regulation
Several members of the TGF␤ superfamily, in-
cluding BMPs, TGF␤1, activins, inhibins, and
myostatins, have been shown to promote osteo-
blast differentiation, proliferation, and function.
Most attention has been paid to BMPs, a large
subfamily of different proteins of which several
have been found to be able to stimulate new
bone formation.13,23 However, receptors for
BMPs are expressed on many different cell types,
which, together with short half-life, limit their
use for systemic administration.
Another signaling system controlling osteo-
blasts is the Wnt/␤-catenin signaling pathway,
which has been the focus for a lot of interest
during recent years as a potential target for drug
development.24 Two groups, independently and
most unexpectedly, have shown that high bone
mass in certain families and low bone mass in
patients with osteoporosis pseudoglioma syn-
drome are explained by gain-of-function and
loss-of-function mutations, respectively, in the
low-density lipoprotein receptor-related protein
5 (Lrp5) gene.25,26 This gene codes for a trans-
membrane protein, which can bind extracellular
peptides in the large Wnt family, and the com-
plex activates a 7 transmembrane signaling pro-
tein called Frizzled (Fig 1, inset).27,28 Down-
stream signaling intracellularly involves the
canonical pathway activating the transcription
241
factor ␤-catenin, which leads to increased osteo-
blastic proliferation and function. The anabolic
effect of PTH might be mediated by this path-
way. Lrp5 can bind also to sclerostin and to
Dickkopf1 (Dkk1), which antagonize the bind-
ing of Lrp5 to Wnt. Interestingly, sclerostin
seems to be exclusively expressed in osteocytes,
and its expression is regulated by mechanical
loading (see later in the text). Antibodies neu-
tralizing sclerostin is currently in clinical trials
for patients with osteoporosis with promising
results.
Neuronal Regulation
Using immunohistochemical analysis, it has be-
come evident during the last 2 decades that
bone tissue is more widely innervated than pre-
viously thought. The skeletal nerve fibers ex-
press a restricted panel of different signaling
molecules.29,30 We, and others, have shown that
osteoblasts and osteoclasts are equipped with
functional receptors for the neuropeptides vaso-
active intestinal peptide, calcitonin gene-related
peptide, substance P, and neuropeptide Y, as
well as for catecholamines, glutamate, serotonin,
dopamine, and neurotrophins.
Compelling evidence for the role of the ner-
vous system was provided by studies demonstrating
that the adipocytic hormone, leptin, through re-
ceptors in the central nervous system and medi-
ated by the sympathetic system and ␤2-adrenergic
receptors in osteoblasts,31 affects bone formation.
Studies have showed that type 2 receptors for neu-
ropeptide Y in hypothalamus also affect bone for-
mation through effects on osteoblasts.32 It has
more recently been shown that the receptors for
leptin in the brain regulating bone mass are
located in the brain stem and that these receptors
on stimulation inhibit the expression of the sero-
tonin-synthesizing enzyme, tryptophan hydroxy-
lase 2, leading to decreased release of serotonin
and less activation of the serotonin receptor in
hypothalamus (Htr2c).33 Consequently, the sym-
pathetic system becomes activated and bone mass
decreases owing to enhanced signaling through
the ␤2-adrenergic receptors in osteoblasts. Ac-
cording to this view, central serotonin is a posi-
tive factor for bone mass. In contrast, it seems as
peripheral serotonin, mainly synthesized in en-
terochromaffin cells in duodenum, through the
enzyme tryptophan hydroxylase 1, has negative
242
effects on the skeleton.34 However, these data
are controversial, and most recently, Cui et al
have reported that serotonin expression in en-
terochromaffin cells is not important for bone
mass.35 Future studies are needed to clarify how
the 2 groups have come to opposite results.
Osteocytes—Cells Embedded in Bone
Matrix with Para- and Endocrine
Functions
Osteocytes that make up ⬎90% of all bone cells
are deeply embedded in both cortical and tra-
becular bone tissue. The role of osteocytes in
bone biology and pathology was elusive for many
years, although it was suggested in old literature
that these cells were involved in bone remodel-
ing and were sensitive to PTH. However, the
development of new techniques has now shown
that these cells seem to be important for regu-
lation of both osteoblasts and osteoclasts as well
as for systemic control of phosphate metabolism
(Fig 1).
Osteocyte Differentiation
Osteocytes originate from osteoblasts of which
some become buried in the matrix and trans-
formed from a polygonal cell to cells with many
dendritic processes, which communicate with
dendritic processes extending from the osteo-
blasts into the matrix.36
Osteocyte Functions
It was suggested initially by Pead et al that osteo-
cytes were the cells in bone that responded to
mechanical loading,37 and since then several
lines of evidence suggest that osteocytes exhibit
mechanoreceptors involved in the sensitivity of
bone to mechanical loading. At a molecular
level, enhanced prostaglandin production, nitric
oxide, estrogen receptors, and Wnt/␤-catenin
have been shown to be involved.36
Osteocytes express the components of the
Wnt/␤-catenin pathway including the Lrp5 an-
tagonists, Dkk1, and sclerostin.38 In contrast,
Dkk1 is expressed by many different cell types,
and sclerostin seems to be exclusively expressed
by osteocytes. Interestingly, increased bone mass
owing to mechanical loading is associated with
decreased expression of sclerostin in osteo-
cytes.39 It is likely that decreased production of
Lerner
sclerostin is the mechanism by which osteocytes
increase osteoblastic bone formation through
increased Wnt/␤-catenin signaling (Fig 1). It
has recently been shown that down regulation of
sclerostin by mechanical loading is mediated by
prostaglandin E2 acting on EP4 receptors.40
Recently, phosphate metabolism has been
shown to be regulated by FGF23, a phosphaturic
protein, highly expressed by osteocytes. This
protein acts on proximal renal tubule and de-
creases renal reabsorption of phosphate.41 Phos-
phate-regulating endopeptidase, dentin matrix
acidic phosphoprotein 1 (Dmp-1), matrix extra-
cellular phosphoglycoprotein, and FGF23 are all
highly expressed in osteocytes and have been
associated with mineralization.36
Osteocytes might also be important for the
initiation of the remodeling process. It is sug-
gested that microcracks in bone is one mecha-
nism by which remodeling is induced (Fig 1).
Microcrack formation in bone tissue leads to
osteocytic apoptosis, and these cells have been
found to express RANKL, which could be a
mechanism to induce osteoclastogenesis needed
to resorb the damaged bone and subsequently
substitute old bone with new bone.2
Osteoclasts—Responsible for
Physiological and Pathologic Bone
Breakdown
Osteoclasts are the only cells in nature that can
degrade mineralized bone tissue and are impor-
tant for physiological remodeling and modeling
processes, calcium homeostasis, tooth eruption,
and orthodontic tooth movement. Excessive os-
teoclast formation and activity is an important
pathogenetic mechanism for bone loss in dis-
eases, such as osteoporosis, marginal and apical
periodontitis, peri-implantitis, rheumatoid ar-
thritis, loosening of joint prostheses, and meta-
static tumor disease.
Osteoclast Function
Mature osteoclasts are present only on mineral-
ized bone surfaces to which they attach by a
sealing zone (Fig 2, inset down to the right). The
attachment involves expression of the dimeric
integrin ␣v␤3 in the sealing zone, which binds to
exposed aginine-glycine-aspartic acid (RAG) se-
quences in bone matrix proteins such as osteo-
 Osteoblasts, Osteoclasts, and Osteocytes 243

HEMATOPOETIC
OSTEOBLASTS/
STEM CELL
STROMAL CELLS

M- RANK
OPG
CSF L

PROLIFERATION DIFFERENTIATION FUSION

MYELOID
STEM CELL
OSTEOCLAST OSTEOCLAST
PROGENITOR CELLS

OSTEOCLAST
IMMUNOGL-LIKE
RANKL cell nuclei
M-CSF RECEPTORS
CELL MEMBR.
c-Fms
RANK FcRγ sealing
DAP12 ruffled border
zone
+ release of
&O
pH 4.5 enzymes
KINASES
TRANSCRIPT. Mineral Matrix
FACTORS dissolu
on degrada
on
NF-κB AP-1 NFATc1 IRF8
BONE
prolifera
on, differen
a
on and fusion RESORPTION

Figure 2. Osteoclasts are derived from hematopoietic stem cells of the myeloid lineage. Differentiation requires
stimulation of the early progenitor cells by the cytokine macrophage colony-stimulating factor (M-CSF) pro-
duced by osteoblasts on the surface of bone or by stromal cells in bone marrow. M-CSF enhances proliferation
and survival of progenitors and upregulates the receptor RANK. Through increased production of RANKL and
decreased release of OPG, the receptor RANK on the progenitor cells will be activated, causing differentiation
of the cells to osteoclast progenitor cells. RANKL and OPG are expressed by osteoblasts and stromal cells and
are regulated by several bone-resorbing hormones and cytokines. Eventually, differentiated osteoclast progenitor
cells will fuse to the mature, multinucleated, and bone-resorbing osteoclasts. Inset to the left: for osteoclast
differentiation to occur, M-CSF activates its cognate receptor called c-Fms and 3 RANKL molecules activate the
trimeric RANK receptor. In addition, it is required that signals either through FcR␥ or DAP12 are activated.
These 2 molecules cannot recognize ligands but have to dimerize with immunoglobulin-like receptors, a large
family of which several, including OSCAR, are expressed in osteoclast progenitors. It is neither known which of
these receptors are important in osteoclastogenesis nor which ligands are involved. Downstream events include
activation of several different phosphorylating enzymes (kinases), which then activate a wide variety of transcrip-
tion factors, including NF-␬B, AP-1, and NFATc1, that induce many genes involved in osteoclast proliferation,
differentiation, and fusion. In contrast, some transcription factors important for macrophage differentiation are
down regulated such as IRF8. Inset to the right: The mature osteoclasts are multinucleated giant cells that attach
to bone through interaction between the sealing zone and extracellular matrix proteins at the bone surface such
as osteopontin. A characteristic feature of osteoclasts is the development of an extensively folded cell membrane,
ruffled border, in the part of the osteoclasts facing bone. In this area, proton pumps and chloride channels are
expressed, which are important for the extracellular acidification and demineralization of bone. Then, proteo-
lytic enzymes are released, which degrade the extracellular matrix proteins.

pontin and bone sialoprotein.42 The cell mem-
brane area surrounded by the sealing zone
becomes extensively folded and develops into
the ruffled border expressing proton pumps and
chloride channels by which osteoclasts create an
acid extracellular milieu in the resorption lacu-
nae, and, thereby, resorption is initiated by dis-
solution of the mineral crystals.43 Subsequently,
the decalcified bone matrix is degraded by pro-
teolytic enzymes released into the resorption la-
244 Lerner
cunae. It is likely that several proteolytic en-
zymes are required to degrade the different
bone matrix proteins, but the cysteine protei-
nase cathepsin K is particularly important.44 Dif-
ferent inhibitors of this enzyme are in clinical
trials in patients with excessive bone resorption.
Osteoclast Differentiation
The formation of multinucleated mature oste-
oclasts is because of fusion of mononuclear pro-
genitor cells originating from myeloid hematopoi-
etic stem cells. These progenitors can differentiate
either to macrophages, dendritic cells in the im-
mune system, or osteoclasts.45 The differentiation
of all these cell types requires stimulation of the
receptor c-Fms by the ligand macrophage colony-
stimulating factor (M-CSF), which is needed for
proliferation and survival of the mononucleated
progenitor cells (Fig 2). The specific differenti-
ation to osteoclasts is because of activation of the
receptor RANK by RANKL expressed by stromal
cells in bone marrow and osteoblasts.1,45,46 The
interaction between RANKL and RANK is inhib-
ited by osteoprotegerin (OPG), which is a decoy
receptor with large homologies to RANK. How-
ever, RANKL and RANK are restricted to some
few cell types; OPG is ubiquitously expressed, for
reasons unknown. Osteoclastogenesis is regu-
lated by the relative expression of RANKL and
OPG, and stimulators of osteoclast formation
such as PTH and 1,25(OH)2-vitamin D3 increase
RANKL and decrease OPG expression. The
most novel antiresorptive therapy in skeletal dis-
eases associated with increased bone resorption
is based on inhibition of RANKL. Thus, human-
ized monoclonal antibodies neutralizing human
RANKL, injected only twice per year, results in
increased bone mass and decreased fractures in
patients with postmenopausal osteoporosis.
For osteoclastogenesis to occur, it has been
found that signals mediated through the trans-
membrane adapter proteins FcR␥ and/or DAP12
also are required.47 FcR␥ or DAP12 cannot bind
ligands, but heterodimerize with different immu-
noglobulin-like receptors (Fig 2, inset down to
the left). It is not known which of these recep-
tors are crucial in osteoclastogenesis, which li-
gands are important for FcR␥/DAP12 signaling
in bone, or which cells produce the ligands for
these costimulatory receptors needed for oste-
oclast differentiation.
The knowledge of the crucial roles for c-Fms,
RANK, and FcR␥/DAP12 in osteoclast differen-
tiation has lead to extensive investigations on
downstream signaling events (Fig 2, inset down
to the left).47-49 Early events include activation of
different kinases leading to activation of a variety
of transcription factors, including NF-␬B, c-Fos–
containing activator protein 1 (AP-1), PU.1, mi-
crophthalmia-associated transcription factor
(MITF), and nuclear factor of activated T-cells 1
(NFATc1), the latter being regarded as the mas-
ter regulator of osteoclastogenesis. In addition,
transcription factors such as MafB and inter-
feron regulatory factor 8 (IRF8), which are im-
portant for macrophage differentiation, are
down regulated during osteoclastogenesis.
We have found that osteoclast differentiation
is regulated also by cysteine proteinases. Thus, 2
endogenous inhibitors of cysteine proteinases,
cystatin C and cystatin D, and the fungal cysteine
proteinase inhibitor E-64, potently inhibit oste-
oclast formation induced by RANKL in oste-
oclast progenitors from mouse bone marrow
and spleen, as well as in RANKL-stimulated hu-
man osteoclast progenitors from peripheral
blood.50,51
Osteoclast Heterogeneity
Most of the studies on osteoclast differentiation
have been performed in vitro using cells from
mouse bone marrow. However, mature oste-
oclasts are not formed within the bone marrow
in vivo. It is assumed that properly stimulated
bone marrow stromal cells expressing RANKL in
vivo initiate osteoclast differentiation in bone
marrow and those partially differentiated mono-
nucleated osteoclast progenitors, like other he-
matopoietic cells, are released into circulation.
These cells are then recruited to periosteal and
endosteal sites in the skeleton by a homing pro-
cess that is not fully understood, but may involve
stromal cell-derived factor-1 (SDF-1 or CXCL12)
produced by osteoblasts and CXCR4 expressed
by osteoclast progenitors.52 In the periosteum
and endosteum, the partially differentiated oste-
oclast progenitors are latent until osteoblasts at
the bone surface express RANKL and induce
their further differentiation to mononucleated
progenitors, which eventually fuse to latent
multinucleated osteoclasts. In parallel, osteo-
blasts degrade the unmineralized bone matrix,
 Osteoblasts, Osteoclasts, and Osteocytes
which is present between the cells and the min-
eralized bone, and then retract from the area
giving the latent osteoclasts a possibility to mi-
grate into the area and attach to mineralized
bone through the sealing zone.43
There are several lines of evidence suggesting
that osteoclasts in different areas of the skeleton
are not identical. Although the skeletal pheno-
type in mice with a wide variety of global knock-
outs of certain genes is identical in all parts of
the skeleton, there are examples in knockout
mice that osteoclasts in some bones, but not in
others, are affected. Everts et al have recently
shown that osteoclast formation in bone marrow
cultures from long bones and jaw bones in mice
is different, and the osteoclasts derived from the
different bone marrows are different depending
on whether the osteoclasts are incubated on
bone or on dentin. The view that osteoclasts in
different parts of the skeleton are not all the
same is discussed in more detail by Everts et al53
and Henriksen et al.54
Regulation of Osteoclastogenesis
As described earlier, formation of mature oste-
oclasts is regulated by RANKL expressed by cells
in the vicinity of the osteoclast progenitors,
which means that it is the capacity to regulate
RANKL/OPG ratio, which is crucial for hor-
mones or cytokines stimulating bone resorption.
Hormones known to stimulate osteoclastogen-
esis through RANKL are PTH, 1,25(OH)2-vita-
min D3, thyroid hormones, glucocorticoids, and
retinoids.45 Some hormones can act as inhibitors
of osteoclast formation through receptors on
the osteoclasts and calcitonin, estrogen, and reti-
noids belong to this group.
Several cytokines also can either stimulate or
inhibit osteoclast formation. Interleukin 1 (IL-
1), IL-6, IL-11, IL-15, IL-17, IL-23, IL-32␥, car-
diotrophin-1, tumor necrosis factor ␣ (TNF)-␣,
leukemia inhibitory factor, and oncostatin M are
cytokines that can stimulate osteoclastogenesis
through increased RANKL, whereas IL-4, IL-10,
IL-12, IL-13, IL-18, IL-27, interfern-␣ (IFN-␣),
and IFN-␤ are inhibitory cytokines.45,55 The
stimulatory cytokines act indirectly through in-
creased RANKL, whereas some inhibitory cyto-
kines act on osteoblasts to decrease RANKL (eg,
IL-4 and IL-13), and others act directly on oste-
oclast progenitors (eg, IFN-␣ and IFN-␤). Cyto-
245
kines are mainly involved in inflammation-in-
duced osteoclast formation, but the relative
importance of cytokine-driven RANKL versus
RANKL expressed by T-, B-, or natural killer cells
(NK-cells) is not known. Other inflammatory
mediators with capacity to enhance osteoclast
formation through RANKL in osteoblasts are
prostaglandins, especially PGE2, which also may
act downstream of some of the osteoclast-stimu-
lating cytokines. In addition, we have shown that
bradykinin, in concert with IL-1 and TNF, syn-
ergistically enhances periosteal RANKL forma-
tion.30
Orthodontic Forces and Bone Modeling
Movement of teeth induced by orthodontic
treatment involves reactions in alveolar bone, as
well as in periodontal ligament, gingiva, blood
vessels, and nerves as elegantly summarized in a
recent review.56 A prerequisite for the teeth to
move in response to orthodontic treatment is
osteoclast formation and bone resorption. This
is illustrated by experimental studies demon-
strating that bisphosphonates, powerful inhibi-
tors of osteoclasts, inhibit orthodontic tooth
movement in mice57 and by case observations in
osteoporotic patients treated with bisphospho-
nates showing decreased response to orthodon-
tic treatment.58 The osteoclasts appear on both
the periodontal ligament side and the bone mar-
row side, and crucial biological questions are: 1)
what drives osteoclastogenesis at these 2 sites?
and 2) where do the progenitors come from? In
the bone marrow side, it is apparent that pro-
genitors exist within the hematopoietic cells and
may easily reach trabecular and endosteal sites.
In the alveolar side, the progenitors may either
be recruited from the circulation or be preexist-
ing in the periodontal ligament. It is most likely
that RANKL is needed also for osteoclast forma-
tion during orthodontic treatment and, there-
fore, an interesting issue is which cells express
RANKL and what drives the expression. The role
of RANKL is indicated by the observation show-
ing that tooth movement and osteoclast forma-
tion are enhanced in mice deficient in OPG.59
Although local factors are most important for
RANKL expression and osteoclast formation, an
interesting issue is whether systemic factors like
hormones and drugs can affect the process. The
fact that not only drugs60 but also systemic fac-
246
tors are involved in osteoclast formation in-
duced by orthodontic treatment is suggested by
experimental studies showing that decreased es-
trogen levels in ovariectomized rats is associated
with enhanced tooth movement61 and that in-
duction of type 1 diabetes in rats is associated
with increased osteoclast formation and faster
tooth movement.62
Further supporting the role of RANKL are
observations showing that orthodontic tooth
movement is associated with increased expres-
sion of RANKL mRNA in human periodontal
ligament63 and RANKL protein in cells present
in the periodontal ligament of rats.64 Given the
fact that fibroblasts in periodontal ligament can
express RANKL, it is likely that these cells are
involved in the upregulation of RANKL. How-
ever, the increased expression of RANKL in peri-
odontal ligament is probably not involved in
osteoclast formation at the endosteal and trabec-
ular sites during orthodontic tooth movement.
An alternative possibility to the view that fibroblasts
in the periodontal ligament are driving osteoclast
formation could be that treatment-induced micro-
cracks in the alveolar cortical bone and apoptotic
osteocytes expressing RANKL induce osteoclast
formation at both the periodontal ligament and
bone marrow site, similar to what is thought being
an initiating mechanism in physiological bone re-
modeling.3-5
It has long been considered that a local in-
flammatory response induces osteoclast forma-
tion during orthodontic treatment, although
histologic studies have shown only restricted
number of inflammatory cells. However, it has
also to be kept in mind that resident cells can
produce pro- and anti-inflammatory cytokines.55
Experimentally induced orthodontic treatment
has demonstrated an increased expression of
osteotropic cytokines such as TNF-␣ in the peri-
odontal ligament,63,65 and it is possible that this
cytokine is expressed by the periodontal liga-
ment fibroblasts, which then, in an autocrine
manner, induce their own RANKL expression.
Another possibility is that TNF-␣ increases
RANKL in the osteoblasts present at the surface
of the cortical bone in teeth alveoli. The finding
that tooth movement is reduced in mice lacking
TNF receptor type 2 shows that TNF signaling is
involved in orthodontic tooth movement.65 It is
also well known that tooth movement induces
increased formation of prostaglandins and that
Lerner
inhibition of their synthesis decreases the rate of
tooth movement.66 Because TNF-␣ is a potent
stimulator of PGE2 formation in most cells by
upregulating the rate-limiting enzyme cyclooxy-
genase-2, and because PGE2 can stimulate
RANKL expression in periodontal ligament fi-
broblasts and osteoblasts, it is possible that a
cascade of TNF-␣–induced PGE2 is responsible
for the RANKL increase in the periodontal liga-
ment. Given the fact that osteoclast formation
can be controlled by a wide variety of stimulatory
and inhibitory cytokines, as discussed earlier, it
is likely that stimulation of RANKL during orth-
odontic treatment is more complicated than sug-
gested previously and, in fact, is dependent on a
balance between several stimulatory and inhibi-
tory signaling molecules acting in concert to
balance the expression of RANKL and OPG.
Essentially nothing is known about which fac-
tors drive bone formation during orthodontic
tooth movement. This means that the mecha-
nisms regulating anabolic activities in bone mod-
eling are not known. However, it is not unlikely
that interactions between osteocytes and osteo-
blasts are important and the Wnt/␤-catenin
pathway and sclerostin are involved, like in phys-
iological remodeling of bone and in the ana-
bolic response to mechanical loading.
Conclusions
It is apparent that the detailed knowledge of
bone tissue reactions in response to orthodontic
treatment is still elusive, and that understanding
the biology of tooth movement and the outcome
of treatment in individual patients is a complex
process requiring knowledge in many different
areas of biomedicine. The rapid development of
molecular biology along with translational stud-
ies in humans, as well as in experimental sys-
tems, are likely to give much more detailed in-
sight in cellular and molecular mechanisms
involved in the modeling processes induced by
orthodontic forces. This is a prerequisite to the
understanding of responses in different individ-
uals and to generate ideas of mechanisms by
which tooth movement could be regulated not
only by mechanical forces but also by biologics,
if needed.
 Osteoblasts, Osteoclasts, and Osteocytes
Acknowledgments
Studies performed in the author’s laboratories were sup-
ported by grants from the Swedish Research Council, Swed-
ish Dental Society, the Royal 80 year Fund of King Gustav V,
the Swedish Rheumatism Association, Medical Faculty at
Umeå University, Combine, and ALF/LUA grants from the
Sahlgrenska University Hospital and the County Council of
Västerbotten.
References
1. Takayanagi H: Osteoimmunology and the effects of the
immune system on bone. Nat Rev Rheumatol 5:667-676,
2009
2. Martin TJ, Seeman E: Bone remodelling: Its local regu-
lation and the emergence of bone fragility. Best Pract
Res Clin Endocrinol Metab 22:701-722, 2008
3. Martin TJ, Sims NA: Osteoclast-derived activity in the
coupling of bone formation to resorption. Trends Mol
Med 11:76-81, 2005
4. Boyce BF, Yao Z, Xing L: Osteoclasts have multiple roles
in bone in addition to bone resorption. Crit Rev Eu-
karyot Gene Expr 19:171-180, 2009
5. Martin T, Gooi JH, Sims NA: Molecular mechanisms in
coupling of bone formation to resorption. Crit Rev Eu-
karyot Gene Expr 19:73-88, 2009
6. Zhao C, Irie N, Takada Y, et al: Bidirectional ephrinB2-
EphB4 signaling controls bone homeostasis. Cell Metab
4:111-121, 2006
7. Karsenty G: Transcriptional control of skeletogenesis.
Annu Rev Genomics Hum Genet 9:183-196, 2008
8. Khosla S, Melton LJ 3rd, Riggs BL: The unitary model
for estrogen deficiency and the pathogenesis of osteo-
porosis: Is a revision needed? J Bone Miner Res 26:441-
450, 2011
9. Parfitt AM: Parathyroid hormone and periosteal bone
expansion. J Bone Miner Res 17:1741-1743, 2002
10. Pevsner-Fischer M, Levin S, Zipori D: The origins of
mesenchymal stromal cell heterogeneity. Stem Cell Res
7:560-568, 2011
11. Nombela-Arrieta C, Ritz J, Silberstein LE: The elusive
nature and function of mesenchymal stem cells. Nat Rev
Mol Cell Biol 12:126-131, 2011
12. Yamaza T, Ren G, Akiyama K, et al: Mouse mandible
contains distinctive mesenchymal stem cells. J Dent Res
90:317-324, 2011
13. Canalis E: Update in new anabolic therapies for osteo-
porosis. J Clin Endocrinol Metab 95:1496-1504, 2010
14. Rodan GA, Martin TJ: Role of osteoblasts in hormonal
control of bone resorption—A hypothesis. Calcif Tissue
Int 34:311, 1982
15. Takahashi N, Yamana H, Yoshiki S, et al: Osteoclast-like
cell formation and its regulation by osteotropic hor-
mones in mouse bone marrow cultures. Endocrinology
122:1373-1382, 1988
16. Kong YY, Boyle WJ, Penninger JM: Osteoprotegerin li-
gand: A common link between osteoclastogenesis,
lymph node formation and lymphocyte development.
Immunol Cell Biol 77:188-193, 1999
247
17. Takahashi N, Udagawa N, Suda T: A new member of
tumor necrosis factor ligand family, ODF/OPGL/
TRANCE/RANKL, regulates osteoclast differentiation
and function. Biochem Biophys Res Commun 256:449-
455, 1999
18. Lee NK, Sowa H, Hinoi E, et al: Endocrine regulation of
energy metabolism by the skeleton. Cell 130:456-469,
2007
19. Kraenzlin ME, Meier C: Parathyroid hormone analogues
in the treatment of osteoporosis. Nat Rev Endocrinol
7:647-656, 2011
20. Bashutski JD, Eber RM, Kinney JS, et al: Teriparatide
and osseous regeneration in the oral cavity. N Engl
J Med 363:2396-2405, 2010
21. Bouillon R, Bischoff-Ferrari H, Willett W: Vitamin D and
health: Perspectives from mice and man. J Bone Miner
Res 23:974-979, 2008
22. Bouillon R, Carmeliet G, Verlinden L, et al: Vitamin D
and human health: Lessons from vitamin D receptor
null mice. Endocr Rev 29:726-776, 2008
23. Rider CC, Mulloy B: Bone morphogenetic protein and
growth differentiation factor differentiation factor cyto-
kine families and their protein antagonists. Biochem J
429:1-12, 2010
24. Marie PJ, Kassem M: Osteoblasts in osteoporosis: Past,
emerging, and future anabolic targets. Eur J Endocrinol
165:1-10, 2011
25. Gong Y, Slee RB, Fukai N, et al: LDL receptor-related
protein 5 (LRP5) affects bone accrual and eye develop-
ment. Cell 107:513-523, 2001
26. Little RD, Carulli JP, Del Mastro RG, et al: A mutation in
the LDL receptor-related protein 5 gene results in the
autosomal dominant high-bone-mass trait. Am J Hum
Genet 70:11-19, 2002
27. Milat F, Ng KW: Is Wnt signalling the final common
pathway leading to bone formation? Mol Cell Endocri-
nol 310:52-62, 2009
28. Case N, Rubin J: Beta-catenin—A supporting role in
skeleton. J Cell Biochem 110:545-553, 2010
29. Warden SJ, Bliziotes MM, Wiren KM, et al: Neural reg-
ulation of bone and the skeletal effects of serotonin
(5-hydroxytryptamine). Mol Cell Endocrinol 242:1-9,
2005
30. Lerner UH, Persson E, Lundberg P: Kinins and neuro-
osteogenic factors, in: Bilezikian JP, Martin TJ, Raisz LG
(eds): Principles of Bone Biology (ed 3). San Diego, CA,
Academic Press, 2008, pp 1025-1057
31. Ducy P, Amling M, Takeda S, et al: Leptin inhibits bone
formation through a hypothalamic relay: A central con-
trol of bone mass. Cell 100:197-207, 2000
32. Baldock PA, Sainsbury A, Couzens M, et al: Hypotha-
lamic Y2 receptors regulate bone formation. J Clin Invest
109:915-921, 2002
33. Yadav VK, Oury F, Suda N, et al: A serotonin-dependent
mechanism explains the leptin regulation of bone mass,
appetite, and energy expenditure. Cell 138:976-989,
2009
34. Yadav VK, Ryu JH, Suda N, et al: Lrp5 controls bone
formation by inhibiting serotonin synthesis in the duo-
denum. Cell 135:825-837, 2008
248
35. Cui Y, Niziolek PJ, Macdonald BT, et al: Lrp5 functions
in bone to regulate bone mass. Nat Med 17:684-691,
2011
36. Bonewald LF: The amazing osteocyte. J Bone Miner Res
26:229-238, 2011
37. Pead MJ, Suswillo R, Skerry TM, et al: Increased 3H-
uridine levels in osteocytes following a single short pe-
riod of dynamic bone loading in vivo. Calcif Tissue Int
43:92-96, 1988
38. Poole KE, van Bezooijen RL, Loveridge N, et al: Scleros-
tin is a delayed secreted product of osteocytes that in-
hibits bone formation. FASEB J 19:1842-1844, 2005
39. Robling AG, Niziolek PJ, Baldridge LA, et al: Mechanical
stimulation of bone in vivo reduces osteocyte expression
of Sost/sclerostin. J Biol Chem 283:5866-5875, 2008
40. Galea GL, Sunters A, Meakin LB, et al: Sost down-regu-
lation by mechanical strain in human osteoblastic cells
involves PGE2 signaling via EP4. FEBS Lett 585:2450-
2454, 2011
41. Lu Y, Feng JQ: FGF23 in skeletal modeling and remod-
eling. Curr Osteoporos Rep 9:103-108, 2011
42. Zou W, Teitelbaum SL: Integrins, growth factors, and
the osteoclast cytoskeleton. Ann N Y Acad Sci 1192:27-
31, 2010
43. Lerner UH: Osteoclast formation and resorption. Matrix
Biol 19:107-120, 2000
44. Brömme D, Lecaille F: Cathepsin K inhibitors for osteo-
porosis and potential off-target effects. Expert Opin In-
vestig Drugs 18:585-600, 2009
45. Lorenzo J, Horowitz M, Choi Y: Osteoimmunology: In-
teractions of the bone and immune system. Endocr Rev
29:403-440, 2008
46. Edwards JR, Mundy GR: Advances in osteoclast biology:
Old findings and new insights from mouse models. Nat
Rev Rheumatol 7:235-243, 2011
47. Negishi-Koga T, Takayanagi H: Ca2⫹-NFATc1 signalling
is an essential axis of osteoclast differentiation. Immunol
Rev 231:241-256, 2009
48. Boyce BF, Yao Z, Xing L: Functions of nuclear factor
kappaB in bone. Ann N Y Acad Sci 1192:367-375, 2010
49. Nemeth K, Schoppet M, Al-Fakhri N, et al: The role of
osteoclast-associated receptor in osteoimmunology. J Im-
munol 186:13-18, 2011
50. Brage M, Ransjö M, Kasprzykowski F, et al: Osteoclasto-
genesis is decreased by cysteine proteinase inhibitors.
Bone 34:412-424, 2004
51. Strålberg F, Henning P, Gjertson I, et al: Human and
mouse osteoclastogenesis are inhibited by cysteine pro-
teinase inhibitors through interference with RANK sig-
naling. Presented at the Annual Meeting of the Ameri-
can Society for Bone and Mineral Research. San Diego,
CA, 2011
Lerner
52. Kollet O, Dar A, Shivtiel S, et al: Osteoclasts degrade
endosteal components and promote mobilization of he-
matopoietic progenitor cells. Nat Med 12:657-664, 2006
53. Everts V, de Vries TJ, Helfrich MH: Osteoclast heteroge-
neity: Lessons from osteopetrosis and inflammatory con-
ditions. Biochim Biophys Acta 1792:757-765, 2009
54. Henriksen K, Bollerslev J, Everts V, et al: Osteoclast
activity and subtypes as a function of physiology and
pathology—Implications for future treatments of osteo-
porosis. Endocr Rev 32:31-63, 2011
55. Liu YC, Lerner UH, Teng YT: Cytokine responses against
periodontal infection: Protective and destructive roles.
Periodontol 2000 52:163-206, 2010
56. Krishnan V, Davidovitch Z: On a path to unfolding the
biological mechanisms of orthodontic tooth movement.
J Dent Res 88:597-608, 2009
57. Fujimura Y, Kitaura H, Yoshimatsu M, et al: Influence of
bisphosphonates on orthodontic tooth movement in
mice. Eur J Orthod 31:572-577, 2009
58. Ghoneima AA, Allam ES, Zunt SL, et al: Bisphospho-
nates treatment and orthodontic considerations. Orthod
Craniofac Res 13:1-10, 2010
59. Oshiro T, Shiotani A, Shibasaki Y, et al: Osteoclast in-
duction in periodontal tissue during experimental
movement of incisors in osteoprotegerin-deficient mice.
Anat Rec 266:218-225, 2002
60. Krishnan V, Davidovitch Z: The effect of drugs on orth-
odontic tooth movement. Orthod Craniofac Res 9:163-
171, 2006
61. Salazar M, Hernandes L, Ramos AL, et al: Effect of
teriparatide on induced tooth displacement in ovariec-
tomized rats: A histomorphometric analysis. Am J Or-
thod Dentofacial Orthop 139:e337-e344, 2011
62. Braga SM, Taddei SR, Andrade I, et al: Effect of diabetes
on orthodontic tooth movement in a mouse model. Eur
J Oral Sci 119:7-14, 2011
63. Garlet TP, Coelho U, Silva JS, et al: Cytokine expression
pattern in compression and tension sides of the peri-
odontal ligament during orthodontic tooth movement
in humans. Eur J Oral Sci 115:355-362, 2007
64. Brooks PJ, Nilforoushan D, Manolson MF, et al: Molec-
ular markers of early orthodontic tooth movement. An-
gle Orthod 79:1108-1113, 2009
65. Yoshimatsu M, Shibata Y, Kitaura H, et al: Experimental
model of tooth movement by orthodontic force in mice
and its application to tumor necrosis factor receptor-
deficient mice. J Bone Miner Metab 24:20-27, 2006
66. Walker JB, Buring SM: NSAID impairment of orthodon-
tic tooth movement. Ann Pharmacother 35:113-115,
2001