CE: D.C.

; SCS-16-01512; Total nos of Pages: 10;

SCS-16-01512

SCIENTIFIC FOUNDATION

Biology of Bone Formation, Fracture Healing, and

Distraction Osteogenesis
Christopher M. Runyan, MD, PhD and Kyle S. Gabrick, MD
1950s to 1980s the Russian orthopedist Gavril Ilizarov carefully
Abstract: Distraction osteogenesis is a bone-regenerative process characterized and perfected the technique. His contributions include
in which an osteotomy is followed by distraction of the surrounding development of a rigid external ring fixator and determination of
vascularized bone segments, with formation of new bone within the optimal pin placement and stability within the fixator, demon-
distraction gap. Distraction osteogenesis is efficacious for recon- stration of the feasibility of corticotomies rather than osteotomies,
structing critical sized bony defects in the appendicular and cranio- determination of ideal latency and activation periods, and a rigorous
facial skeleton. To provide opportunity to expand applications of histologic assessment of the distraction site, including a description
distraction osteogenesis, it is important to have a thorough under- of the neo-physis.3,4 Distraction osteogenesis was first applied to
the craniofacial skeleton by Snyder et al.5 His group surgically
standing of the underlying molecular biology and physiology of bone
shortened 1 side of a canine mandible, and then corrected the
development and fracture healing. To accomplish these objectives a resultant crossbite with an external screw-driven device. McCarthy
review of the literature was performed using search terms ‘‘endo- et al conducted a series of further canine experiments to identify
chondral ossification, intramembranous ossification, craniofacial optimal latency, activation, and consolidation phases within the
skeleton, appendicular skeleton, fracture healing, bone development, craniofacial skeleton, before applying it to reconstruct the human
and distraction osteogenesis.’’ Bones of the craniofacial and appen- hypoplastic mandible.6– 9 Distraction osteogenesis has since pro-
dicular skeleton have distinct mechanisms of embryonic develop- vided craniofacial surgeons with a particularly useful tool to address
ment. The former develops from growth centers of mesenchymal bony defects and deficits. While distraction has been well described
precursors through intramembranous ossification. The latter forms in the surgical literature, advances in regenerative medicine may
though endochondral ossification in growth plates. However, both warrant a second look at the biology of distraction and its appli-
cation to bone tissue engineering.
endochondral and intramembranous bone share similar master regu-
Tissue engineering requires 3 primary components: a progenitor
latory transcription factors and downstream growth factors. Fracture or stem cell to produce the desired tissue, growth factors to provide
healing mirrors the pathway by which these bones developed embry- the necessary inductive signals to the progenitor cells, and a
onically. In contrast, bone formed by distraction osteogenesis does so scaffold to guide appropriate 3-dimensional configuration of the
by intramembranous ossification, regardless of whether it occurs growing tissue. Clinical use of distraction osteogenesis is essen-
within the appendicular or craniofacial skeleton. Understanding tially a form of bone tissue engineering. During distraction osteo-
molecular pathway differences between bone formation by these genesis, the bone-anchored distractor device provides the rigidity
mechanisms may allow for optimization and expansion of skeletal and necessary space that would normally be provided by a scaffold.
reconstruction by distraction osteogenesis Progenitor cells and growth factors are conveniently provided by
the niche surrounding the distraction site. To the reconstructive
surgeon hoping to generate new, vascularized bone, these cellular
Key Words: Appendicular skeleton, craniofacial skeleton, and molecular interactions may be a black box. Bone is unique
distraction osteogenesis, endochondral ossification, fracture among all tissues in the body, as it is the only tissue to heal or
healing, intramembranous ossification regenerate without scar formation and to regain its full premorbid
strength and function. The complex molecular interactions of
(J Craniofac Surg 2017;00: 00–00) healing bone reflect how they formed during development.10,11
An understanding of the molecular biology and physiology of bone

D istraction osteogenesis is a bone-regenerative process in which

an osteotomy is followed by gradual distraction of the sur-
rounding vascularized bone segments, with formation of new bone
formation and fracture healing will provide insights into how bone
is produced during distraction osteogenesis. This will then allow the
reconstructive surgeon to optimally utilize distraction osteogenesis
within the distraction gap. First described by Alessandro Codivilla for bone production and potentially offer new therapeutic appli-
at the turn of the 20th century, its clinical use was initially limited by cations of the technology
poor predictability and high rates of complication.1,2 From the

From the Department of Plastic Surgery, Wake Forest Baptist Medical

PATHWAYS OF BONE DEVELOPMENT
Center, Winston-Salem, NC. During embryonic development bone forms by 1 of 2 pathways:
Received October 27, 2016. endochondral or intramembranous ossification.12 The former
Accepted for publication December 20, 2016. requires a cartilaginous intermediate, and is responsible for the
Address correspondence and reprint requests to Christopher M. Runyan, formation of the entire appendicular (limbs and pelvis) and much of
MD, PhD, Department of Plastic Surgery, Wake Forest Baptist Health, the axial skeleton, including the ribs, scapulae, and skull base.
Medical Center Blvd., Winston Salem, NC 27157; Endochondral bone forms from either paraxial mesoderm (axial
E-mail: [email protected] skeleton) or from lateral plate mesoderm, which contributes to the
The authors report no conflicts of interest.
Copyright # 2017 by Mutaz B. Habal, MD limb buds (appendicular skeleton). Intramembranous ossification
ISSN: 1049-2275 does not involve a cartilaginous intermediate but instead relies on
DOI: 10.1097/SCS.0000000000003625 direct differentiation of mesenchymal precursor or neural crest cells

The Journal of Craniofacial Surgery  Volume 00, Number 00, Month 2017 1
Copyright © 2017 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
CE: D.C.; SCS-16-01512; Total nos of Pages: 10;
SCS-16-01512

Runyan and Gabrick The Journal of Craniofacial Surgery  Volume 00, Number 00, Month 2016

into osteoblasts, and is the mechanism for development of most of Some of the caudal-most bones of the skull (occipital, ethmoid,
the craniofacial skeleton. Intramembranous bones within the cra- petrous portion of the temporal, and portions of the sphenoid
niofacial skeleton (Fig. 1) are derived from either neural crest cells bones) develop by endochondral ossification.
for the more cephalad structures and facial bones, or from paraxial Endochondral and intramembranous bone both are first ident-
mesoderm for the more caudal structures and skull base.13,14 ified as clusters of undifferentiated cells known as mesenchymal
condensations which by an unknown mechanism coalesce in the
areas of future skeletal development.12,15 Neural crest cells are
derived from neuroectoderm of the developing neural tube, but
undergo an epithelial-to-mesenchymal transition followed by dela-
mination and ventral migration into craniofacial structures within
the developing embryo. As with mesoderm-derived cells within
mesenchymal condensations, neural crest cells similarly may lead
to bone production via either intramembranous or endochondral
ossification16,17 (Fig. 2). The progression and differentiation of
these cells are guided by signaling pathways, many of which are
relevant for fracture healing.
Figure 3 depicts the possible fates of cells within the mesench-
ymal condensations. In the craniofacial skeleton mesenchymal
condensations may undergo intramembranous ossification, produ-
cing bone directly without a cartilaginous intermediate. In the
remainder of the axial and appendicular skeleton mesenchymal
precursor cells give rise to an intermediate tissue, so-called imma-
ture cartilage. This immature cartilage is then destined to 1 of 2
pathways: persistent and replacement cartilages. Persistent cartilage
remains relatively avascular, and eventually forms the cartilages of
the nose, ear, intervertebral discs, and ribs. In contrast, replacement
cartilage undergoes chondrocyte hypertrophy and vascularization
allowing progression to endochondral ossification. During this
process, chondrocytes enter a tightly controlled program of pro-
liferation, prehypertrophy, hypertrophy, apoptosis, and replacement
by osteoblasts.18
Many of the signal transduction pathways regulating the pro-
gression of mesenchymal condensations to bone and cartilage are
understood, and are recapitulated in fracture healing. The proosteo-
genic factor runt-related transcription factor 2 (Runx2) is expressed
among both preosteoblasts in mesenchymal condensations and later
in immature cartilage.19 Mice deficient in both alleles of Runx2
form no bone demonstrating its requirement for both intramem-
branous and endochondral bone formation.20–22 Further, a mutation
in one copy of Runx2 in humans leads to cleidocranial dysplasia
which is marked by hypoplastic clavicles, supernumerary teeth,
enlarged fontanelles, and eventual osteoporosis.21 A similarly
important prochondrogenic transcription factor, Sox9 (SRY [sex-
determining region Y]-related high mobility group box gene 9) is
essential for cartilage development. The absence of Sox9 in mice
results in a complete absence of cartilage formation,23–25 and
partial loss in humans leads to campomelic dysplasia,26–28 which
is marked by craniofacial defects, bowing and angulation of the
long bones, and tracheobronchial hypoplasia that frequently leads to
perinatal respiratory distress and lethality. Together Runx2 and

Neural Crest Cells

Intramembranous Endochondral
FIGURE 1. Derivation of bones of the calvarium (adapted from Percival).14 (A, Ossification Ossification
B) Two views of the human craniofacial skeleton, including (A) frontal, (B) lateral
depicting both the cell source and mechanism of bone formation. Light blue—
intramembranous ossification. Yellow—endochondral ossification. Green—
both intramembranous and endochondral ossification. Dotted—neural crest Mesenchymal
cell-derived. Diagonal lines—paraxial mesoderm-derived. Cross hatched—both (mesoderm)Cells
neural crest- and paraxial mesoderm-derived. Eth, ethmoid; Fro, frontal; Lac,
lacrimal; Man, mandible; Max, maxilla; Nas, nasal; Occ, occipital; Par, parietal; FIGURE 2. Relative contributions of neural crest cells and paraxial mesoderm
Squ, squamous; Tem, temporal; Vom, vomer; Zyg, zygoma. cells to the 2 types of bone within the craniofacial skeleton.

2 # 2017 Mutaz B. Habal, MD

Copyright © 2017 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
CE: D.C.; SCS-16-01512; Total nos of Pages: 10;
SCS-16-01512

The Journal of Craniofacial Surgery  Volume 00, Number 00, Month 2016 Bone Biology

into prehypertrophic and hypertrophic chondrocytes.38 Parathyroid-

related peptide is expressed within perichondrial cells and chon-
drocytes located in the distal aspect of the epiphysis. A PTHrP
gradient is established with the lower concentrations extending
toward the metaphysis. Parathyroid-related peptide stimulates
chondrocyte proliferation, and when levels are low chondrocytes
stop proliferating, begin expressing Ihh, and undergo hypertrophy.
The transition of replacement cartilage to bone via endochondral
ossification requires a supporting cast of other important factors
including Wingless-related integration (Wnt) proteins, fibroblast
growth factor (FGF), vascular endothelial growth factor (VEGF),
matrix metalloproteinases (MMPs), and hypoxia-inducible factor
(HIF). Mice lacking Wnt-signaling within mesenchymal conden-
sations form cartilage but no endochondral or intramembranous
bone.39 Wingless-related integration activity is controlled by Ihh
signaling during bone formation,35,39 and Wnt signaling increases
chondrocyte proliferation and inhibits chondrocyte apoptosis.35
Fibroblast growth factor receptor 3 (FGFR3) is the receptor for
multiple FGF proteins including FGF1, FGF2, and FGF18, and is
highly expressed within the growth plate.40 Achondroplasia is
associated with activating mutations of FGFR3. Binding of
FGF18 from perichondrium to FGFR3 within the growth plate
FIGURE 3. Pathways for bone formation. Adapted from Eames et al.11 inhibits bone growth by inhibition of chondrocyte differentiation
and proliferation.41,42 Matrix metalloproteinases are extracellular
matrix-degrading enzymes that help promote neovascularization
among other roles. Within the growth plate MMP9 and MMP14 are
Sox9 are master regulatory transcription factors for osteogenic and produced by hypertrophic chondrocytes, and promote endochondral
chondrogenic specification, respectively. bone formation by facilitating invasion of blood vessels into the
Sox9 promotes expression of essential cartilage-related collagen primary ossification centers of the growth plate (reviewed in 43).
genes including Coll II,29 Coll IX,30 Coll XI,31 which together help Hypoxia-inducible factor -1a is the central regulator of the hypoxic
generate an extracellular collagen matrix. Within immature carti- response. When cells are hypoxic, Hif-1a activates VEGF to
lage chondrocytes rapidly divide and remain undifferentiated. Key stimulate vasculogenesis. Loss of Hif-1a in chondrocytes causes
factors in stimulating chondrocyte proliferation and Sox9 activity massive apoptosis of those cells in the center of growth plates,
are bone morphogenetic proteins 2 (BMP2) and 4.16 This is perhaps where oxygen levels are lowest.44 Vascular endothelial growth
counterintuitive because exogenous BMP2 is clinically utilized as a factor is released from hypertrophic chondrocytes where it binds
powerful morphogen for bone formation. Within persistent carti- to extracellular matrix proteins within the growth plate, recruiting
lage Sox9 stimulates similar prochondrogenic factors Sox5, and endothelial cells to induce blood vessel formation.45 This couples
Sox6,32 which together prevent chondrocytes from maturing and resorption of cartilage with bone formation.
undergoing hypertrophy. In contrast the transition of immature to Development of calvarial bones by intramembranous ossifica-
replacement cartilage involves chondrocyte maturation through tion occurs as presumptive bone cells proliferate and migrate
distinct prehypertrophic and hypertrophic stages, as well as vascular outward from mesenchymal condensations.46 Growth plate for-
invasion and activation of bone markers. This requires additional mation is specific to endochondral bone. Intramembranous bone
signaling pathways, the most important of which is Hedgehog instead relies upon ossification centers that add bone in a radial
(reviewed in 33). fashion moving away from the center. Many of the proosteogenic
The Hedgehog gene is evolutionarily conserved and mammalian molecular pathways essential for endochondral bone formation are
homologues include Sonic, Desert , and Indian (Ihh) hedgehogs. essential for intramembranous bone formation, including Runx2,
Indian hedgehog is essential for endochondral bone formation as Wnt, Ihh, and BMP pathways. A lack of BMP signaling within the
knockout mice lacking this gene have a complete absence of cranial mesenchymal condensations is permissive for osteoblast
osteoblasts in the endochondral skeleton.34 It is expressed by formation, whereas at later stages BMP signaling is essential for
prehypertrophic chondrocytes within replacement cartilage and neural-crest-derived calvarial bone formation.13,16,47 Indian hedge-
accelerates their hypertrophy and promotes osteoblast differen- hog also has an important role in this process. Indian hedgehog is
tiation. Indian hedgehog does this by activating Runx2, which then expressed at the leading edge of growing cranial bones, promoting
activates Osterix;35 without either of these transcription factors no bone formation by BMP-2 and BMP-4 mediated direct osteogenic
bone can form. Indian hedgehog also decreases BMP2 activity, differentiation rather than proliferation.48 Its loss results in signifi-
which leads to downregulation of Sox5, Sox6, Sox9, and Coll II.36 cantly decreased calvarial bone formation.16 Deletion of repressors
Recent experiments performed in a bone organ culture system of Hh signaling (Gli3 and Rab23) results in high Hh activity with
further demonstrated that although BMP2 has potent proosteoblast associated increased ossification of calvarial bones and craniosy-
properties, Hh signaling is required; without the presence of Hh nostosis.49,50 Runt-related transcription factor 2 is expressed within
activity BMP2 promotes ectopic chondrogenesis within the peri- calvarial osteoblasts during the process and promotes osteogenesis.
chondrium.37 Indian hedgehog also stimulates expression of the Loss of one allele of Runx2 is associated with delayed suture
hypertrophic cartilage marker, type-X collagen. Perhaps the best closure and persistent fontanels,51 whereas a duplication of Runx2
understood Ihh-mediated pathway in developing bone is that of was identified in a set of twins with metopic suture synostosis.52
parathyroid-related peptide (PTHrP). Within growth plates of The proosteogenic effects of Runx2 in intramembranous bone are
endochondral bone Ihh and PTHrP participate in a feedback loop, mediated through Wnt signaling. Activation of the Wnt pathway
regulating the rate of chondrocyte proliferation and differentiation promotes specification of the osteogenic lineage and represses the

# 2017 Mutaz B. Habal, MD 3

Copyright © 2017 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
CE: D.C.; SCS-16-01512; Total nos of Pages: 10;
SCS-16-01512

Runyan and Gabrick The Journal of Craniofacial Surgery  Volume 00, Number 00, Month 2016

chondrogenic lineage within calvarial mesenchyme.13,53,54 TGFb the BMPs), and angiogenic factors. A number of other pathways are
signaling is also important as it promotes calvarial osteocyte implicated in the healing process as their loss results in significant
proliferation.55 Nearly all studies of intramembranous bone devel- perturbations in the ability to heal, although their specific roles in the
opment examine the frontal or parietal bones, and relatively little is 4 phases of bone healing are not well defined. These include the
understood of the process within intramembranous bones of the Hedgehog75 and Wnt signaling pathways.76–79
facial skeleton.56 In the absence of rigid fixation, fracture healing of the appendi-
cular skeleton occurs through formation of a cartilage scaffold, which
PATHWAYS OF APPENDICULAR BONE is gradually replaced with bone. This healing closely resembles steps
of embryonic endochondral ossification.10 Mesenchymal precursors
FRACTURE HEALING coalesce in the shape and location of the bone to be formed both for
Fractures of bones of the appendicular skeleton heal by both endochondral ossification and fracture healing.80,81 Both pro-
intramembranous and endochondral ossification. Endochondral cesses also involve mesenchymal cell proliferation and differen-
bone formation predominates outside the periosteum in mechani- tiation and hypertrophy along a cartilaginous or osteogenic
cally unstable regions and immediately adjacent to the fracture site. pathway. An obvious difference between the processes is the
Intramembranous bone formation occurs subperiosteally at the presence of the inflammatory step in fracture healing that facili-
proximal and distal edges of the callus and forms hard callus.57 tates recruitment of the mesenchymal stem cells. However, once
Bridging of the hard callus across the fracture gap provides initial these cells are present some of the same signaling pathways are
stabilization and leads to restoration of biomechanical function.58 involved including Ihh, VEGF, and MMP.10 It is perhaps the
As endochondral ossification is the mechanism of bone formation in preservation of many of these embryonic pathways that allow
the appendicular skeleton, it is also the mechanism primarily fractured bone to avoid forming scar as other tissues do, but to heal
responsible for appendicular skeletal repair. through a truly regenerative process.
Four overlapping phases of fracture healing may be evident
histologically (reviewed in 59):
PATHWAYS OF CRANIOFACIAL SKELETAL
1. Immediate inflammatory response. This occurs over the initial
24 to 48 hours post fracture and is marked by hematoma FRACTURE HEALING
formation, hemostasis, inflammation, and recruitment of An early rabbit mandible fracture model demonstrated that in the
mesenchymal stem cells. absence of rigid fixation mandible fracture healing has some
2. Cartilage formation with early endochondral ossification and histologic similarities with long bone fractures.82 Within 2 weeks
periosteal response. During this period mesenchymal stem cells of fracture a large subperiosteal callus develops containing both
differentiate into chondrocytes, which then produce a cartila- chondroid and immature osteoid. Within the subsequent 2 weeks
ginous callus rich in collagen and proteoglycans.60,61 The soft, this callus is gradually replaced with trabecular bone and is
cartilaginous callus grows inversely proportional to the stability completely bridged with new neovascular channels and Haversian
of the fracture, and does so asymmetrically within the fracture. systems. Paccione et al83 similarly observed in their mouse mand-
For example, femur fractures produce larger distal calluses and ible fracture model that the sequential presence of islands of
tibial fractures larger proximal calluses, suggesting a recapi- rudimentary cartilage matrix formation, vascular ingrowth, osteo-
tulation of bone development with the calluses forming nearest blast activation, mineralization and lamellar bone formation,
the growth plates.58,62 The soft callus growth peaks between 7 together resembled secondary bone endochondral bone healing.
and 9 days following the fracture.61 The periosteal response They suggest that the contribution of a cartilage intermediate in
results in early intramembranous ossification, and is associated their mandible fracture model (and that of others) was simply due to
with cell proliferation and early vascular ingrowth and neo- bony instability. Indeed, the presence of instability in long bone
angiogenesis. fractures results in increased motion at the fracture site, which
3. Cartilage resorption and primary bone formation. During this promotes cartilaginous callus formation during the primary bone
phase chondrocytes proliferate, mature, become hypertrophic, healing phase.
and increase synthesis of collagen, which accumulates within Rigorous animal studies have not been performed to examine the
the extracellular matrix. As the chondrocytes then begin to histologic and molecular changes of facial bone fractures treated
undergo apoptosis, additional mesenchymal progenitor cells are with rigid fixation. There are a number of reasons for this. The small
recruited and differentiate into osteoblasts. This leads to callus size of rodent facial bones precludes plate fixation. Microplates
mineralization, as osteoblasts use the soft callus as a template to were not available when bone healing studies were commonly
deposit woven bone in place of the mineralized cartilage. This is performed. The lack of a robust, straight, marrow cavity precludes
initially manifest as a thin shell of bone around the periphery of the use of intramedullary stabilization. Despite this, clinical experi-
the callus. Neo-angiogenesis also continues during this phase. ence provides overwhelming evidence that bones that develop by
4. Secondary bone formation and remodeling. During this final intramembranous ossification heal by the same mechanisms, and
phase the bony callus grows and is reshaped by osteoclastic generally not through a cartilaginous intermediate. Skull fractures
resorption and osteoblastic bone formation, resulting in illustrate this principle. The scalp provides a tight soft tissue
regeneration of the original cortical and trabecular arrangement envelope to promote calvarial fracture reduction, while the con-
with a marrow-containing medullary cavity. vexity of the calvarium forms a sturdy keystone arch, which
provides natural rigid fixation. Most of the bones of the facial
The molecular physiology of these 4 phases of fracture healing is skeleton similarly have a stabilizing periosteum and soft tissue
well understood and shares many molecular similarities with endo- envelope, and are not subject to repeated forces. In contrast, the
chondral bone development. A comprehensive description of these mandible is subject to cyclic mechanical loading associated with
factors is beyond the scope of this review; however, an updated, mastication. However with immobilization or rigid load-bearing or
concise summary is presented in Table 1.59,63–74 Of the many load-sharing fixation it heals by direct ossification.
cytokines and growth factors involved, 3 groups have complex, Hasegawa et al84 provided experimental evidence opposing a
well-defined overlapping roles during the 4 stages of bone healing: role for chondrogenesis in membranous bone healing. They initially
proinflammatory cytokines, TGF-b-superfamily members (including identified a multipotent mesenchymal progenitor cell within

4 # 2017 Mutaz B. Habal, MD

Copyright © 2017 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
CE: D.C.; SCS-16-01512; Total nos of Pages: 10;
SCS-16-01512

The Journal of Craniofacial Surgery  Volume 00, Number 00, Month 2016 Bone Biology

59
TABLE 1. Molecular Pathway Activation During Endochondral Bone Fracture Healing (Adapted From )

Stage of Fracture Repair Biologic Process Signaling Molecule Activation and Proposed Functions

Inflammation Hematoma IL-1, IL-6, and TNF-a release by circulating granulocytes and lymphocytes recruits
inflammatory cells, enhances extracellular matrix synthesis, and stimulates
angiogenesis.63
Inflammation and recruitment of TGF-b, PDGF, and BMP-2 expression promote extracellular matrix formation and
progenitor cells initial callus formation.64,65 MMP-9 regulates the distribution of inflammatory
cells.66
Cartilage formation Collagen deposition Collagen type-II and type-III accumulate shortly after inflammation, produced by
chondrocytes in the cartilaginous callus, and periosteal osteoblasts.
Chondrogenesis and endochondral TGF-b2 and TGF-b3 stimulate chondrogenesis, corresponding with Collagen type-II
ossification synthesis.67 BMP-2 promotes chondrocyte differentiation.68 PTH also promotes
cartilaginous and bony callus formation,69 whereas Opg prevents
chondroclastogenesis by inhibiting RANKL.70
Vascular in-growth MMP-9 promotes vascular invasion of hypertrophic cartilage, by promoting VEGF
bioavailability.71 VEGF directly stimulates angiogenesis, and is maximally
expressed when resorption is initiated.59
Primary bone formation Chondrocyte apoptosis and cartilage TNF-a stimulates mineralized chrondocyte apoptosis and cartilage resorption, and
resorption helps recruit osteoprogenitor cells.72,73 RANKL activity increases while Opg
decreases, stimulating chondroclastogenesis.70
Changes in collagen expression Collagen type-II and type-III are removed as cartilage callus resorbs. Collagen type-I
accumulates as bony trabeculae develop. Collagen type-X expression by
hypertrophic chondrocytes provides a template for bone formation.
Mesenchymal cell differentiation to Stimulated by BMP-2, -6, and -9.74
osteoblasts
Osteoblast recruitment and maturation Stimulated by BMP-3, -4, -7, and -8.67,74
Neo-angiogenesis VEGF and PDGF expression continue to promote angiogenesis.
Secondary bone Formation Bone remodeling TNF-a, IL-1 and RANKL activity promote bone remodeling by osteoclast remodeling
of woven bone for lamellar bone formation.

BMP, bone morphogenetic proteins; FGF, fibroblast growth factor; HIF, hypoxia-inducible factor; IGF, insulin-derived growth factor; IL, interleukin; MMP, matrix
metalloproteinases; VEGF, vascular endothelial growth factor.

fracture hematomas of long bones, and demonstrated their ability to bridge oriented in the direction of distraction called the fibrous
differentiate into osteocytes, adipocytes, and chondrocytes in interzone (FIZ, see Fig. 4).90,91 The FIZ is rich in chondrocyte-like
vitro.85 They subsequently cultured human mandible fracture hema- cells, fibroblasts, and oval cells, which are morphologically inter-
toma cells and found that although these cells had a similar mediates between fibroblasts and chondrocytes.90– 92 As the dis-
mesenchymal cell surface expression profile and had good osteo- traction gap increases the FIZ remains 4-mm thick, and at the
genic and adipogenic potential, they had a significantly reduced conclusion of the process the FIZ is the last region to ossify.
ability to differentiate into chondrocytes compared to progenitors Adjacent to the FIZ on either side is the primary mineralization
isolated from long bone fracture hematomas. front (PMF), which contains a high density of proliferating osteo-
Compared with long bone fractures, our knowledge of the blasts. These osteoblasts undergo primary mineralization in regions
molecular physiology of healing craniofacial fractures is extremely of newly formed capillaries and vascular sinuses, leading to the
sparse. Experiments in a rat model of mandible fracture healing formation of columns of bone resembling stalagmites and stalac-
implicate TGF-b superfamily members, including TGF-b1, tities, known as the zone of microcolumn formation (MCF). When
BMP-2, -4, and -7, in osteoblast migration, differentiation, and distraction ends the PMF advances from each end toward the center,
proliferation.86,87 bridging the FIZ. Sequential mineralization of osteoid occurs during
the activation and especially during the consolidation phase, start-
PHYSIOLOGIC EFFECTS OF DISTRACTION ing within the surrounding MCF, which then proceeds to bridge the
FIZ. During the consolidation period mineralization of new bone is
OSTEOGENESIS ON BONE HEALING completed and bony remodeling occurs resulting in the formation of
Bones undergoing distraction osteogenesis share similar histologic mature, lamellar bone with marrow.
characteristics of healing, regardless of whether they are within
the craniofacial or appendicular skeleton.7,88 However, there are
significant histologic differences between bone distraction osteo-
genesis and fracture healing. The latency period of distraction
resembles early fracture healing with hematoma formation and
recruitment of inflammatory cells and mesenchymal stem cells.9,59
Endochondral bone formation may be observed during latency and
early during distractor activation, although the endochondral bone
is not found within the distraction gap but is limited to areas
adjacent to the periosteum. Jazrawi et al89 proposed that this
observation suggests that the distraction environment may suppress
cartilage development.
FIGURE 4. Neo-physis of bone healing with distraction osteogenesis. FIZ,
Rather than forming a cartilaginous callus within the distraction fibrous interosseous zone; MCF, micro column formation; PMF, primary
gap, a physis-like structure of cells organizes into a fibrovascular mineralization front. See the text for description. (Adapted from 14).

# 2017 Mutaz B. Habal, MD 5

Copyright © 2017 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
CE: D.C.; SCS-16-01512; Total nos of Pages: 10;
SCS-16-01512

Runyan and Gabrick The Journal of Craniofacial Surgery  Volume 00, Number 00, Month 2016

The predominant mechanisms of bone formation within this TABLE 2. Differential Expression of Signaling Molecules During Distraction
niche are 2-fold. First, Yasui et al93 observed that the FIZ of Osteogenesis
distracted rat femurs contained chondrocyte-appearing cells within Latency Active Distraction Consolidation
a bony matrix, but without capillary ingrowth as is found in
endochondral ossification. Similar to chondrocytes, these chon- Signaling Molecules Early Late Early Late Early Late
droid cells expressed type-II collagen; however, they transition to
type-I collagen expression, suggestive of direct transformation of Cytokines
the chondrocyte-like cells into osteoblasts.94 Yasui named this IL-159 þþþ
phenomenon ‘‘transchondroid bone formation,’’ and proposed that IL-698 þþþ þþþ þþþ
it represents a new type of bone formation. However, Yasui et al TNF-a59 þþþ
observed that the predominant mechanism of bone formation during RANKL/OPG ratio59 þþ þþþ þþþ þ
distraction osteogenesis is intramembranous ossification, which TGF-b superfamily
may be distinguished from the other mechanisms by the histologic BMP-299,101,103 þþ þþþ þþþ þ
absence of cartilage and the expression of only type-I col- BMP-499,101,103 þþ þþþ þþþ þ
lagen.3,4,7,95 At the ultrastructural level, disorganized bundles of BMP-699,101,103 þþþ þþ þ
type-I collagen are found at the end of the latency period.96 As TGF-b99–101,103 þ þþ þþþ þþþ þ
distraction begins, these bundles increase in size and become Angiogenic factors
oriented in a plane parallel with the distraction force.7,95,97 Osteoid VEGF A104 þþþ þþþ þ
is then deposited along these collagen bundles by osteoblasts VEGF B104 þ þ þ
located at corticotomy edges and within the distraction gap.97 VEGF C104 þþ þ þ þ
VEGF D104 þþ þþ þ
MOLECULAR EFFECTS OF DISTRACTION Angiopoietin 1104 þþ þ þ
Angiopoietin 2104 þþ þ
OSTEOGENESIS ON BONE HEALING
HIF-1 a105 þþþ þþþ
Bone undergoing distraction osteogenesis initially receives an
Other osteogenic factors
osteotomy. The molecular profile during the immediate postosteot-
FGF-2 (bFGF)99,101,103 þþ þþ þ þ
omy (latency) phase thus resembles that of fracture healing
IGF99,101,103 þþ þþ
(Table 2).59,98–106 Proinflammatory cytokines interleukin (IL)-1
Collagen I100,101 — —  þ þþþ
and IL-6 are upregulated in the initial period, promoting extra-
Osteocalcin92,100,101,106 — —  þ þ þ
cellular matrix synthesis and inflammatory cell recruitment.59,98
Osteopontin92,102 /þþþ102 þþþ ? ?
Osteogenic and chondrogenic differentiation of these progenitors is
similarly stimulated by early BMP-2 expression. A separate proin- A ‘‘þ’’ indicates gene upregulation, whereas ‘‘’’ indicates gene downregulation.
flammatory marker, TNF-a, is not expressed during latency, likely Empty squares indicate a lack of data, or lack of differential gene expression beyond
because its induction requires a greater traumatic insult than a baseline.
simple osteotomy.98 BMP, bone morphogenetic proteins; FGF, fibroblast growth factor; HIF, hypoxia-
With distractor activation the molecular expression profile inducible factor; IGF, insulin-derived growth factor; IL, interleukin; VEGF, vascular
significantly deviates from that of fracture healing. Interleukin-6 endothelial growth factor.
is upregulated a second time when distraction starts and mechanical Adapted From Ai-Aql.59
strain is applied to the callus. At this time its expression is high in
osteoblasts, chondrocytes, and in oval cells within the FIZ where
tensile strains are the highest. Interleukin-6 upregulation is thought
to contribute to intramembranous ossification by enhancing osteo- Bone morphogenetic protein-6 downregulation occurs as the
genic differentiation, and that IL-6 has an anabolic effect on DO and primary mode of ossification transitions from endochondral to
catabolic effect in fracture repair.98 intramembranous, reflecting its contributions to endochondral bone
TGF-b-superfamily members are also upregulated during dis- formation.99
tractor activation. TGF-b was increased in distracted mandibles Two additional growth factors have been identified which are
compared with those with nondistracted osteotomies,9 and a direct responsive to the increased mechanical strain found during dis-
correlation between an increasing rate of mandibular distraction and tractor activation. Insulin-derived growth factor-1 and FGF-2, or
TGF-b expression has been observed.107 During distraction TGF-b basic-FGF are both highly expressed around the PMF and may
promotes osteoblast proliferation while suppressing their matu- promote osteoblast differentiation before subsequent downregula-
ration, effectively delaying their differentiation, thus promoting tion during consolidation.107,112
new bone formation.108,109 Bone morphogenetic protein 2 and As with fracture healing, osteoclastogenesis is necessary to help
BMP-4 expression are both expressed immediately following the bone formed by distraction osteogenesis to remodel and form
osteotomy, are downregulated, and then highly reexpressed during mature, lamellar bone. The RANKL/OPG system is thought to
distraction activation.99 These BMPs are upregulated specifically be the key regulator for balanced bone turnover during DO.113 As
within chondrogenic cells at the PMF, and within oval cells within with fracture healing, a high RANKL/OPG expression ratio pro-
the FIZ, in response to the application of mechanical strain.99,110 motes osteoclastogenesis. The RANKL/OPG ratio increases late
They are maintained throughout active distraction, but then gradu- during latency and peaks within the consolidation phase, with the
ally disappear during consolidation, further implying a role in greatest turnover occurring at 3 to 4 weeks of consolidation.113,114
proliferation of cells required for completion of bone healing. Activation of osteoclasts by TNF-a occurs throughout fracture
Consistent with this, the addition of exogenous BMP-2 shortens healing; however, it is not expressed in DO until late during
treatment time during DO by accelerating bone formation during consolidation, suggesting that RANKL/OPG play the primary role
the consolidation phase.111 In contrast to other factors, BMP-6 for bone turnover and maturation.72 Osteocalcin is expressed by
expression is limited to chondrocytes within the FIZ, begins during mature osteoblasts and promotes mineralization. Its expression is
the latency phase, and then declines during the activation phase. significantly decreased compared with normal bone during the

6 # 2017 Mutaz B. Habal, MD

Copyright © 2017 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
CE: D.C.; SCS-16-01512; Total nos of Pages: 10;
SCS-16-01512

The Journal of Craniofacial Surgery  Volume 00, Number 00, Month 2016 Bone Biology

latency period. Osteocalcin levels gradually increase early during

distraction, until reaching normal levels toward the end of con-
solidation.92,100 In contrast, osteocalcin in acutely lengthened
mandibles does not significantly increase 6 weeks postdistraction.
This suggests deficiencies in osseous regeneration in acutely
lengthened specimens are due to disturbances in mineralization/
bone turnover in addition to decreased bone scaffold production.

ANGIOGENESIS IN DISTRACTION
OSTEOGENESIS
Angiogenesis is an essential process for distraction osteogenesis.
When angiogenesis is chemically inhibited, a lack of ossified bone
and blood vessels occurs between the 2 cut ends of bone, with a
fibrous nonunion resulting.115 Mechanical distraction induces much
greater angiogenic response than fracture healing.59 Blood flow
during activation increases up to 10 times normal blood flow, as
measured by quantitative technetium scintigraphy.91 Histologically,
periosteal and endosteal vessels form columns alongside newly
developing bone, toward the FIZ.116 Within the FIZ capillaries are
formed by both sinusoidal and transport capillary angiogenesis.
During consolidation the periosteal and medullary vascular net-
works connect at the distraction site, including the FIZ.116 Although
new vessel formation begins during activation, maximal vessel FIGURE 5. Comparison of the progression of healing in fractures and distraction
volume increase occurs during consolidation, suggesting a link osteogenesis. Reproduced with permission.59
between angiogenesis and bone formation.117–119
Among VEGF family members, only VEGF-A and neuropilin (a
VEGF receptor) are significantly upregulated during the activation
phase.105 Vascular endothelial growth factor-D is upregulated bone also has large amounts of unmineralized osteoid in the central
briefly during the latency period, then diminished thereafter.105 region of distraction gap, whereas the fracture callus of endochon-
Vascular endothelial growth factor-A is expressed in maturing dral bone calcifies rapidly as it undergoes primary bone healing.
osteoblasts within the PMF and within osteoclasts in the MCF Bragdon speculates that the lack of cartilage formation during
zone, directing angiogenesis in this region of the distraction gap.116 distraction is due to the population of precursor cells that reside
Partial blockade of VEGF signaling in a tibial model of DO results within the endosteum.118 Endosteal cells are restricted to the
in blockade of intramembranous ossification but allows for chon- osteogenic lineage, whereas the periosteum, which contributes to
drogenesis, whereas complete VEGF blockade inhibits both osteo- both fracture healing and distraction osteogenesis, has precursor
genesis and chondrogenesis.120 The primary source of VEGF-A cells capable of differentiating into both chondrocytes and osteo-
during DO is mesenchymal cells within the surrounding muscle. blasts.123
These blood vessels then synthesize morphogens (eg, BMP-2) that Angiogenesis is critical for both fracture healing and distraction
promote bone formation in distracted bone.119 An upstream acti- osteogenesis. Vascular endothelial growth factors are expressed
vator of VEGF-A, HIF-1a, is significantly upregulated in bone during both processes but have higher relative expression during
undergoing distraction compared with fracture healing,104 fracture healing. Vascular endothelial growth factor-receptor
suggesting many of the downstream genes that are targets of knockout studies showed that both angiogenesis and osteogenesis
HIF-1a (eg, VEGF-A) play a major role in promoting new bone during distraction osteogenesis were dependent on activity of both
formation during DO. Deferoxamine enhancement of MDO is VEGF receptors 1 and 2.120 Also, inhibition of VEGF in a fracture-
thought to be by upregulation of HIF-1a activity.121,122 Morgan healing model showed delayed healing and failure to progress from
et al117 found that the period of active distraction is characterized a cartilaginous to bony callus.124 In fracture healing, angiogenesis
primarily by arteriogenesis in surrounding muscle, during conso- begins between days 7 and 14 as chondrogenic tissues undergo
lidation, angiogenesis predominates in the intraosteal region, vessel resorption.59 However, during distraction osteogenesis, angiogen-
formation proceeds from the surrounding muscle into the regener- esis is initiated only after active distraction has begun and is thought
ate. Periods of intense osteogenesis are concurrent with those to be driven by the distraction process rather than by signals
of angiogenesis. elaborated from chondrocytes.59,118 The observation that the
majority of new vessels occur within the medullary space of the
distraction regenerate supports this theory.104,120 This is in contrast
CONTRASTING BONE FORMATION BY to fracture healing, wherein new vessel formation occurs within
FRACTURE HEALING AND DISTRACTION the external callus and is associated with the cartilage-to-bone
OSTEOGENESIS transition.125
Distraction osteogenesis shares aspects of some of the physiologic In certain respects, DO more closely resembles embryonic bone
pathways of fracture healing, but is clearly a distinct process. This development than fracture healing. The rate of bone formation
can be easily appreciated by comparing the 2 processes histologi- during distraction osteogenesis is 200 to 400 uM/d, which is 4–8
cally (Fig. 5).59 Shortly after fracture of the appendicular skeleton a faster than fastest physeal growth in adolescence, and equivalent to
robust cartilage callus forms outside the bone, stabilizing the that of the fetal femur.88,116 There is also circumstantial evidence
fracture. In distraction osteogenesis much less cartilage formed that pathways that are important for bone development are differ-
and its presence is temporally restricted to the early periods after entially regulated during distraction osteogenesis. Shibazaki et al126
distraction is initiated, after which it is rapidly resorbed. Distracted reported increased PTHrP activity within distracted mandibular

# 2017 Mutaz B. Habal, MD 7

Copyright © 2017 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
CE: D.C.; SCS-16-01512; Total nos of Pages: 10;
SCS-16-01512

Runyan and Gabrick The Journal of Craniofacial Surgery  Volume 00, Number 00, Month 2016

condyles. Kasaai et al127 found significant increases in Wnt signal- 8. McCarthy JG, Schreiber JS, Karp NS, et al. Lengthening the human
ing factors in a mouse tibial distraction model. Hedgehog signaling mandible by gradual distraction. Plast Reconstr Surg 1992;89:1–8
is also altered in a rabbit model of calvarial distraction.128 However, 9. McCarthy JG, Stelnicki EJ, Mehrara BJ, et al. Distraction osteogenesis
there is not enough understood of DO to determine whether it is a of the craniofacial skeleton. Plast Reconstr Surg 2001;107:1812–1827
physiologic recapitulation of embryonic bone development. This is 10. Ferguson C, Alpern E, Miclau T, et al. Does adult fracture repair
certainly an area of future study. recapitulate embryonic skeletal formation? Mech Dev 1999;87:57–66
11. Eames BF, De la Fuente L, Helms JA. Molecular ontogeny of the
skeleton. Birth Defects Res Part C Embryo Today Rev 2003;69:93–101
CONCLUSION 12. Karaplis AC. Embryonic development of bone and regulation of
Bones of the appendicular and craniofacial skeleton have distinct intramembranous and endochondral bone formation. In: Principles of
mechanisms of development. The former relies upon growth plates Bone Biology, Two-Volume Set. Vol 1. Cambridge, MA: Academic
and endochondral formation. The majority of craniofacial bone Press; 2008:53–84.
forms from growth centers or nuclei, via direct intramembranous 13. Ishii M, Sun J, Ting M-C, et al. The dsevelopment of the calvarial
ossification. Despite differences, both pathways share important bones and sutures and the pathophysiology of craniosynostosis. Curr
Top Dev Biol 2015;115:131–156
master regulatory transcription and downstream growth factors,
14. Percival CJ, Richtsmeier JT. Angiogenesis and intramembranous
including Runx2,Wnt, and Ihh. The type of bone formation that osteogenesis. Dev Dyn 2013;242:909–922
occurs during fracture healing largely mirrors the pathway by which 15. Eames BF, Helms JA. Conserved molecular program regulating cranial
the affected bone developed. Endochondral bone fractures of the and appendicular skeletogenesis. Dev Dyn 2004;231:4–13
appendicular skeleton heal by endochondral ossification. Cranio- 16. Abzhanov A, Rodda SJ, McMahon AP, et al. Regulation of
facial fractures heal primarily by intramembranous ossification, skeletogenic differentiation in cranial dermal bone. Development
with a contribution of endochondral bone formation in areas of bony 2007;134:3133–3144
instability. Regardless of whether distraction osteogenesis occurs 17. Bhatt S, Diaz R, Trainor PA, et al. Signals and switches in mammalian
within the appendicular or craniofacial skeleton, it resembles neural crest cell differentiation signals and switches in mammalian
formation and fracture healing of the latter—intramembranous neural crest cell differentiation. Cold Spring Harb Perspect Biol
2013;5:a008326
ossification. Intramembranous bone forming during distraction 18. Thorogood PV, Hinchliffe JR. An analysis of the condensation process
osteogenesis resembles that of developing or healing craniofacial during chondrogenesis in the embryonic chick hind limb. J Embryol
bone, but with heightened angiogenesis. Exp Morphol 1975;33:581–606
A thorough understanding of bone development, fracture heal- 19. Ducy P, Karsenty G. Genetic control of cell differentiation in the
ing, and distraction osteogenesis will provide opportunities to skeleton. Curr Opin Cell Biol 1998;10:614–619
improve and expand indications of the technique. Distraction 20. Komori T, Yagi H, Nomura S, et al. Targeted disruption of Cbfa1
osteogenesis is a natural form of bone engineering which employs results in a complete lack of bone formation owing to maturational
favorable osteoinductive signaling to generate intramembranous arrest of osteoblasts. Cell 1997;89:755–764
bone with rapid angiogenesis. The technique has already demon- 21. Mundlos S, Otto F, Mundlos C, et al. Mutations involving the
transcription factor CBFA1 cause cleidocranial dysplasia. Cell
strated clinical efficacy in lengthening bones of the appendicular 1997;89:773–779
and craniofacial skeleton. Critical-sized defects can be a significant 22. Otto F, Thornell AP, Crompton T, et al. Cbfa1, a candidate gene for
challenge to the reconstructive surgeon. Distraction osteogenesis is cleidocranial dysplasia syndrome, is essential for osteoblast
a safe and less invasive alternative to standard osteotomies and bone differentiation and bone development. Cell 1997;89:765–771
lengthening with grafts. It is associated with more rapid vascular- 23. Bi W, Deng JM, Zhang Z, et al. Sox9 is required for cartilage
ization and lower failure rate. Additionally, distraction osteogenesis formation. Nat Genet 1999;22:85–89
gradually expands the overlying soft tissue and does not require 24. Akiyama H, Chaboissier MC, Martin JF, et al. The transcription factor
complete dissection of bone from dura or periosteum prior to Sox9 has essential roles in successive steps of the chondrocyte
differentiation pathway and is required for expression of Sox5 and
activation. These advantages have allowed this technique to gain
Sox6. Genes Dev 2002;16:2813–2828
wide acceptance for addressing congenital and traumatic defects in 25. Yan YL, Miller CT, Nissen RM, et al. A zebrafish sox9 gene required
the appendicular and craniofacial skeleton. for cartilage morphogenesis. Development 2002;129:5065–5079
26. Foster JW, Dominguez-Steglich MA, Guioli S, et al. Campomelic
ACKNOWLEDGMENTS dysplasia and autosomal sex reversal caused by mutations in an SRY-
The authors are indebted to Dr Joseph McCarthy and Dr Roberto related gene. Nature 1994;372:525–530
27. Kwok C, Weller PA, Guioli S, et al. Mutations in SOX9, the gene
Flores for their guidance in preparing this review.
responsible for Campomelic dysplasia and autosomal sex reversal. Am
J Hum Genet 1995;57:1028–1036
REFERENCES 28. Wagner T, Wirth J, Meyer J, et al. Autosomal sex reversal and
1. Codivilla A. On the means of lengthening, in the lower limbs, the campomelic dysplasia are caused by mutations in and around the SRY-
muscles and tissues which are shortened through deformity. Am J related gene SOX9. Cell 1994;79:1111–1120
Orthop Surg 1905:353–369 29. Lefebvre V, Huang W, Harley VR, et al. SOX9 is a potent activator of
2. Jordan CJ, Goldstein RY, Mclaurin TM, et al. The evolution of the the chondrocyte-specific enhancer of the pro alpha1(II) collagen gene.
ilizarov technique: part 1: the history of limb lengthening. Bull NYU Mol Cell Biol 1997;17:2336–2346
Hosp Jt Dis 2013;71:89–95 30. Zhang P, Jimenez SA, Stokes DG. Regulation of human COL9A1 gene
3. Ilizarov GA. The tension-stress effect on the genesis and growth of expression. Activation of the proximal promoter region by Sox9. J Biol
tisues. Part II. Clin Othop Relat Res 1989:263–285 Chem 2003;278:117–123
4. Ilizarov GA. The Tension-stress effect on the genesis and growth of 31. Liu Y, Li H, Tanaka K, et al. Identification of an enhancer sequence
tisues. Part I. Clin Othop Relat Res 1989:249–281 within the first intron required for cartilage-specific transcription of the
5. Snyder CC, Levine GA, Swanson HM, et al. Mandibular lengthening a2(XI) collagen gene. J Biol Chem 2000;275:12712–12718
by gradual distraction. Plast Reconstr Surg 1973;51:506–508 32. Ikeda T, Kawaguchi H, Kamekura S, et al. Distinct roles of Sox5, Sox6,
6. Karp NS, Thorne CHM, McCarthy JG, et al. Bone lengthening in the and Sox9 in different stages of chondrogenic differentiation. J Bone
craniofacial skeleton. Ann Plast Surg 1990;24:231–237 Miner Metab 2005;23:337–340
7. Karp NS, McCarthy JG, Schreiber JS, et al. Membranous bone 33. Yang J, Andre P, Ye L, et al. The Hedgehog signalling pathway in bone
lengthening: a serial histological study. Ann Plast Surg 1992;29:2–7 formation. Int J Oral Sci 2015;7:73–79

8 # 2017 Mutaz B. Habal, MD

Copyright © 2017 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
CE: D.C.; SCS-16-01512; Total nos of Pages: 10;
SCS-16-01512

The Journal of Craniofacial Surgery  Volume 00, Number 00, Month 2016 Bone Biology

34. St-Jacques B, Hammerschmidt M, McMahon AP. Indian hedgehog 59. Ai-Aql ZS, Alagl AS, Graves DT, et al. Molecular mechanisms
signaling regulates proliferation and differentiation of chondrocytes controlling bone formation during fracture healing and distraction
and is essential for bone formation. Genes Dev 1999;13:2072–2086 osteogenesis. J Dent Res 2008;87:107–118
35. Mak KK, Chen M-H, Day TF, et al. Wnt/beta-catenin signaling 60. Hankenson K, Zimmermann G, Marcucio RS. Biologic perspectives
interacts differentially with Ihh signaling in controlling endochondral of delayed fracture healing. Injury 2014;45(suppl 2):S8–S15
bone and synovial joint formation. Development 2006;133:3695–3707 61. Marsell R, Einhorn TA. The biology of fracture healing. Injury
36. Long F, Chung U, Ohba S, et al. Ihh signaling is directly required for 2011;42:551–555
the osteoblast lineage in the endochondral skeleton. Development 62. Morgan EF, Mason ZD, Chien KB, et al. Micro-computed
2004; 131:1309–1318 tomography assessment of fracture healing: relationships among
37. Hojo H, Ohba S, Taniguchi K, et al. Hedgehog-Gli activators direct callus structure, composition, and mechanical function. Bone
osteo-chondrogenic function of bone morphogenetic protein toward 2009;44:335–344
osteogenesis in the perichondrium. J Biol Chem 2013;288:9924–9932 63. Kon T, Cho TJ, Aizawa T, et al. Expression of osteoprotegerin, receptor
38. Kronenberg HM. PTHrP and skeletal development. Ann N Y Acad Sci activator of NF-kappaB ligand (osteoprotegerin ligand) and related
2006;1068:1–13 proinflammatory cytokines during fracture healing. J Bone Miner Res
39. Hu H, Hilton MJ, Tu X, et al. Sequential roles of Hedgehog and Wnt 2001;16:1004–1014
signaling in osteoblast development. Development 2005;132:49–60 64. Sandberg MM, Hannu TA, Vuorio EI. Gene expression during bone
40. Peters K, Ornitz D, Werner S, et al. Unique expression pattern of the repair. Clin Orthop Relat Res 1993;289:292–312
FGF receptor 3 gene during mouse organogenesis. Dev Biol 1993; 65. Bostrom M. Expression of bone morphogenetic proteins in fracture
155:423–430 healing. Clin Orthop Relat Res 1998; (355 suppl):S116–S123
41. Murakami S, Balmes G, McKinney S, et al. Constitutive activation of 66. Wang X, Yu YY, Lieu S, et al. MMP9 regulates the cellular response to
MEK1 in chondrocytes causes Stat1-independent achondroplasia-like inflammation after skeletal Injury. Bone 2013;52:111–119
dwarfism and rescues the Fgfr3-deficient mouse phenotype. Genes 67. Cho T-J, Gerstenfeld LC, Einhorn TA. Differential temporal
Dev 2004;18:290–305 expression of members of the transforming growth factor beta
42. Ohbayashi N, Shibayama M, Kurotaki Y, et al. FGF18 is required for superfamily during murine fracture healing. J Bone Miner Res
normal cell proliferation and differentiation during osteogenesis and 2002;17:513–520
chondrogenesis. Genes Dev 2002;16:870–879. 68. Wang Q, Huang C, Xue M, et al. Expression of endogenous BMP-2 in
43. Vu TH, Werb Z. Matrix metalloproteinases: effectors of development periosteal progenitor cells is essential for bone healing. Bone 2011;
and normal physiology. Genes Dev 2000;14:2123–2133 48:524–532
44. Schipani E, Ryan HE, Didrickson S, et al. Hypoxia in cartilage: HIF- 69. Ren Y, Liu B, Feng Y, et al. Endogenous PTH deficiency impairs
1a is essential for chondrocyte growth arrest and survival. Genes Dev fracture healing and impedes the fracture-healing efficacy of
2001;15:2865–2876 exogenous PTH(1–34). PLoS One 2011;6:e23060
45. Zelzer E, Mamluk R, Ferrara N, et al. VEGFA is necessary for 70. Ota N, Takaishi H, Kosaki N, et al. Accelerated cartilage resorption by
chondrocyte survival during bone development. Development chondroclasts during bone fracture healing in osteoprotegerin-deficient
2004;131:2161–2171 mice. Endocrinology 2009;150:4823–4834
46. Yoshida T, Vivatbutsiri P, Morriss-Kay G, et al. Cell lineage in 71. Colnot C, Thompson Z, Miclau T, et al. Altered fracture repair in the
mammalian craniofacial mesenchyme. Mech Dev 2008;125:797–808 absence of MMP9. Development 2003;130:4123–4133
47. Bonilla-Claudio M, Wang J, Bai Y, et al. Bmp signaling regulates a 72. Gerstenfeld LC, Cho TJ, Kon T, et al. Impaired fracture healing in
dose-dependent transcriptional program to control facial skeletal the absence of TNF-alpha signaling: the role of TNF-alpha in
development. Development 2012;139:709–719 endochondral cartilage resorption. J Bone Miner Res 2003;18:
48. Lenton K, James AW, Manu A, et al. Indian hedgehog positively 1584–1592
regulates calvarial ossification and modulates bone morphogenetic 73. Glass GE, Chan JK, Freidin A, et al. TNF-alpha promotes fracture
protein signaling. Genesis 2011;49:784–796 repair by augmenting the recruitment and differentiation of muscle-
49. Jenkins D, Seelow D, Jehee F, et al. RAB23 mutations in Carpenter derived stromal cells. Proc Natl Acad Sci U S A 2011;108:1585–1590
syndrome imply an unexpected role for hedgehog signaling in cranial- 74. Cheng H, Jiang W, Phillips FM, et al. Osteogenic activity of the
suture development and obestiy. Am J Hum Genet 2007;80:1162–1170 fourteen types of human bone morphogenetic proteins (BMPs). J Bone
50. Rice DPC, Connor EC, Veltmaat JM, et al. Gli3Xt-J/Xt-J mice exhibit Joint Surg Am 2003;85-A:1544–1552
lambdoid suture craniosynostosis which results from altered 75. Wang Q, Huang C, Zeng F, et al. Activation of the Hh pathway in
osteoprogenitor proliferation and differentiation. Hum Mol Genet periosteum-derived mesenchymal stem cells induces bone formation
2010;19:3457–3467 in vivo: implication for postnatal bone repair. Am J Pathol
51. Otto F, Kanegane H, Mundlos S. Mutations in the RUNX2 gene in 2010;177:3100–3111
patients with cleidocranial dysplasia. Hum Mutat 2002;19:209–216 76. Jilka RL, Brien CAO, Ali AA, et al. Intermittent PTH stimulates
52. Mefford HC, Shafer N, Antonacci F, et al. Copy number variation periosteal bone formation by actions on post-mitotic preosteoblasts.
analysis in single-suture craniosynostosis: multiple rare variants Bone 2010;44:275–286
including RUNX2 duplication in two cousins with metopic 77. Bodine PVN, Seestaller-Wehr L, Kharode YP, et al. Bone anabolic
craniosynostosis. Am J Med Genet A 2010;152A:2203–2210 effects of parathyroid hormone are blunted by deletion of the Wnt
53. Goodnough LH, DiNuoscio GJ, Ferguson JW, et al. Distinct antagonist secreted frizzled-related protein-1. J Cell Physiol
requirements for cranial ectoderm and mesenchyme-derived Wnts in 2007;210:352–357
specification and differentiation of osteoblast and dermal progenitors. 78. Guo J, Liu M, Yang D, et al. Suppression of Wnt signaling by Dkk1
PLoS Genet 2014;10:12–14 attenuates PTH-mediated stromal cell response and new bone
54. Tran TH, Jarrell A, Zentner GE, et al. Role of canonical Wnt signaling/ formation. Cell Metab 2010;11:161–171
ß-catenin via Dermo1 in cranial dermal cell development. 79. Minear S, Leucht P, Jiang J, et al. Wnt proteins promote bone
Development 2010;137:3973–3984 regeneration. Sci Transl Med 2010;2:29ra30
55. Sasaki T. TGF-mediated FGF signaling is crucial for regulating cranial 80. Hall BK, Miyake T. The membranous skeleton: the role of cell
neural crest cell proliferation during frontal bone development. condensations in vertebrate skeletogenesis. Anat Embryol (Berl)
Development 2005;133:371–381 1992;186:107–124
56. Sperber G, Sperber SM, Guttmann GD. Craniofacial Embryogenetics 81. Hiltunen A, Vuorio E, Aro HT. A standardized experimental fracture in
and Development. 2nd ed. Shelton, CT: PMPH USA, Ltd; 2010 the mouse tibia. J Orthop Res 1993;11:305–312
57. Dimitriou R, Tsiridis E, Giannoudis PV. Current concepts of molecular 82. Craft PD, Mani MM, Pazel J, et al. Experimental study of healing
aspects of bone healing. Injury 2005;36:1392–1404 in fractures of membranous bone. Plast Reconstr Surg 1974;55:
58. Gerstenfeld LC, Alkhiary YM, Krall EA, et al. Three-dimensional 321–325
reconstruction of fracture callus morphogenesis. J Histochem 83. Paccione MF, Warren SM, Spector JA, et al. A mouse model of
Cytochem 2006;54:1215–1228 mandibular osteotomy healing. J Craniofac Surg 2001;12:444–450

# 2017 Mutaz B. Habal, MD 9

Copyright © 2017 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
CE: D.C.; SCS-16-01512; Total nos of Pages: 10;
SCS-16-01512

Runyan and Gabrick The Journal of Craniofacial Surgery  Volume 00, Number 00, Month 2016

84. Hasegawa T, Miwa M, Sakai Y, et al. Mandibular hematoma cells as a growth factor in distraction osteogenesis of the mandible. J Craniofac
potential reservoir for osteoprogenitor cells in fractures. J Oral Surg 1999;10:80–86
Maxillofac Surg 2012;70:599–607 108. Lammens J, Liu Z, Aerssens J, et al. Distraction bone healing versus
85. Oe K, Miwa M, Sakai Y, et al. An in vitro study demonstrating that osteotomy healing: a comparative biochemical analysis. J Bone Miner
haematomas found at the site of human fractures contain progenitor Res 1998;13:279–286
cells with multilineage capacity. J Bone Jt Surg Br 2007;89-B:133–138 109. Holbein O, Neidlinger-Wilke C, Suger G, et al. Ilizarov callus
86. Steinbrech DS, Mehrara BJ, Rowe NM, et al. Gene expression of TGF- distraction produces systemic bone cell mitogens. J Orthop Res
beta, TGF-beta receptor, and extracellular matrix proteins during 1995;13:629–638
membranous bone healing in rats. Plast Reconstr Surg 110. Rauch F, Lauzier D, Croteau S, et al. Temporal and spatial expression
2000;105:2028–2038 of bone morphogenetic protein-2, -4, and -7 during distraction
87. Spector JA, Luchs JS, Mehrara BJ, et al. Expression of bone osteogenesis in rabbits. Bone 2000;27:453–459
morphogenetic proteins during membranous bone healing. Plast 111. Yonezawa H, Harada K, Ikebe T, et al. Effect of recombinant human
Reconstr Surg 2001;107:124–134 bone morphogenetic protein-2 (rhBMP-2) on bone consolidation on
88. Aronson J, Good B, Stewart C, et al. Preliminary studies of distraction osteogenesis: a preliminary study in rabbit mandibles. J
mineralization during distraction osteogenesis. Clin Orthop Relat Res Craniomaxillofac Surg 2006;34:270–276
1990:43–49 112. Tavakoli K, Yu Y, Shahidi S, et al. Expression of growth factors in the
89. Jazrawi LM, Majeska RJ, Klein ML, et al. Bone and cartilage mandibular distraction zone: a sheep study. Br J Plast Surg
formation in an experimental model of distraction osteogenesis. J 1999;52:434–439
Orthop Trauma 1998;12:111–116 113. Pérez-Sayáns M, Somoza-Martı́n JM, Barros-Angueira F, et al.
90. Vauhkonen M, Peltonen J, Karaharju E, et al. Collagen synthesis and RANK/RANKL/OPG role in distraction osteogenesis. Oral Surg Oral
mineralization in the early phase of distraction bone healing. Bone Med Oral Pathol Oral Radiol Endodontol 2010;109:679–686
Miner 1990;10:171–181 114. Zhu W-Q, Wang X, Wang X-X, et al. Temporal and spatial expression
91. Aronson J. Temporal and spatial increases in blood flow during of osteoprotegerin and receptor activator of nuclear factor-kappaB
distraction osteogenesis. Clin Orthop Relat Res 1994:124–131 ligand during mandibular distraction in rats. J Craniomaxillofac Surg
92. Sato M, Yasui N, Nakase T, et al. Expression of bone matrix proteins 2007;35:103–111
mRNA during distraction osteogenesis. J Bone Miner Res 115. Fang TD, Salim A, Xia W, et al. Angiogenesis is required for
1998;13:1221–1231 successful bone induction during distraction osteogenesis. J Bone
93. Yasui N, Sato M, Ochi T, et al. Three modes of ossification during Miner Res 2005;20:1114–1124
distraction osteogenesis in the rat. J Bone Joint Surg Br 1997;79:824– 116. Choi IH, Chung CY, Cho TJ, et al. Angiogenesis and mineralization
830 during distraction osteogenesis. J Korean Med Sci 2002;17:435–447
94. Li G, Virdi AS, Ashhurst DE, et al. Tissues formed during distraction 117. Morgan EF, Hussein AI, Al-Awadhi BA, et al. Vascular development
osteogenesis in the rabbit are determined by the distraction rate: during distraction osteogenesis proceeds by sequential intramuscular
localization of the cells that express the mRNAs and the distribution of arteriogenesis followed by intraosteal angiogenesis. Bone
types I and II collagens. Cell Biol Int 2000;24:25–33 2012;51:535–545
95. Karaharju EO, Aalto K, Kahri A, et al. Distraction bone healing. Clin 118. Bragdon B, Lybrand K, Gerstenfeld L. Overview of biological
Orthop Relat Res 1993;297:38–43 mechanisms and applications of three murine models of bone repair:
96. Hamanishi C, Yoshii T, Totani Y, et al. Lengthened callus activated by closed fracture with intramedullary fixation, distraction osteogenesis,
axial shortening. Clin Orthop Relat Res 1994;307:250–254 and marrow ablation by reaming. Curr Protoc Mouse Biol 2015;5:21–
97. Ilizarov GA. Transosseous Osteosynthesis. Vol 1. (Green SA, ed.). 34
Berlin: Springer-Verlag; 1992. 119. Matsubara H, Hogan DE, Morgan EF, et al. Vascular tissues are a
98. Cho T-J, Kim JA, Chung CY, et al. Expression and role of interleukin-6 primary source of BMP2 expression during bone formation induced by
in distraction osteogenesis. Calcif Tissue Int 2007;80:192–200 distraction osteogenesis. Bone 2012;51:168–180
99. Sato M, Ochi T, Nakase T, et al. Mechanical tension-stress induces 120. Jacobsen KA, Al-Aql ZS, Wan C, et al. Bone formation during
expression of bone morphogenetic protein (BMP)-2 and BMP-4, but distraction osteogenesis is dependent on both VEGFR1 and VEGFR2
not BMP-6, BMP-7, and GDF-5 mRNA, during distraction signaling. J Bone Miner Res 2008;23:596–609
osteogenesis. J Bone Miner Res 1999;14:1084–1095 121. Donneys A, Farberg AS, Tchanque-Fossuo CN, et al. Deferoxamine
100. Mehrara BJ, Longaker MT. New developments in craniofacial surgery enhances the vascular response of bone regeneration in mandibular
research. Cleft Palate Craniofac J 1999;36:377–387 distraction osteogenesis. Plast Reconstr Surg 2012;129:850–856
101. Nuntanaranont T, Suttapreyasri S, Vongvatcharanon S. Quantitative 122. Farberg AS, Sarhaddi D, Donneys A, et al. Deferoxamine enhances
expression of bone-related cytokines induced by mechanical tension- bone regeneration in mandibular distraction osteogenesis. Plast
stress during distraction osteogenesis in a rabbit mandible. J Investig Reconstr Surg 2014;133:666–671
Clin Dent 2014;5:255–265 123. Colnot C. Skeletal cell fate decisions within periosteum and bone
102. Perrien DS, Brown EC, Aronson J, et al. Immunohistochemical study marrow during bone regeneration. J Bone Miner Res 2009;24:274–282
of osteopontin expression during distraction osteogenesis in the rat. J 124. Street J, Bao M, deGuzman L, et al. Vascular endothelial growth factor
Histochem Cytochem 2002;50:567–574 stimulates bone repair by promoting angiogenesis and bone turnover.
103. Khanal A, Yoshioka I, Tominaga K, et al. The BMP signaling and its Proc Natl Acad Sci 2002;99:9656–9661
Smads in mandibular distraction osteogenesis. Oral Dis 2008;14:347– 125. Duvall CL, Taylor WR, Weiss D, et al. Impaired angiogenesis, early
355 callus formation, and late stage remodeling in fracture healing of
104. Pacicca DM, Patel N, Lee C, et al. Expression of angiogenic factors osteopontin-deficient mice. J Bone Miner Res 2006;22:286–297
during distraction osteogenesis. Bone 2003;33:889–898 126. Shibazaki R, Maki K, Tachikawa T, et al. Changes in parathyroid
105. Carvalho RS, Einhorn TA, Lehmann W, et al. The role of angiogenesis hormone-related protein and 3-dimensional trabecular bone structure
in a murine tibial model of distraction osteogenesis. Bone of the mandibular condyle following mandibular distraction
2004;34:849–861 osteogenesis in growing rats. J Oral Maxillofac Surg 2005;63:505–512
106. Meyer U, Meyer T, Vosshans J, et al. Decreased expression of 127. Kasaai B, Moffatt P, Al-Salmi L, et al. Spatial and temporal
osteocalcin and osteonectin in relation to high strains and decreased localization of WNT signaling proteins in a mouse model of distraction
mineralization in mandibular distraction osteogenesis. osteogenesis. J Histochem Cytochem 2012;60:219–228
J Craniomaxillofac Surg 1999;27:222–227 128. Nott RL, Stelnicki EJ, Mack JA, et al. Changes in the protein
107. Farhadieh RD, Dickinson R, Yu Y, et al. The role of transforming expression of hedgehog and patched-1 in perisutural tissues induced by
growth factor-beta, insulin-like growth factor I, and basic fibroblast cranial distraction. Plast Reconstr Surg 2002;110:523–532

10 # 2017 Mutaz B. Habal, MD

Copyright © 2017 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.