Nihms-1641529 Mechanisms of Bone Development and Repair

HHS Public Access
Author manuscript
Nat Rev Mol Cell Biol. Author manuscript; available in PMC 2020 November 28.
Author Manuscript
Published in final edited form as:

Nat Rev Mol Cell Biol. 2020 November ; 21(11): 696–711. doi:10.1038/s41580-020-00279-w.
Mechanisms of bone development and repair

Ankit Salhotra1,2,4, Harsh N. Shah1,2,4, Benjamin Levi3,✉, Michael T. Longaker1,2,✉
1Divisionof Plastic and Reconstructive Surgery, Department of Surgery, Stanford University
School of Medicine, Stanford, CA, USA.
2Institute
for Stem Cell Biology and Regenerative Medicine, Stanford University School of
Medicine, Stanford, CA, USA.
Author Manuscript
3Department of Surgery, University of Michigan, Ann Arbor, MI, USA.

4These authors contributed equally: Ankit Salhotra, Harsh N. Shah.
Abstract
Bone development occurs through a series of synchronous events that result in the formation of the
body scaffold. The repair potential of bone and its surrounding microenvironment — including
inflammatory, endothelial and Schwann cells — persists throughout adulthood, enabling
restoration of tissue to its homeostatic functional state. The isolation of a single skeletal stem cell
population through cell surface markers and the development of single-cell technologies are
enabling precise elucidation of cellular activity and fate during bone repair by providing key
insights into the mechanisms that maintain and regenerate bone during homeostasis and repair.
Author Manuscript
Increased understanding of bone development, as well as normal and aberrant bone repair, has
important therapeutic implications for the treatment of bone disease and ageing-related
degeneration.
The musculoskeletal system provides the physical scaffold for the mammalian body. Bones
of the human skeleton provide attachment sites for muscles, tendons and ligaments, enabling
locomotion. Bones also contain the microenvironments for adult haematopoiesis to occur.
The crucial role of bone in mammalian physiology is further highlighted by the body’s
unique ability to repair bone through regeneration, restoring it to a fully functional, pre-
injury state. Studies have shown that a regulated balance of activity between bone-forming
osteoblasts and bone-resorbing osteoclasts — the two main cellular constituents of bone —
is responsible for this repair capacity. Previous research on the role of osteoblasts has
Author Manuscript
highlighted the importance of gradients of morphogens, such as bone morphogenetic protein

(BMP) and sonic hedgehog (SHH), during bone repair. These morphogen gradients, among
others, are also essential during bone development (osteogenesis).
✉
blevi@med.umich.edu; longaker@stanford.edu.
Author contributions
The authors contributed equally to all aspects of the article.
Competing interests
The authors declare no competing interests.
Salhotra et al. Page 2
The osteoblast lineage is of great interest in medicine owing to its implications in bone
Author Manuscript
development and disease. Although a certain degree of repair capacity is maintained

throughout adulthood, the ability to repair bone diminishes substantially during ageing,
potentially leading to osteoporosis. Therefore, this Review examines areas of synergy and
diversity in the bone developmental and repair processes. We discuss the cell types involved
in osteogenesis and the molecular signalling pathways that are essential for bone formation.
This Review also explores the function of critical genes and transcription factors during
bone development. Additionally, the functions of different cells and signalling pathways
during bone repair are described, as well as their role in bone development. Finally, we
assess the dysfunctional cellular and molecular signalling that results in clinical bone
disease, thus informing the current state of science and potential gaps in knowledge.
Cell types involved in osteogenesis

Author Manuscript
The skeletal lineage includes a diverse group of cells that maintain and repair bone during
homeostasis and injury, respectively. This lineage of cells includes osteoblasts, osteocytes
and chondrocytes1–4. These skeletal cell types are involved mainly in the formation of bone
and cartilage, whereas the cells that are responsible for bone resorption, known as
osteoclasts, are derived from the haematopoietic lineage. Normal bone homeostasis is
maintained through a balance between osteoblast and osteoclast activity; however, during the
ageing process, especially in postmenopausal women, osteoclast activity surpasses
osteoblast activity, resulting in increased overall bone resorption and weaker bones5.
Osteoblasts
Osteoblasts are the main cells responsible for bone formation. These cells secrete
extracellular matrix proteins such as type I collagen, osteopontin, osteocalcin and alkaline
Author Manuscript
phosphatase; multiple osteoblasts interact with one another to create a unit of bone known as
an osteon3. The deposition of calcium, in the form of hydroxyapatite, with type I collagen
provides structural support to the skeleton3.
The specification of osteoblasts towards the skeletal lineage can be divided into three
distinct stages of increasing differentiation: osteoprogenitor, preosteoblast and osteoblast1,2
(FIG. 1). Initially, expression of the transcription factor SOX9 marks the commitment to an
osteoprogenitor cell. SOX9 expression also directs cell differentiation towards a chondrocyte
cell fate. Chondrocytes are the only cell type found in healthy cartilage, where they produce
a cartilaginous matrix consisting of collagen and proteoglycans. The subsequent expression
of Runt-related transcription factor 2 (RUNX2) in the osteoprogenitor cell signifies the
commitment to a preosteoblast6. During the maturation stage, WNT-β-catenin signalling acts
Author Manuscript
on preosteoblasts to induce the expression of osterix (OSX; also know as SP7), which
defines the cell’s differentiation to an osteoblast6. Ultimately, the expression of RUNX2 and
OSX marks the commitment to a mature osteoblast.
A fraction of these osteoblasts will undergo apoptosis, whereas a subset of osteoblasts

secrete extracellular matrix components and embed into the matrix of the bone, forming
osteocytes7. Osteocytes account for most of the cells found in mature mineralized bone and
coordinate bone maintenance through interactions between osteoblasts and osteoclasts7–9
(FIG. 1). The process of bone maintenance is sensitive to mechanical forces; during
Author Manuscript
mechanical unloading, osteocytes express receptor activator of nuclear factor-κB ligand

(RANKL), which promotes bone resorption through the activation of osteoclasts10.
Conversely, in response to mechanical loading, osteocytes decrease the expression of
Dickkopf-related protein 1 (DKK1) and sclerostin, leading to increased bone formation
through activation of WNT-β-catenin signalling in osteoblasts9. Osteocytes respond to
hormonal and mechanical signals to tightly control bone remodelling through signalling
pathways that are discussed later.
Osteoclasts
Osteoclasts are large multinucleated cells whose primary function is bone resorption. These
cells originate from the haematopoietic lineage and differentiate to mature osteoclasts
through the interaction of macrophage colony-stimulating factor (M-CSF) and RANKL11
Author Manuscript
(FIG. 1). M-CSF promotes proliferation of osteoclast precursors, whereas RANKL promotes
differentiation of osteoclast precursors to mature osteoclasts11. The expression of RANKL is
necessary for osteoclast function as mice lacking RANKL are unable to resorb bone12,13.
During bone remodelling, osteoclastogenesis begins with recruitment of osteoclast
precursors by osteocytes that express RANKL. The expression of RANKL and M-CSF
within the bone marrow compartment initiates the differentiation of osteoclast precursors to
osteoclasts, leading to the start of bone remodelling. Under homeostasis, the ratio of bone
formation and bone resorption follows a tightly controlled programme to ensure consistency
in bone mass. For instance, the release of active transforming growth factor-β (TGFβ) and
insulin-like growth factor 1 (IGF1) after resorption of the bone matrix triggers osteoblast
differentiation to replace the resorbed matrix with new bone matrix14. An imbalance of this
control leads to osteoporosis, the most common disease associated with upregulation of
Author Manuscript
osteoclast activity, which occurs when bone resorption exceeds bone formation (discussed in
detail later)14. Conversely, pathologically increased osteoblast activity leads to heterotopic
ossification, which is signified by bone formation at an extraskeletal site (discussed in detail
later)15.
Stem and progenitor cells

Osteoblast differentiation requires a multitude of steps from a stem cell differentiating into a
mature osteoblast. Mesenchymal stem cells (MSCs) and skeletal stem cells (SSCs) have both
been shown, separately, to give rise to osteoblasts; however, the relationship between these
two stem cell populations has not been precisely determined. In this Review, both
populations are considered separately to discuss their role in osteoblast differentiation.
Furthermore, throughout osteoblast differentiation, specific genes are expressed to mark
Author Manuscript
their maturation at each stage during development and repair. Therefore, to study the
function of each intermediary cell type, transgenic mice are used to understand the lineage
specification process. The Cre recombinase (Cre)-loxP system enables conditional gene
inactivation by which Cre excises the ‘target’ DNA sequence, corresponding to the gene of
interest, that has been flanked by two 34-bp DNA sequences termed the ‘loxP sites’16.
TABLE 1 outlines the numerous transgenic mouse models that can be used to study specific
cell types during osteoblast differentiation, along with the advantages and disadvantages of
Author Manuscript
each model.
Mesenchymal stem cells

MSCs, first discovered in the bone marrow, are a multipotent, non-haematopoietic stem cell
population. MSCs have the capacity to differentiate into mature cell types of mesenchymal
tissues such as bone, cartilage and fat17,18. Specifically, seminal work established the
osteogenic potential of MSCs through heterotopic transplantation of bone marrow cells19–21.
Through the addition of exogenous factors, MSCs have the ability to be directed towards an
osteogenic cell differentiation fate17,18. For example, the addition of dexamethasone, β-
glycerol phosphate and ascorbate to cultured human MSCs results in osteogenic cell
differentiation. The cells form nodules with increased expression of alkaline phosphatase17.
Studying the differentiation path of MSCs towards osteoblasts provides insight into key
Author Manuscript
lineage specification moments during the differentiation process.
Transcription factors enable the initiation and promotion of MSC differentiation towards an
osteogenic fate. Specifically, RUNX2 and OSX are the main transcription factors whose
activation commits the cells to the osteogenic lineage. The expression of RUNX2 is
preceded by the upregulation of GLUT1, which results in feedforward regulation22. GLUT1
is a glucose transporter, and its upregulation facilitates increased glucose uptake by cells.
This sequence of events indicates that osteoblast differentiation requires a high energy
demand, which is met by increased glucose transport and uptake through upregulation of
GLUT1. This feedforward regulation explains impaired bone healing in patients with
diabetes, as their cells become insensitive to glucose uptake22,23.
MSCs have been shown to maintain expression of genes characteristic of embryonic

Author Manuscript
development, such as the Hox genes. Hox genes encode evolutionarily conserved
transcription factors that control skeletal patterning24,25. Hox gene expression is regionally
restricted and regulates the morphology of specific vertebral and long bone elements24,25.
For example, Hox6 expression is present in the ribs, and Hox11 (also known as Tlx1)
expression is present in the zeugopod (radius, ulna, tibia and fibula) of mice postnatally to
adulthood25. Furthermore, Hoxa11+ cells from postnatal development of the zeugopod to
adulthood display characteristics of osteoblast progenitors26–28. When the function of the
Hoxa11 alleles in ulnar fracture healing in adult mice was studied, Hoxa11 mutant mice
exhibited perturbed fracture repair28. Specifically, cells of the Hoxa11+ mesenchymal
population had decreased osteoblast differentiation potential25,29. Taken together, these
studies indicate that mesenchymal cells regionally restricted to the skeleton maintain
expression of developmental genes, whose regulation has a role in bone repair.
Author Manuscript
Skeletal stem cells

The isolation of SSCs in mice and humans on the basis of distinctive immunophenotypic cell
surface markers is a relatively new concept30–32 (FIG. 2). Thus, we focus here on the ability
to isolate mouse and human skeletal progenitors and their role during repair1,4,33,34. Of note,
the term ‘skeletal stem cells’ has evolved over time and continues to be refined.
A subpopulation of cells isolated from the fetal mouse long bone growth plate has the ability
to differentiate into bone, cartilage and bone stroma30. This cell population was identified
Author Manuscript
with use of a ubiquitous actin rainbow reporter system that randomly marks dividing cells
and their progeny, allowing fluorescent tracing of ‘clonal’ cell clusters arising from a single
parent cell30,35. Clonal regions were observed in the growth plate of long bones, indicating
the presence of a restricted progenitor cell. Additionally, isolation of cells from the growth
plate was achieved by fluorescence-activated cell sorting. In vitro and in vivo studies showed
that CD45−TER119−TIE2−ITGAV+CD200+ single sorted cells have the ability to generate
serial colony-forming units, differentiate into bone, cartilage and stromal cells, and support
haematopoiesis30.
The fetal human growth plate is of particular interest owing to clonal observations made in
the fetal mouse growth plate. With use of a single-cell RNA sequencing approach, analysis
revealed a set of 76 gene candidates expressed in mouse SSCs, mouse bone, cartilage and
Author Manuscript
stromal progenitor cells (BCSPs) and their respective human orthologues31. By focusing on
the cell surface markers that were specifically enriched in the growth plate and not in the
diaphysis, the study authors selected a list of potential markers. Flow analysis and
immunofluorescent staining of the growth plate revealed that CD45−CD235a
−TIE2−CD31−PDPN+CD73+CD164+ human cells had the capability to generate colony-
forming units from single cells in vitro and have the ability to differentiate into bone,
cartilage and stroma in the sub-renal space of a mouse. In addition, co-transplantation of
haematopoietic cells with CD45−CD235a−TIE2−CD31−PDPN+CD73+CD164+ human cells
in irradiated immunodeficient NSG mice supported haematopoiesis31. The role of SSCs has
been implicated in development, but to date no studies have successfully demonstrated how
SSCs contribute to the developing skeleton36. A transgenic mouse model specifically
marking SSCs would enable these studies; however, such a model is not yet available.
Author Manuscript
The identification of mouse and human SSCs was based on the combination of transgenic
mouse models, flow cytometry and single-cell sequencing30,31,37. Although these
advancements enabled the discovery of SSCs, it is important to highlight the limitations of
the known SSC hierarchy. One of the most important technical and conceptual limitations of
mouse and human SSCs is the loss of spatial information on cell isolation, which limits the
ability to precisely locate and understand their role in the native bone niche. In addition, the
SSCs were identified in the long bones of mice and humans, bones that form through
endochondral ossification. However, different skeletal compartments develop through
distinct processes; for example, bones of the skull use intramembranous ossification in
development and repair38. Whether the identity of the SSC is shared across two different
skeleton compartments (cranial versus long bones) is yet to be defined. In addition, the cell
Author Manuscript
surface marker profiles across mice and humans are known to be different. Indeed, mouse
and human SSCs might be different cell types that merely happen to behave similarly in the
in vitro and in vivo systems studied. Further work is required to help explain the discrepancy
between the immunophenotypic cell surface profile across the two species so as to evaluate
the relevance of these SSCs to the broader biological context of bone development and
repair.
In the context of injury, postnatal SSCs have an important role in enacting a bone repair
paradigm to promote healing4. For instance, in response to fracture injury, mouse BCSPs
Author Manuscript
express CD49f and are activated to contribute to bone repair39. By contrast, these fracture-
elicited mouse BCSPs are absent in the uninjured bone. The injury-induced activation of
SSCs is also captured in humans. With use of a novel human xenograft model, a unicortical
injury on human fetal phalanges displayed an elevated frequency of human SSCs and
exhibited increased osteogenic potential in vitro31. In diabetic mice, SSCs have decreased
expansion and differentiation abilities, resulting in poor fracture healing through the
repression of Indian hedgehog (IHH) signalling40. Downregulation of IHH signalling is also
recapitulated in femoral and knee specimens of human patients with diabetes40. However,
this effect can be rescued in mice with the exogenous delivery of IHH or SHH, which
successfully improves bone healing by inducing SSC expansion40.
A unique SSC-driven repair process has also been observed in the craniofacial region, a
Author Manuscript
skeletal component that heals through intramembranous ossification. During mandibular

distraction osteogenesis in mice, focal adhesion kinase (FAK)-responsive SSCs acquire an
embryonic neural crest identity and promote regeneration41. With inhibition of FAK, SSC-
driven bone regrowth is severely impaired, resulting in a fibrous intermediate in place of
bone41. These studies highlight the importance of SSCs in postnatal bone repair. However,
no studies to date have successfully demonstrated how SSCs contribute to the developing
skeleton36. Further studies are required to elucidate their specific roles during embryonic
development.
Many groups have made significant progress in understanding the role of skeletal
progenitors in bone remodelling and maintenance by combining two approaches: the Cre-
loxP system (TABLE 1), to enable lineage tracing, and fluorescence-activated cell sorting, to
Author Manuscript
select for the SSC population on the basis of its cell surface marker profile. These studies
have identified compartment-specific skeletal progenitors and highlight the ability to lineage
trace these skeletal progenitors through development42,43. Studies building on the capacity
to lineage-trace skeletal progenitors will help elucidate their role during bone development.
In addition, future advancements should address the rigor of methods for isolating cells from
freshly harvested tissue, which can be subject to the loss of fragile cell types. Improved
isolation methods, preventing the deterioration of sparse cell populations and surface
antigens that might be necessary for understanding development, might reveal additional
subpopulations. Coupled with emerging novel single-cell transcriptional analyses and assay
for transposase-accessible chromatin sequencing methods, the relationships of these cell
populations to existing skeletal progenitors can be established44,45.
Author Manuscript
Regulation of cell lineage specification

Skeletal formation occurs through orchestrated, temporal events enabling the bone to form
and ossify over the period of development. The expression of transcription factors at certain
stages in bone development guides the progenitor cells to differentiate towards an osteoblast
fate. Along with the genetic changes, signalling pathways enable the cells to communicate
with their environment. The external cues given by the signalling pathways synchronize
cellular differentiation. The transcription factors and signalling pathways necessary for bone
development become activated as bone regrows after an injury. This section discusses the
Author Manuscript
essential transcription factors and signalling pathways for osteoblast differentiation in the
context of bone development and injury.
Embryonic origins of bones

During skeletal development, osteoblasts arise from multiple embryonic germ layers.
Osteoblasts of the craniofacial region originate from neural crest cells, which are derived
from the neural ectoderm38,46. The neural ectoderm develops into the tissues of the
craniofacial region, including the skull, dentin of teeth and bones of the face. By contrast,
the long bones of the skeleton originate from paraxial mesoderm (somites) (which gives rise
to the axial skeleton) and lateral plate mesoderm (which gives rise to the appendicular
skeleton)38.
Bone formation during embryogenesis occurs in two distinct processes: intramembranous

Author Manuscript
ossification or endochondral ossification. Intramembranous ossification begins with the

condensation of mesenchymal populations that directly differentiate into bone38. The flat
bones of the body, including the skull, mandible, maxilla and clavicle, originate from this
process. By contrast, endochondral ossification is an intricate process signified by the
development of bone through a cartilage intermediate38,47. During endochondral
ossification, the cells in the middle of the mesenchymal condensations develop into
chondrocytes, which begin to secrete cartilage matrix. The cells surrounding the newly
differentiated chondrocytes form the perichondrium, which defines the border of the
developing skeleton38. The cells defining the border withdraw from the cell cycle and
undergo hypertrophy, prompting differentiation of osteoblasts from the perichondrium.
Chondrocyte hypertrophy is essential to activate osteoblast differentiation. Blood vessels
begin to infiltrate the hypertrophic cartilage, which triggers osteoblast differentiation and
Author Manuscript
eventual formation of the bone marrow cavity. The infiltration of blood vessels serves
multiple functions: it facilitates recruitment of chondro-resorptive cells (which degrade
existing cartilage) and osteoprogenitors (which promote osteoblastogenesis) and enables
perichondrial cells to enter the developing bone marrow cavity47,48. Blood vessel infiltration
into the hypertrophic cartilage is signified by the development of a primary ossification
centre.
The primary ossification centre continues to expand during embryonic development and is
eventually succeeded by the formation of secondary ossification centres. The secondary
ossification centres develop in the growing ends of the bones (epiphyses)49. The
compartments of the growing bones can be distinguished by their locations within the bone.
Between the epiphysis and the diaphysis (midshaft) is the metaphysis, which contains the
Author Manuscript
epiphyseal growth plate and can be further categorized into different zones of
development38,49 (FIG. 3). The reserve zone contains quiescent chondrocytes. The
proliferative zone is a site of rapid replication of chondrocytes; as these cells divide, they
orient themselves parallel to the growing bone, thus becoming arranged in a columnar
fashion38,47. As the chondrocytes migrate further away from the epiphysis, they halt
proliferation and begin to enlarge (hypertrophy) to contribute to skeletal growth. Most of
these hypertrophic chondrocytes undergo apoptosis, followed by calcification of the
cartilaginous matrix, while the remaining chondrocytes become osteoblasts and contribute to
the growing skeleton47,48. The invasion of vessels into the bone cavity recruits osteoclasts to
Author Manuscript
resorb the calcified cartilage matrix and osteoblasts to lay down new mineralized bone
tissue, promoting the ossification of the newly formed bone38. Postnatal bone growth
continues into adolescence until bone remodelling is complete and maturity is achieved,
marked by the closure of the growth plate-containing metaphysis and fusion of the epiphysis
and diaphysis.
Transcriptional regulation
The activation of transcription factors at specific moments during differentiation provides
the necessary cues to specify the functions of the osteoprogenitor as the cell commits to an
osteoblast. This section discusses the essential transcription factors for osteoblast
differentiation such as SOX9, RUNX2, OSX and activated transcription factor 4 (ATF4).
Author Manuscript
Furthermore, TABLE 2 highlights the transcription factors and protein-coding genes

involved throughout the differentiation process. These specific transcription factors and
protein-coding genes were chosen because they interact with the essential transcription
factors discussed in the following sections to ensure proper osteoblast differentiation.
SOX9.—SOX9, a member of the SRY-related high mobility group box-containing (SOX)

transcription factor family, controls skeletal development during endochondral
Ossification50. Studies in mice have emphasized the importance of SOX9 as the integral
transcription factor for chondrogenic commitment49, which is essential for establishing the
intermediate primordium for eventual ossification by osteoblasts. SOX9 also drives cartilage
differentiation by activating chondrogenic genes such as Col2a1 (REFS50,51). Mouse
embryo chimaeras consisting of Sox9−/− and Sox9+/+ pluripotent cells were revealed to
contain only Sox9+/+ cells in the cartilage condensation52. Furthermore, loss of Sox9
Author Manuscript
perturbs cartilage differentiation and inactivates chondrogenic marker expression, eventually

causing cell death53,54.
In humans, heterozygous mutations within the SOX9 locus lead to the development of an
early lethal skeletal syndrome named ‘campomelic dysplasia’55. Individuals with
campomelic dysplasia are born with skeletal defects such as micrognathia, dwarfism and
other skeletal malformations56. Normally, the expression of SOX9 throughout adulthood
enables continual maintenance of developing cartilage and bone growth51,52.
RUNX2.—RUNX2 is a transcription factor that drives skeletal development (TABLE 2).

RUNX2 binds to core-binding factor subunit-β to form a heterodimer that regulates the
expression of osteoblast genes such as those encoding osteopontin (Spp1), bone sialoprotein
Author Manuscript
2 (Ibsp) and osteocalcin 2 (Bglap2)57–59. In mice, Runx2 is strongly expressed in immature

osteoblasts but expression decreases in mature osteoblasts57. Runx2 deletion in mice results
in complete osteoblast absence and loss of Spp1, Ibsp and Bglap2 expression60–62.
Therefore, RUNX2 activity during early skeletal development is essential for osteoblast
differentiation.
By contrast, the role of RUNX2 in mature osteoblasts is not well understood and requires
further investigation. Conditional Runx2-knockout studies under the mature osteoblast
promoter Col1a1 led to conflicting results. Deletion of the Runt domain in exon 4 led to no
phenotypic changes62, whereas truncation of Runx2 through exon 8 deletion resulted in
Author Manuscript
reduced bone formation63. These findings suggest that Runx2 maintains osteoblasts in the
immature state, thereby arresting osteoblast maturation and the completion of bone
formation63. Furthermore, these findings are consistent with the observed reduction of
Runx2 expression in mature osteoblasts64.
OSX.—OSX, an osteoblast transcription factor, is required for normal skeletal development

(TABLE 2). OSX triggers differentiation of immature osteoblasts to mature osteoblasts and
eventually into osteocytes65–67. In mice, as Osx expression increases during osteoblast
maturation, Runx2 expression decreases simultaneously68. The deletion of Osx in mouse
embryos leads to failure of bone formation and lack of expression of osteoblast genes such
as Sparc (which encodes osteonectin) and Spp1 (REF.68). Postnatal inactivation of Osx leads
to perturbed bone formation with the absence of trabeculae and more porous cortical bone,
Author Manuscript
abnormal cartilage accumulation, and osteocyte dysfunction along with a decrease in

osteocyte gene expression such as expression of Sost and Dkk1 (REFS69,70) (TABLE 2).
In bones undergoing endochondral ossification, the heterogeneous population of immature

osteoblasts, osteoclasts and blood vessels is able to invade the cartilage primordial matrix;
however, without the presence of OSX, ossification does not occur68. Furthermore, the cells
in the condensed mesenchyme of the membranous skeletal elements are unable to
differentiate into osteoblasts without the expression of Osx68. Runx2 expression is still
present in skeleton-forming cells even in the absence of Osx expression. Therefore, OSX
acts downstream of RUNX2, indicating that during the lineage specification of osteoblasts,
RUNX2 promotes immature osteoblast differentiation, whereas OSX is required for mature
osteoblast differentiation.
Author Manuscript
ATF4.—ATF4 is a leucine zipper-containing transcription factor and a component of the

cAMP response element-binding protein (CREB) family71,72. Originally, ATF4 was
identified as a nuclear binding protein enriched in osteoblasts that was required for the
activation of BGLAP2 and terminal differentiation of osteoblasts. Mutations in ATF4 result
in skeletal abnormalities, such as decreased bone mass associated with Coffin-Lowry
syndrome73. Furthermore, an Atf4-knockout mouse model exhibited decreased bone mass
and bone strength resulting from delayed formation of bone trabeculae71,74. This knockout
model has provided insights into the role of ATF4 in osteoblast differentiation and bone
homeostasis71,74.
The regulatory effects of ATF4 on bone homeostasis occur through two mechanisms. First,
Author Manuscript
ATF4 regulates osteoblast differentiation through post-transcriptional modification, enabling

the synthesis of type I collagen, an essential role of the osteoblast74. Second, ATF4 interacts
cooperatively with RUNX2 by forming a complex with SATB2, enhancing the activity of
both transcription factors and promoting osteogenesis75. Furthermore, ATF4 activity is
suppressed by factor inhibiting ATF4-mediated transcription (FIAT). ATF4 and FIAT (also
known as TXLNG) are expressed in osteoblasts72. The negative feedback between ATF4 and
FIAT regulates activation of BGLAP2: ATF4 binds to osteocalcin-specific element 1,
leading to BGLAP2 expression and further extending the role of ATF4 in bone homeostasis
Author Manuscript
(TABLE 2).
AP-1.—The activator protein 1 (AP-1) transcription factor complex has been implicated as
an important regulator in bone development, particularly for osteoblast homeostasis (TABLE
2). AP-1 activity can be induced through TGFβ, parathyroid hormone and 1,25-
dihydroxyvitamin D76. The AP-1 complex is composed of a variety of members from the
FOS, JUN and ATF families76. Mutations of the genes encoding these transcription factors
result in skeletal defects. Specifically, mutations in Junb result in osteopenia owing to
osteoblast defects; however, mutations in Jund result in increased bone mass from increased
bone formation76,77. Furthermore, the AP-1 complex preferentially binds to an osteoblast-
specific enhancer, Ce1, in the Runx2 promoter78. For instance, Fosb in the AP-1 complex
binds to Ce1, and ΔFosb, the naturally occurring short isoform of Fosb, causes the
development of osteosclerosis owing to increased osteoblast differentiation78.
Author Manuscript
Overexpression of FOS-related antigen 1 (FRA1), a FOS-related protein encoded by the

FOS target gene Fosl1, also results in osteosclerosis79,80. The increase in FRA1 expression
accelerated the differentiation of osteoprogenitors into osteoblasts, resulting in an increased
number of active osteoblasts79. Therefore, the regulation of the JUN, FOS and ATF factors
within the AP-1 complex functions to modulate the activity of osteoblasts in bone formation
and homeostasis.
Signalling pathways in bone development

Differentiation of the osteoblast lineage (discussed earlier) is regulated by various signalling
pathways (FIG. 4). Although the signalling pathways are discussed separately in this section,
the pathways function in a coordinated manner to ensure appropriate bone development and
repair.
Author Manuscript
Hedgehog signalling.—In 1980, Hedgehog (HH) was identified as an important gene for
creating the differences between the development of the anterior and posterior sections of
individual body segments in Drosophila81. The control of the anterior-posterior body axis by
HH has been shown to be conserved throughout multiple species, including mammals82,83.
HH is a secreted protein that functions as a morphogen through a paracrine signalling

mechanism. The pathway consists of three homologues — SHH, IHH and desert hedgehog
(DHH) — with the SHH pathway being the best studied84. Patched homologue 1 (PTCH1)
and Smoothened homologue (SMO) are transmembrane proteins that are the receptors for
the HH signalling pathway. In the absence of HH, PTCH inhibits SMO85,86. When HH binds
to PTCH, its inhibition of SMO is removed, activating downstream transcriptional gene
Author Manuscript
targets such as the GLI family of transcription factors84–86 (FIG. 4a).
In limb development, SHH functions as an important morphogen in anteroposterior

patterning of the developing limb bud. In addition to SHH itself, GLI2 and GLI3, the
transcription factor targets of SHH signalling, are required for normal skeletal
development87,88. Absence of either Gli2 or Gli3 results in abnormal skeletal development
and embryonic lethality in mice; GLI1 is not necessary for skeletal development89–91 but it
does, however, act synergistically with GLI2 and GLI3 in osteogenesis. In an in vitro
system, overexpression of Gli1 upregulated the transcription of early osteogenesis-related

genes such as Runx2 and Osx92.
Author Manuscript
Along with its importance in development, the HH pathway also has a vital role in bone
formation during endochondral ossification. Osteoblast differentiation requires the
regulation of Gli1 or Gli2 through HH signalling93. Furthermore, osteoblast differentiation is
lost in the absence of IHH signalling, resulting in incomplete endochondral ossification and
incomplete subsequent bone formation94,95. Specifically, downregulation of IHH signalling
decreases the osteoblast differentiation potential of mouse SSCs, which further results in
poor fracture healing40. Additionally, microarray analysis indicates the upregulation of
Ptch1 in mouse BCSPs during the initial 3 days of femoral fracture healing39. Therefore, HH
signalling is important for osteoblast differentiation of mouse SSCs enabling the cells to
undergo endochondral ossification and repair bone. Postnatally, osteoblasts express IHH,
with the transcriptional target GLI2 having a key role in activating osteoblast-specific genes.
Author Manuscript
HH signalling targets the activation of the transcription factor GLI2, which in turn activates
RUNX2, the master regulator of osteoblast differentiation93.
Furthermore, HH signalling interacts with the WNT and BMP pathways to regulate bone
formation. The WNT pathway is required downstream of HH signalling to regulate
osteoblast differentiation; additionally, the presence of β-catenin is required for HH
signalling96,97. Finally, HH signalling interacts with the BMP pathway during endochondral
ossification. The BMP pathway acts downstream of HH signalling to regulate osteoblast
differentiation from perichondrial cells98.
Notch signalling.—Notch signalling requires direct cell-to-cell contact. Notch receptors

(NOTCH1, NOTCH2, NOTCH3 and NOTCH4) expressed on cells interact with the Jagged
Author Manuscript
(JAG1 and JAG2) and Delta-like (DLL1, DLL3 and DLL4) families to initiate intracellular
signalling99. This binding leads to proteolytic cleavage of the γ-secretase complex with the
help of presenilin 1 (PS1) or PS2, which releases Notch intracellular domain (NCID)100.
This domain translocates to the nucleus to interact Hairless LAG-2 family transcription
factor RBPJ to initiate the Mastermind-like protein 1 (MAML1) activator complex99,100.
The activator complex induces expression of Notch target genes, including the genes
encoding Hairy and Enhancer of Split (HES) and HES-related with YRPW motif
(HEY)99–101 (FIG. 4b).
Genetic studies in mice have revealed the importance of Notch signalling during skeletal
development. In Prrx1Cre mice, inactivation of PS1, NOTCH1 and NOTCH2 in the
developing limb bud resulted in an increase in the abundance of hypertrophic chondrocytes
in the growth plate, leading to increased mass of the trabecular bone101,102. Notch enhances
Author Manuscript
cell replication of the osteoblast lineage and suppresses osteoblast differentiation101,102. A

possible explanation for the mechanism of action is an effect secondary to the inhibition of
RUNX2 function100,103. NCID can directly interact with Runx2 and inhibit the
transactivation of Bglap2100,103. HES and HEY proteins also inhibit RUNX2
function100,103. Increased expression of Notch1 under an osteoblast-specific promoter (Osx)
hindered osteoblast lineage cells from differentiating to specialized osteoblasts, leading to
osteopenia in mice104. Conversely, inactivation of Notch1 and Notch2 under the control of
the Osx promoter led to an increased number of mature osteoblasts and increased cancellous
bone volume105. These studies suggest the importance of Notch signalling in osteoblast
Author Manuscript
lineage differentiation.
Finally, the Notch signalling pathway has been shown to be upregulated in mouse SSCs after
mandibular distraction41. One hypothesis, based on previous studies of Notch signalling and
osteoblast differentiation, posits that the upregulation of Notch signalling on injury might
allow mouse SSCs to replicate and ensure the appropriate number of osteoblast progenitors
are present for bone repair before osteoblast differentiation. However, additional research is
needed to determine the relationship between Notch signalling and SSCs.
Canonical WNT signalling.—WNT ligands communicate by attaching to a cell surface

receptor, eliciting an intracellular signalling cascade106. The canonical WNT pathway is
characterized by the stabilization of β-catenin on activation by WNT ligands through their
Author Manuscript
binding to the receptor Frizzled and low-density lipoprotein receptor-related protein 5

(LRP5) or LRP6 (REFS107–109). Binding of WNT ligands leads to accumulation of β-
catenin in the cytoplasm, thus enabling it to translocate to the nucleus. In the nucleus, β-
catenin associates with T cell factor (TCF)/lymphoid enhancing factor (LEF) to stimulate
transcriptional activity (FIG. 4c).
Mutations causing loss of function of LRP5 lead to low bone mass and the development of
osteoporosis-pseudoglioma syndrome, an autosomal recessive disorder characterized by
osteoporosis and eye abnormalities110. By contrast, mutations that increase the function of
LRP5 have been linked to increased bone mass in humans111,112. Although the mechanism
by which LRP5 (or LRP6) and WNT ligands regulate bone mass has not been elucidated
fully, both components are required to stimulate osteoblast proliferation and osteoblast
Author Manuscript
maturation113. When liposomal vesicles of purified WNT3A were delivered to the site of a
fracture in mice, rapid healing occurred owing to increased proliferation and earlier
differentiation of osteoblast lineage cells114.
WNT antagonists have been shown to be upregulated in mouse SSCs and downstream
progenitors after injury, probably to ensure the correct timing for osteoblast differentiation in
the bone repair process. For example, microarray analysis of mouse BCSPs showed the
upregulation of DKK1, a WNT antagonist, 3 days after a femoral fracture39. However,
DKK1 was downregulated 7 days after the injury, indicating the temporality of WNT
signalling for osteoblast differentiation of mouse SSCs39. Furthermore, under normal
conditions, SOST, which encodes sclerostin and is upregulated in human SSCs, binds to
LRPs, suppressing receptor and WNT activity8,115. Therefore, SOST acts as a negative
regulator of bone formation by opposing the effects of LRP activation to ensure the bone
Author Manuscript
does not overgrow. Missense mutations in the Lrp5 gene cause the receptor to have low
binding affinity for sclerostin and DKK1, thus leading to accumulation of bone mass in mice
exhibiting phenotypes similar to the human disease known as autosomal dominant high bone
mass116. Knockout of the Lrp5 gene in mice led to decreased bone mass reminiscent of the
human osteoporotic phenotype111,117. Additionally, haploinsufficiency of Lrp6 in Lrp5−/−
mice results in reduced bone mass118. Whereas the lower bone mass in Lrp5-knockout mice
is attributed to reduced bone formation, the lower bone mass observed in Lrp6 hypomorphic
mice is due to increased bone resorption119.
Author Manuscript
Finally, the canonical WNT signalling pathway has an important role in interacting with
other signalling pathways. Activation of the Notch pathway inhibits the canonical WNT
pathway, causing an arrest in osteoblast differentiation120. As outlined earlier, Notch
pathway activation increases osteoprogenitor proliferation while suppressing differentiation
into mature osteoblasts. However, the canonical WNT pathway enables osteoblast lineage
cells to progress to the final, committed osteoblast. Therefore, the balance between Notch
signalling and canonical WNT signalling determines the commitment of lineage cells to
osteoblasts. Additionally, BMP signalling complements WNT-induced osteogenic
differentiation as both signalling pathways share common targets, such as connective tissue
growth factor121–124. For example, osteogenic effects are enhanced in the presence of
WNT3A and BMP9; furthermore, the induction of ectopic bone formation by BMP2 is
Author Manuscript
antagonized through Dkk1 overexpression and the conditional knockout of Ctnnb1 (which
encodes β-catenin)125,126. Furthermore, BMP2 might promote osteogenic differentiation
through increased expression of Lrp5 and stabilization of β-catenin127.
BMP signalling.—The TGFß family consists of a large group of structurally similar

proteins. TGFß is a multi-functional peptide that regulates cellular processes such as
proliferation and differentiation. These proteins form TGFß complexes, which interact with
cell surface serine/threonine-specific protein kinase receptors128. Subsequent intracellular
signals are propagated by SMAD proteins, ultimately resulting in changes in target gene
expression128. The TGFß superfamily contains the TGFß subfamily, the activin subfamily
and BMPs. Specifically, BMP2 and BMP4 have been shown to be essential in osteoblast
differentiation (FIG. 4d).
Author Manuscript
BMP binds to BMP receptor (BMPR) located on the cell surface. BMPR is a transmembrane
protein belonging to the serine/threonine kinase family, and the protein consists of a type 1
receptor and a type 2 receptor129,130. The interaction between BMP and BMPR stabilizes
the complex, resulting in the type 2 receptor phosphorylating the type 1 receptor129,131,132.
The phosphorylated type 1 receptors result in propagation of the intracellular signal through
the phosphorylation of the receptor-regulated SMAD family of proteins (SMAD1, SMAD5
and SMAD8)129,130. The phosphorylated SMAD protein interacts with SMAD4, which
contains a nuclear import sequence, enabling the protein to be localized to the nucleus and
regulate gene expression129,131,132.
Genetic studies have determined the role of Bmp2 in promoting osteoblast differentiation by
targeting Runx2 downstream. Conditional knockout of Bmp2 resulted in developmental
Author Manuscript
deficiencies affecting prenatal and postnatal bone formation. Specifically, bone mass and
trabeculation were significantly decreased in Bmp2-knockout mice133. Studies have shown
that a loss of Bmp2 alone does not stop limb osteoblast differentiation; however, a
combinational knockout of Bmp2 and Bmp4 or Bmp2 and Bmp7 resulted in abnormal
osteoblast differentiation133. Although Bmp2 might be dispensable for osteoblast formation
during development, it is required for fracture repair. During the healing period, Bmp2-
deficient mice expressed lower expression levels of Runx2, Osx, Bglap2 and Col1a1, genes
which are normally upregulated during bone formation and repair134. Both Bmp2 and
Smad3 were upregulated in mouse SSCs after mandibular fracture41. The gene regulation
Author Manuscript
changes support the necessity of BMP signalling and SMAD protein signal transduction for
osteoblast differentiation and subsequent bone repair. Furthermore, analysis of the
transcriptional expression of Bmp2 in mouse SSCs and their downstream progenitors,
without any prior injury, showed a decrease in Bmp2 expression from the mouse SSC to a
committed osteoblast30.
As the BMP signalling cascade contains many components, another target of interest to
modulate BMP signalling is BMPR type 1A (BMPR1A; also known as ALK3). BMPR1A is
present on preosteoblasts and osteoblasts, and constitutively active Bmpr1a has been shown
to have a role in cell differentiation135,136. In the context of osteoblasts, Bmpr1a has been
shown to have a dual role in restricting preosteoblast proliferation while stimulating
osteoblast activity135,137. By deletion of Bmpr1a, an increase in trabecular bone formation
Author Manuscript
was observed owing to an increase in the number of osteoblasts resulting from the
hyperproliferation of preosteoblasts136,138. Furthermore, BMPR1A has a key role in
determining the mechanical properties of bone. The presence of the receptor is essential for
bone quality and mechanical integrity as it leads to increased collagen crosslink maturation
in osteoblasts139.
FGF signalling.—Fibroblast growth factors (FGFs) are a family of cell signalling proteins
that are crucial for normal development. The growth factors’ activating cell surface receptors
are called ‘fibroblast growth factor receptors’ (FGFRs)140. The binding of FGF to the
extracellular ligand-binding domain of FGFR results in the phosphorylation of tyrosine
residues in the FGFR intracellular domain. Subsequently, this phosphorylation results in the
activation of downstream signalling pathways such as the Ras-MAPK, phophoinositide 3-
Author Manuscript
kinase-AKT and protein kinase C pathways141,142. FGF signalling involves crucial elements
of normal development, such as mesoderm induction, neural development and limb
development. FGF2 is expressed in the developing limb bud and contributes to limb growth
and patterning142. Overexpression of human FGF2 in mice results in dwarfism with
shortened and flattened long bones143. Deletion of Fgf2 in mice leads to decreased bone
mass, bone formation and bone mineralization144 (FIG. 4e).
In addition to FGF2, both FGF8 and FGF10 have a role in limb development. Targeted Fgf8
deletion results in failed limb development in mice, with a substantial reduction in limb bud
size and hypoplasia of skeletal elements145,146. A positive-feedback loop exists between
FGF8 and FGF10, which is essential for limb development146. Similarly to Fgf8 deletion in
mice, Fgf10-knockout mice show a complete lack of forelimb and hindlimb development;
Author Manuscript
limb bud initiation still occurs in these mice but limb bud outgrowth is impaired147–149.
FGF8 is also expressed in osteoblasts, specifically in the cortical bone of the
calvarium150,151. Addition of FGF8 to bone cell culture medium results in increased
differentiation of the cells into osteoblasts and increased in vitro bone formation152. After
mandibular distraction, Fgf8 expression increases during the healing process41. Initially,
Fgf8 expression in mouse SSCs is upregulated; however, as the cells differentiate into
osteoblasts, Fgf8 expression becomes downregulated41. Therefore, FGF8 might regulate the
ability of cells to differentiate into osteoblasts and their subsequent osteoblastic activity.
Along with understanding the role of FGF in skeletal development and homeostasis,
Author Manuscript
elucidating the role of FGFRs in these same contexts can give further insight into the role of
the signalling pathway. FGFR1 has an important role in limb development. Disruption of
FGFR1 in mice at the early stage of development, before limb mesenchyme thickening,
results in a severe defect characterized by malformation of the apical ectodermal ridge153. In
conditional Fgfr1-knockout mice, in which the disruption of Fgfr1 can be temporally
controlled and confined to the late stage of development, the mice show a diminished albeit
functional appendicular skeleton and malformed forelimbs and hindlimbs154. FGFR1 also
acts as a negative regulator of long bone growth. Conditional deletion of Fgfr1 in
osteochondroprogenitor cell lineages leads to an increased height of the hypertrophic zone
owing to delayed degradation or maturation of the hypertrophic chondrocytes during
endochondral ossification155,156. Furthermore, Fgfr1 deletion studies in mice show an
increase in osteoblast proliferation and delay in differentiation and matrix mineralization.
Finally, Fgfr1 expression increases during the healing process after mandibular distraction41.
Author Manuscript
Fgfr1 expression is downregulated in mouse SSCs; however, expression levels increase as

the cells became more committed to the osteoblast fate41. Therefore, the increase in Fgfr1
expression during osteoblast lineage commitment further indicates the importance of the
receptor in differentiation and matrix mineralization to form bone.
FGFR2 is another important receptor in the FGF signalling pathway regulating limb
development. FGFR2 is expressed in the condensing mesenchyme of the early limb bud,
with expression localized to the perichondrial tissue and periosteal tissue of the long bones
and cranial sutures157,158. Selective deletions of the transmembrane domains of FGFR2
result in mouse embryonic lethality. Specifically, deletion of domain III results in the failure
of mutant embryos to form limb buds, indicating that Fgfr2 domain III is essential for limb
initiation159,160. The role of FGFR2 in cranial suture development has generated several
Author Manuscript
gain-of-function mutant mouse models. One such model is the Fgfr2+/S252W mouse, which
develops craniosynostosis, resembling human Apert syndrome161. This model elucidated the
role of FGFR2 in the abnormal osteoblast proliferation and differentiation associated with
Apert syndrome. Other mutant mouse models, such as the Fgfr2−/− mouse, show delayed
differentiation and mineralization of the skull vault and premature coronal suture formation
owing to decreased osteoblast differentiation and mineralization159,161. Furthermore, these
observations correlate with decreased expression of the osteoblast markers osteopontin and
RUNX2 (REF.159).
The role of the niche in bone repair

Osteoblast functionality during bone repair is dependent on the signals of the surrounding
Author Manuscript
niche cells. Niche cells provide a specific microenvironment and integrate signals to mediate
the appropriate stem cell response according to the needs of the organism. The niche
interacts with osteoprogenitors to maintain their undifferentiated properties during
homeostasis. However, after a bone injury, the changing niche environment causes the
precursor cells to differentiate into osteoblasts and repair the damaged tissue. Therefore, to
understand the molecular regulation of osteoblasts that enables their function in bone repair,
the influence of niche cells such as inflammatory, endothelial and Schwann cells must be
understood.
Inflammatory cells become activated after acute bone injury, resulting in a cascade initiated
Author Manuscript
by a haematoma (from ruptured blood vessels inside the bone and surrounding soft
tissue)162,163. The haematoma, characterized by hypoxia and low pH, acts as a temporary
scaffold enabling the active invasion of local tissue macrophages and polymorphonuclear
neutrophils. Specifically, in fracture healing, the resident macrophage populations of the
endosteal and periosteal surfaces have a pivotal role in intramembranous ossification, while
recruited inflammatory macrophages have an important role in endochondral
ossification162,164,165. The influx of these inflammatory cells results in the secretion of
chemokines such as IL-6 and chemokine ligand 2 (CCL2)166,167. Along with these
proinflammatory chemokines, BMP4 and VEGF are released into the microenvironment at
the site of the acute bone injury130,165,168. The excreted factors BMP4, VEGF, IL-6 and
CCL2 promote osteogenic differentiation by interacting with the nascent osteoprogenitors
and commit their fate towards the osteoblast lineage to enable bone repair165–168.
Author Manuscript
Revascularization is essential for bone repair, as new vasculature brings nutrients to the site
of the injury. These blood vessels are composed of endothelial cells, which interact with
osteoprogenitor cells to create a microenvironment supportive of osteoblast lineage fate
specification169. Osteogenesis has been linked to type H endothelial cells, a subtype of
capillary endothelial cells169–171. The type H cell is found in the metaphysis and endosteum
of postnatal long bones171. These cells express PECAM1 and endomucin, which are key
endothelial cell surface markers. In addition to promoting angiogenesis, type H cells provide
molecular signals that target osteoprogenitor cells through the Notch signalling pathway172.
Endothelial Notch signalling regulates osteogenesis, as indicated by the finding that
disruption of Notch pathway signalling reduced overall osteogenesis172. On a molecular
level, endothelial Notch signalling disruption decreases the expression of Spp1, Runx2 and
Osx172. From the findings taken together, Notch signalling mediated by endothelial cells
Author Manuscript
alters the niche at the injury site, enabling osteoprogenitor cells to differentiate into
osteoblasts.
Sensory and sympathetic nerves have a crucial role in skeletal homeostasis and bone repair.
Nerve growth factor (NGF), a neurotrophin, is involved in the development, maintenance
and regeneration of sensory and sympathetic nerves46,173,174. In vivo studies applied
distraction osteogenesis in the rabbit mandible; this bone is unique as the inferior alveolar
nerve runs through the mandible175. The exogenous application of NGF reduced the
consolidation period for bone repair in this model175. Therefore, the presence of NGF, which
is natively secreted in the niche by nerves, accelerated osteogenesis. Additionally, the
mandibular distraction osteogenesis model found elevated NGF and VEGF expression
during the bone repair period175,176. NGF was expressed by cells in the nerve, whereas
Author Manuscript
VEGF was expressed by Schwann cells176. From the findings taken together, the presence of
nerve and Schwann cells in the niche supports osteogenic differentiation of osteoprogenitor
cells leading to new bone formation. Overall, osteoblast lineage determination depends on
the niche to provide the necessary signals to induce vascularization and eventual bone matrix
formation to develop new bone.
Dysregulation and clinical relevance

Author Manuscript
The dysregulation of bone biology in the setting of bone repair occurs on a spectrum from
‘lack of bone’ to ‘excessive bone’, with restoration of ‘normal bone’ as the midpoint (FIG.
5). Thus far, this Review has discussed the development and maintenance of normal bone
during homeostasis; however, clinical bone disorders can be found along this continuum.
Non-union of a fracture, namely in long bones, occurs when the healing process is
interrupted, causing insufficient bone formation within the bone gap177,178. To reduce the
occurrence of non-union, clinicians use recombinant human BMP2, resulting in a healing
rate of 86.6% for tibial fractures177,179. Safety and efficacy profiling of recombinant human
FGF2 has shown a beneficial effect on fracture healing in the tibia. However, clinical studies
did not show any significant increases in the healing rate between the therapy group and the
control group177,180.
Author Manuscript
Another example of lack of bone in healing is hip fractures caused by falls in elderly people.
Worldwide annually, 1.7 million hip fractures occur, a number that is projected to increase to
more than six million by 2050 owing to the ageing population181. Most hip fractures in
elderly people occur secondary to underlying osteoporosis.
The final example is a unicortical defect, which occurs frequently in children. Greenstick
fractures account for 12% of all paediatric emergency department visits in the USA182. The
clinical intervention for this fracture type is to immobilize the bone using a cast, allowing it
to naturally heal182. As the fracture still preserves the shape of the bone and the regenerative
potential in a child is robust, the fracture can heal quickly over time with minimal strain on
the affected bone.
The opposite aspect of the spectrum is ‘excessive bone formation’, exemplified by

Author Manuscript
heterotopic ossification. Heterotopic ossification is defined as ectopic (extraskeletal) bone

formation that can occur following soft tissue damage such as a trauma, burn or spinal cord
injury. This ectopic bone formation has been shown to require the innate immune response
and specifically the response of macrophages, which stimulate prochondrogenic and pro-
osteogenic genes and activation of osteoblast progenitor cells in this setting183–185. The
inflammatory response activates neutrophils and macrophages, which secrete
prochondrogenic factors (such as TGFβ1) and pro-osteogenic factors (such as oncostatin M),
driving tissue-resident progenitor cell osteochondral differentiation183,186,187. The
inflammatory response induces a cytokine cascade including IL-6 and VEGF, which are
known to promote angiogenesis and canonical and non-canonical BMP signalling, as
previously discussed74,183,188,189. The elimination of macrophages has been shown to
decrease callus formation in fracture healing190 in addition to decreasing traumatic
Author Manuscript
heterotopic ossification187.
Identifying the cell lineage responsible for non-genetic heterotopic ossification has been of
significant interest. Lineage tracing analyses have identified tissue-resident cells of the
Prx1Cre and ScxCre lineages that mark the presumptive heterotopic ossification progenitor
cells191–194. Other Cre alleles that have been shown to mark heterotopic ossification
progenitors include PdgfraCre and Gli1Cre195,196. Only a small percentage of heterotopic
ossification was shown to express markers consistent with BCSPs197. Similarly to fracture
Author Manuscript
healing, canonical BMP and TGFβ signalling are also central to heterotopic ossification
formation and are potential therapeutic targets185,198. Additionally, similarly to fracture
repair, mechanotransductive signalling and extracellular matrix properties, such as collagen
alignment, have been shown to alter cell fate199. Understanding the cells and pathways
responsible for aberrant cell fate in heterotopic ossification might inform additional disease
processes such as muscle fibrosis and allow improved targeted therapies for bone repair200.
Similarly to bone development and repair, differences exist in heterotopic ossification
formation on the basis of age and sex201,202. Overall, clinical bone disorders provide
perspective on the regulation of bone biology and the consequences for bone homeostasis
when key pathways or molecules become dysregulated.
Conclusions and perspectives

Author Manuscript
Understanding of the osteoblast lineage has significantly increased through identification of

specific cellular and molecular components necessary for bone formation. By application of
the physiological implications of the high cellular activity in the growth plate and the use of
cell sorting technologies, a stem cell population — SSCs - has been isolated, which is
lineage-restricted to bone and has the capability to support haematopoiesis. Through further
high-throughput sequencing studies of SSCs, specific genes and chromatin accessibility
motifs have been shown to be upregulated, including Runx2, Ihh and Fak (also known as
Ptk2). This novel genetic information provides insight into the pathways necessary for the
maintenance of SSCs during homeostasis and activation of the cell population during an
injury-induced regenerative response. Even with the extensive strides made in the field, more
research needs to be conducted to further understand the roles and regulation of the
osteoblast lineage in bone development and repair.
Author Manuscript
First, the relationship, or the lack thereof, between SSCs and MSCs needs to be determined
in the context of bone development and disease. Both cell types have been shown to give rise
to osteoblasts and chondrocytes. However, further research needs to be conducted to
determine if MSCs and SSCs are separate populations or if one cell type is a subset of the
other cell type. Understanding this relationship will reconcile the various viewpoints in the
field of the origin of the apex osteoblast progenitor cell. Second, determining the role of
morphogen gradients in activating the osteoblast differentiation programme during limb bud
development is essential. Currently, various developmental signalling pathways are known to
be necessary for appropriate limb patterning; however, further research needs to be
conducted to determine these signalling interactions and particularly the specific genes
committing multipotent cells to an osteoblast lineage. Finally, additional research needs to
Author Manuscript
be performed to determine a possible therapeutic role for guiding stem cells towards
osteoblast differentiation. By understanding the nuances of the differentiation pathways and
key transcription factors for osteoblast differentiation, therapies can be designed wherein
clinicians can harness the endogenous SSCs and/or MSCs within an injured bone to drive
osteoblast differentiation, thereby facilitating bone repair. The potential for studying the
osteoblast lineage is vast. Driven by the application of new technologies to answer these
fundamental questions, an increased understanding of the osteoblast lineage can benefit both
science and clinical practice.
References
Author Manuscript
1. Ambrosi TH, Longaker MT & Chan CKF A revised perspective of skeletal stem cell biology. Front.
Cell Dev. Biol 7, 189 (2019). [PubMed: 31572721]
2. Murphy MP et al. The role of skeletal stem cells in the reconstruction of bone defects. J. Craniofac.
Surg 28, 1136–1141 (2017). [PubMed: 28665863]
3. Long F Building strong bones: molecular regulation of the osteoblast lineage. Nat. Rev. Mol. Cell
Biol 13, 27–38 (2012).
4. Bianco P & Robey PG Skeletal stem cells. Development 142, 1023–1027 (2015). [PubMed:
25758217]
5. Garnero P, Sornay-Rendu E, Chapuy MC & Delmas PD Increased bone turnover in late
postmenopausal women is a major determinant of osteoporosis. J. Bone Miner. Res 11, 337–349
(2009).
6. Soltanoff CS, Yang S, Chen W & Li YP Signaling networks that control the lineage commitment
and differentiation of bone cells. Crit. Rev. Eukaryot. Gene Expr 19, 1–46 (2009). [PubMed:
19191755]
Author Manuscript
7. Compton JT & Lee FY Current concepts review: a review of osteocyte function and the emerging
importance of sclerostin. J. Bone Joint Surg. Am 96, 1659–1668 (2014). [PubMed: 25274791]
8. Van Bezooijen RL et al. Sclerostin Is an osteocyte-expressed negative regulator of bone formation,
but not a classical BMP antagonist. J. Exp. Med 199, 805–814 (2004). [PubMed: 15024046]
9. Robling AG et al. Mechanical stimulation of bone in vivo reduces osteocyte expression of Sost/
sclerostin. J. Biol. Chem 283, 5866–5875 (2008). [PubMed: 18089564]
10. Tatsumi S et al. Targeted ablation of osteocytes induces osteoporosis with defective
mechanotransduction. Cell Metab. 5, 464–475 (2007). [PubMed: 17550781]
11. Jacome-Galarza CE, Lee SK, Lorenzo JA & Aguila HL Identification, characterization, and
isolation of a common progenitor for osteoclasts, macrophages, and dendritic cells from murine
bone marrow and periphery. J. Bone Miner. Res 28, 1203–1213 (2013). [PubMed: 23165930]
12. Kong YY et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and
lymph-node organogenesis. Nature 397, 315–323 (1999). [PubMed: 9950424]
13. Dougall WC et al. RANK is essential for osteoclast and lymph node development. Genes. Dev 13,
Author Manuscript
2412–2424 (1999). [PubMed: 10500098]

14. Xu F & Teitelbaum SL Osteoclasts: new insights. Bone Res 1, 11–26 (2013). [PubMed: 26273491]
15. Meyers C et al. Heterotopic ossification: a comprehensive review. JBMR Plus 3, e10172 (2019).
[PubMed: 31044187]
16. Dallas SL, Xie Y, Shiflett LA & Ueki Y Mouse Cre models for the study of bone diseases. Curr.
Osteoporos. Rep 16, 466–477 (2018). [PubMed: 29934753]
17. Pittenger MF et al. Multilineage potential of adult human mesenchymal stem cells. Science 284,
143–147 (1999). [PubMed: 10102814] This work establishes the potential for MSCs to
differentiate into bone, cartilage and fat.
18. Chen Q et al. Fate decision of mesenchymal stem cells: adipocytes or osteoblasts? Cell Death
Differ. 23, 1128–1139 (2016). [PubMed: 26868907]
19. Friedenstein AJ, Chailakhjan RK & Lalykina KS The development of fibroblast colonies in
monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Prolif. 3, 393–403 (1970).
20. Friedenstein AJ, Chailakhyan RK & Gerasimov UV Bone marrow osteogenic stem cells: in vitro
Author Manuscript
cultivation and transplantation in diffusion chambers. Cell Prolif. 20, 263–272 (1987).
21. Friedenstein AJ Osteogenic stem cells in the bone marrow. Bone Miner. Res 10.1016/
b978-0-444-81371-8.50012-1 (1990).
22. Wei J et al. Glucose uptake and Runx2 synergize to orchestrate osteoblast differentiation and bone
formation. Cell 161, 1576–1591 (2015). [PubMed: 26091038]
23. Wang T, Zhang X & Bikle DD Osteogenic differentiation of periosteal cells during fracture
healing. J. Cell Physiol 232, 913–921 (2017). [PubMed: 27731505]
24. Ackema KB & Charité J Mesenchymal stem cells from different organs are characterized by
distinct topographic Hox codes. Stem Cell Dev. 17, 979–991 (2008).
25. Rux DR et al. Regionally restricted Hox function in adult bone marrow multipotent mesenchymal
stem/stromal cells. Dev. Cell 39, 653–666 (2016). [PubMed: 27939685]
Author Manuscript
26. Nelson LT, Rakshit S, Sun H & Wellik DM Generation and expression of a Hoxa11eGFP targeted
allele in mice. Dev. Dyn 237, 3410–3416 (2008). [PubMed: 18942146]
27. Swinehart IT, Schlientz AJ, Quintanilla CA, Mortlock DP & Wellik DM Hox11 genes are required
for regional patterning and integration of muscle, tendon and bone. Development 140, 4574–4582
(2013). [PubMed: 24154528]
28. Pineault KM, Song JY, Kozloff KM, Lucas D & Wellik DM Hox11 expressing regional skeletal
stem cells are progenitors for osteoblasts, chondrocytes and adipocytes throughout life. Nat.
Commun 10, 3168 (2019). [PubMed: 31320650]
29. Rux DR & Wellik DM Hox genes in the adult skeleton: novel functions beyond embryonic
development. Dev. Dyn 246, 310–317 (2017). [PubMed: 28026082]
30. Chan CKF et al. Identification and specification of the mouse skeletal stem cell. Cell 160, 285–298
(2015). [PubMed: 25594184] The work is the first to isolate the SSC in mice, which has the
differentiation capacity to be restricted to bone, cartilage,and bone stroma.
31. Chan CKF et al. Identification of the human skeletal stem cell. Cell 175, 43–56 (2018). [PubMed:
Author Manuscript
30241615]
32. Sacchetti B et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a
hematopoietic microenvironment. Cell 131, 324–336 (2007). [PubMed: 17956733]
33. Kassem M & Bianco P Skeletal stem cells in space and time. Cell 160, 17–19 (2015). [PubMed:
25594172]
34. Bianco P Stem cells and bone: a historical perspective. Bone 70, 2–9 (2015). [PubMed: 25171959]
35. Ueno H & Weissman IL Clonal analysis of mouse development reveals a polyclonal origin for yolk
sac blood islands. Dev. Cell 11, 519–533 (2006). [PubMed: 17011491]
36. Worthley DL et al. Gremlin 1 identifies a skeletal stem cell with bone, cartilage, and reticular
stromal potential. Cell 160, 269–284 (2015). [PubMed: 25594183]
37. Chan CKF et al. Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells. Proc.
Natl Acad. Sci. USA 110, 12643–12648 (2013). [PubMed: 23858471]
38. Berendsen AD & Olsen BR Bone development. Bone 80, 14–18 (2015). [PubMed: 26453494]
39. Marecic O et al. Identification and characterization of an injury-induced skeletal progenitor. Proc.
Author Manuscript
Natl Acad. Sci. USA 112, 9920–9925 (2015). [PubMed: 26216955]

40. Tevlin R et al. Pharmacological rescue of diabetic skeletal stem cell niches. Sci. Transl Med 9,
eaag2809 (2017). [PubMed: 28077677]
41. Ransom RC et al. Mechanoresponsive stem cells acquire neural crest fate in jaw regeneration.
Nature 563, 514–521 (2018). [PubMed: 30356216]
42. Mizuhashi K et al. Resting zone of the growth plate houses a unique class of skeletal stem cells.
Nature 563, 254–258 (2018). [PubMed: 30401834]
43. Debnath S et al. Discovery of a periosteal stem cell mediating intramembranous bone formation.
Nature 562, 133–139 (2018). [PubMed: 30250253]
44. Jia G et al. Single cell RNA-seq and ATAC-seq analysis of cardiac progenitor cell transition states
and lineage settlement. Nat. Commun 9, 4877 (2018). [PubMed: 30451828]
45. Baker S, Rogerson C, Hayes A, Sharrocks A & Rattray M Classifying cells with Scasat, a single-
cell ATAC-seq analysis tool. Nucleic Acids Res. 47, e10 (2019). [PubMed: 30335168]
46. Le Douarin NM & Smith J Development of the peripheral nervous system from the neural crest.
Author Manuscript
Annu. Rev. Cell Biol 4, 375–404 (1988). [PubMed: 3058162]

47. Long F & Ornitz DM Development of the endochondral skeleton. Cold Spring Harb Perspect. Biol
5, a008334 (2013). [PubMed: 23284041]
48. Kronenberg HM Developmental regulation of the growth plate. Nature. 423, 332–336 (2003).
[PubMed: 12748651]
49. Maes C & Kronenberg HM Postnatal bone growth: growth plate biology, bone formation, and
remodeling. pediatric. Bone 10.1016/B978-0-12-382040-2.10004-8 (2012).
50. Lefebvr EV & Dvir-Ginzberg M SOX9 and the many facets of its regulation in the chondrocyte
lineage. Connect. Tissue Res 58, 2–14 (2017). [PubMed: 27128146]
51. Lovell-Badge R The early history of the Sox genes. Int. J. Biochem. Cell Biol 42, 378–380 (2010).
[PubMed: 20005971]
Author Manuscript
52. Bi W, Deng JM, Zhang Z, Behringer RR & De Crombrugghe B Sox9 is required for cartilage
formation. Nat. Genet 22, 85–89 (1999). [PubMed: 10319868]
53. Akiyama H, Chaboissier MC, Martin JF, Schedl A & De Crombrugghe B The transcription factor
Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is
required for expression of Sox5 and Sox6. Genes Dev. 16, 2813–2828 (2002). [PubMed:
12414734]
54. Henry SP, Liang S, Akdemir KC & De Crombrugghe B The postnatal role of Sox9 in cartilage. J.
Bone Miner. Res 27, 2511–2525 (2012). [PubMed: 22777888]
55. Schafer AJ et al. Campomelic dysplasia with XY sex reversal: diverse phenotypes resulting from
mutations in a single gene. Ann. N. Y. Acad. Sci 785, 137–149 (1996). [PubMed: 8702120]
56. Gentilin B et al. Phenotype of five cases of prenatally diagnosed campomelic dysplasia harboring
novel mutations of the SOX9 gene. Ultrasound Obstet. Gynecol 36, 315–323 (2010). [PubMed:
20812307]
57. Komori T Regulation of bone development and extracellular matrix protein genes by RUNX2. Cell
Author Manuscript
Tissue Res. 339, 189–195 (2010). [PubMed: 19649655]

58. Ducy P, Zhang R, Geoffroy V, Ridall AL & Karsenty G Osf2/Cbfa1: a transcriptional activator of
osteoblast differentiation. Cell 89, 747–754 (1997). [PubMed: 9182762]
59. Harada H et al. Cbfa1 isoforms exert functional differences in osteoblast differentiation. J. Biol.
Chem 274, 6972–6978 (1999). [PubMed: 10066751]
60. Komori T et al. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to
maturational arrest of osteoblasts. Cell 89, 755–764 (1997). [PubMed: 9182763] This work
establishes RUNX2 as an essential transcription factor for osteoblast differentiation.
61. Otto F et al. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for
osteoblast differentiation and bone development. Cell 89, 765–771 (1997). [PubMed: 9182764]
62. Inada M et al. Maturational disturbance of chondrocytes in Cbfa1-deficient mice. Dev. Dyn 214,
279–290 (1999). [PubMed: 10213384]
63. Takarada T et al. An analysis of skeletal development in osteoblast-specific and chondrocyte-
specific runt-related transcription factor-2 (Runx2) knockout mice. J. Bone Miner. Res 28, 2064–
Author Manuscript
2069 (2013). [PubMed: 23553905]

64. Maruyama Z et al. Runx2 determines bone maturity and turnover rate in postnatal bone
development and is involved in bone loss in estrogen deficiency. Dev. Dyn 236, 1876–1890 (2007).
[PubMed: 17497678]
65. Sinha KM & Zhou X Genetic and molecular control of osterix in skeletal formation. J. Cell
Biochem 114, 975–984 (2013). [PubMed: 23225263]
66. Karsenty G Minireview: tranzscriptional control of osteoblast differentiation. Endocrinology 142,
2731–2733 (2001). [PubMed: 11415989]
67. Nakashima K & De Crombrugghe B Transcriptional mechanisms in osteoblast differentiation and
bone formation. Trends Genet. 19, 458–466 (2003). [PubMed: 12902164]
68. Nakashima K et al. The novel zinc finger-containing transcription factor osterix is required for
osteoblast differentiation and bone formation. Cell 108, 17–29 (2002). [PubMed: 11792318] This
work establishes the temporal coordination between OSX and RUNX2 activation for osteoblast
differentiation.
69. Yang X & Karsenty G Transcription factors in bone: developmental and pathological aspects.
Author Manuscript
Trends Mol. Med 8, 340–345 (2002). [PubMed: 12114114]

70. Zhou X et al. Multiple functions of osterix are required for bone growth and homeostasis in
postnatal mice. Proc. Natl Acad. Sci. USA 107, 12919–12924 (2010). [PubMed: 20615976]
71. Liu TM & Lee EH Transcriptional regulatory cascades in Runx2-dependent bone development.
Tissue Eng. Part B Rev 19, 254–263 (2013). [PubMed: 23150948]
72. St-Arnaud R & Hekmatnejad B Combinatorial control of ATF4-dependent gene transcription in
osteoblasts. Ann. N. Y. Acad. Sci 1237, 11–18 (2011). [PubMed: 22082360]
73. Yang X et al. ATF4 is a substrate of RSK2 and an essential regulator of osteoblast biology:
implication for Coffin-Lowry syndrome. Cell 117, 387–398 (2004). [PubMed: 15109498]
Author Manuscript
74. Jing D et al. The role of microRNAs in bone remodeling. Int. J. Oral. Sci 7, 131–143 (2015).
[PubMed: 26208037]
75. Xiao G et al. Cooperative interactions between activating transcription factor 4 and Runx2/Cbfa1
stimulate osteoblast-specific osteocalcin gene expression. J. Biol. Chem 280, 30689–30696 (2005).
[PubMed: 16000305]
76. Wagner EF Functions of AP1 (Fos/Jun) in bone development. Ann. Rheum. Dis 61, ii40–ii42
(2002). [PubMed: 12379619]
77. Kenner L et al. Mice lacking JunB are osteopenic due to cell-autonomous osteoblast and osteoclast
defects. J. Cell Biol 164, 613–623 (2004). [PubMed: 14769860]
78. Zambotti A, Makhluf H, Shen J & Ducy P Characterization of an osteoblast-specific enhancer
element in the CBFA1. Gene 277, 41497–41506 (2002).
79. Jochum W et al. Increased bone formation and osteosclerosis in mice overexpressing the
transcription factor Fra-1. Nat. Med 6, 980–984 (2000). [PubMed: 10973316]
80. Bozec A et al. Fra-2/AP-1 controls bone formation by regulating osteoblast differentiation and
Author Manuscript
collagen production. J. Cell Biol 190, 1093–1106 (2010). [PubMed: 20837772]

81. Nüsslein-volhard C & Wieschaus E Mutations affecting segment number and polarity in
drosophila. Nature. 287, 795–801 (1980). [PubMed: 6776413]
82. McMahon AP, Ingham PW & Tabin CJ 1 Developmental roles and clinical significance of
Hedgehog signaling. Curr. Top. Dev. Biol 53, 1–114 (2003). [PubMed: 12509125]
83. Ocbina PJR & Anderson KV Intraflagellar transport, Cilia, and mammalian hedgehog signaling:
analysis in mouse embryonic fibroblasts. Dev. Dyn 237, 2030–2038 (2008). [PubMed: 18488998]
84. Riddle RD, Johnson RL, Laufer E & Tabin C Sonic hedgehog mediates the polarizing activity of
the ZPA. Cell 75, 1401–1416 (1993). [PubMed: 8269518]
85. Rohatgi R, Milenkovic L & Scott MP Patched1 regulates hedgehog signaling at the primary cilium.
Science. 317, 372–376 (2007). [PubMed: 17641202]
86. Corbit KC et al. Vertebrate Smoothened functions at the primary cilium. Nature. 437, 1018–1021
(2005). [PubMed: 16136078]
87. Towers M, Mahood R, Yin Y & Tickle C Integration of growth and specification in chick wing
Author Manuscript
digit-patterning. Nature 452, 882–886 (2008). [PubMed: 18354396]

88. Chinnaiya K, Tickle C & Towers M Sonic hedgehog-expressing cells in the developing limb
measure time by an intrinsic cell cycle clock. Nat. Commun 5, 4230 (2014). [PubMed: 25001275]
89. Wang B, Fallon JF & Beachy PA Hedgehog-regulated processing of Gli3 produces an anterior/
posterior repressor gradient in the developing vertebrate limb. Cell 100, 423–434 (2000).
[PubMed: 10693759]
90. Mo R et al. Specific and redundant functions of Gli2 and Gli3 zinc finger genes in skeletal
patterning and development. Development 124, 113–123 (1997). [PubMed: 9006072]
91. Park H et al. Mouse Gli1 mutants are viable but have defects in SHH signaling in combination with
a Gli2 mutation. Development 127, 1593–1605 (2000). [PubMed: 10725236]
92. Hojo H et al. Gli1 protein participates in hedgehog-mediated specification of osteoblast lineage
during endochondral ossification. J. Biol. Chem 287, 17860–17869 (2012). [PubMed: 22493482]
93. Amano K, Densmore M, Nishimura R & Lanske B Indian hedgehog signaling regulates
transcription and expression of collagen type X via Runx2/Smads interactions. J. Biol. Chem 289,
Author Manuscript
24898–24910 (2014). [PubMed: 25028519]

94. Jemtland R, Divieti P, Lee K & Segre GV Hedgehog promotes primary osteoblast differentiation
and increases PTHrP mRNA expression and iPTHrP secretion. Bone 32, 611–620 (2003).
[PubMed: 12810168]
95. Long F & Linsenmayer TF Regulation of growth region cartilage proliferation and differentiation
by perichondrium. Development 125, 1067–1073 (1998). [PubMed: 9463353]
96. Mak KK, Chen MH, Day TF, Chuang PT & Yang Y Wnt/β-catenin signaling interacts
differentially with Ihh signaling in controlling endochondral bone and synovial joint formation.
Development 133, 3695–3707 (2006). [PubMed: 16936073]
97. Day TF & Yang Y Wnt and hedgehog signaling pathways in bone development. J. Bone Joint Surg.
Ser. Am 90, 19–24 (2008). [PubMed: 18292352]
Author Manuscript
98. Hojo H et al. Hedgehog-Gli activators direct osteo-chondrogenic function of bone morphogenetic
protein toward osteogenesis in the perichondrium. J. Biol. Chem 288, 9924–9932 (2013).
[PubMed: 23423383]
99. Schroeter EH, Kisslinger JA & Kopan R Notch-1 signalling requires ligand-induced proteolytic
release of intracellular domain. Nature 393, 382–386 (1998). [PubMed: 9620803]
100. Zanotti S & Canalis E Notch signaling and the skeleton. Endocr. Rev 37, 223–253 (2016).
[PubMed: 27074349]
101. Tu X et al. Physiological Notch signaling maintains bone homeostasis via RBPjk and Hey
upstream of NFATc1. PLoS Genet. 8, e1002577 (2012). [PubMed: 22457635]
102. Hilton MJ et al. Notch signaling maintains bone marrow mesenchymal progenitors by suppressing
osteoblast differentiation. Nat. Med 14, 306–314 (2008). [PubMed: 18297083]
103. Engin F et al. Dimorphic effects of Notch signaling in bone homeostasis. Nat. Med 14, 299–305
(2008). [PubMed: 18297084]
104. Canalis E, Parker K, Feng JQ & Zanotti S Osteoblast lineage-specific effects of notch activation
Author Manuscript
in the skeleton. Endocrinology 154, 623–634 (2013). [PubMed: 23275471]

105. Zanotti S & Canalis E Notch1 and Notch2 expression in osteoblast precursors regulates femoral
microarchitecture. Bone 62, 22–28 (2014). [PubMed: 24508387]
106. Kim JB et al. Bone regeneration is regulated by Wnt signaling. J. Bone Miner. Res 22, 1913–1923
(2007). [PubMed: 17696762]
107. Huelsken J & Birchmeier W New aspects of Wnt signaling pathways in higher vertebrates. Curr.
Opin. Genet. Dev 11, 547–553 (2001). [PubMed: 11532397]
108. Williams BO & Insogna KL Where Wnts went: the exploding field of Lrp5 and Lrp6 signaling in
bone. J. Bone Miner. Res 24, 171–178 (2009). [PubMed: 19072724]
109. Joiner DM, Ke J, Zhong Z, Xu HE & Williams BO LRP5 and LRP6 in development and disease.
Trends Endocrinol. Metab 24, 31–39 (2013). [PubMed: 23245947]
110. Baron R & Kneissel M WNT signaling in bone homeostasis and disease: from human mutations
to treatments. Nat. Med 19, 179–192 (2013). [PubMed: 23389618]
111. Boyden LM et al. High bone density due to a mutation in LDL-receptor-related protein 5. N.
Author Manuscript
Engl. J. Med 346, 1513–1521 (2002). [PubMed: 12015390]

112. Little RD 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). [PubMed: 11741193]
113. Houschyar KS et al. Wnt pathway in bone repair and regeneration - what do we know so far.
Front. Cell Dev. Biol 6, 170 (2019). [PubMed: 30666305]
114. Minear S et al. Wnt proteins promote bone regeneration. Sci. Transl Med 2, 29ra30 (2010).
115. Poole KES et al. Sclerostin is a delayed secreted product of osteocytes that inhibits bone
formation. FASEB J. 19, 1842–1844 (2005). [PubMed: 16123173]
116. Ai M, Holmen SL, Van Hul W, Williams BO & Warman ML Reduced affinity to and inhibition
by DKK1 form a common mechanism by which high bone mass-associated missense mutations
in LRP5 affect canonical Wnt signaling. Mol. Cell Biol 25, 4946–4955 (2005). [PubMed:
15923613]
117. Brunkow ME et al. Bone dysplasia sclerosteosis results from loss of the SOST gene product, a
novel cystine knot-containing protein. Am. J. Hum. Genet 68, 577–589 (2001). [PubMed:
Author Manuscript
11179006]
118. Holmen SL et al. Decreased BMD and limb deformities in mice carrying mutations in both Lrp5
and Lrp6. J. Bone Miner. Res 19, 2033–2040 (2004). [PubMed: 15537447]
119. Kubota T et al. Lrp6 hypomorphic mutation affects bone mass through bone resorption in mice
and impairs interaction with Mesd. J. Bone Miner. Res 23, 1661–1671 (2008). [PubMed:
18505367]
120. Lin GL & Hankenson KD Integration of BMP, Wnt, and notch signaling pathways in osteoblast
differentiation. J. Cell Biochem 112, 3491–3501 (2011). [PubMed: 21793042]
121. Wu M, Chen G & Li YP TGF-β and BMP signaling in osteoblast, skeletal development, and bone
formation, homeostasis and disease. Bone Res. 4, 16009 (2016). [PubMed: 27563484]
Author Manuscript
122. Itasaki N & Hoppler S Crosstalk between Wnt and bone morphogenic protein signaling: a
turbulent relationship. Dev. Dyn 239, 16–33 (2010). [PubMed: 19544585]
123. Luo Q et al. Connective tissue growth factor (CTGF) is regulated by Wnt and bone
morphogenetic proteins signaling in osteoblast differentiation of mesenchymal stem cells. J. Biol.
Chem 279, 55958–55968 (2004). [PubMed: 15496414]
124. Si W et al. CCN1/Cyr61 is regulated by the canonical Wnt signal and plays an important role in
Wnt3A-induced osteoblast differentiation of mesenchymal stem cells. Mol. Cell Biol 26, 2955–
2964 (2006). [PubMed: 16581771]
125. Boland GM, Perkins G, Hall DJ & Tuan RS Wnt 3a promotes proliferation and suppresses
osteogenic differentiation of adult human mesenchymal stem cells. J. Cell Biochem 93, 1210–
1230 (2004). [PubMed: 15486964]
126. Chen Y et al. β-Catenin signaling pathway is crucial for bone morphogenetic protein 2 to induce
new bone formation. J. Biol. Chem 282, 526–533 (2007). [PubMed: 17085452]
127. Zhang M et al. BMP-2 modulates β-catenin signaling through stimulation of Lrp5 expression and
Author Manuscript
inhibition of β-TrCP expression in osteoblasts. J. Cell Biochem 108, 896–905 (2009). [PubMed:
19795382]
128. Wrana JL et al. TGFβ signals through a heteromeric protein kinase receptor complex. Cell 71,
1003–1014 (1992). [PubMed: 1333888]
129. Schmierer B & Hill CS TGFbeta-SMAD signal transduction: molecular specificity and functional
flexibility. Nat. Rev. Mol. Cell Biol 8, 970–982 (2007). [PubMed: 18000526]
130. Salazar VS, Gamer LW & Rosen V BMP signalling in skeletal development, disease and repair.
Nat. Rev. Endocrinol 12, 203–221 (2016). [PubMed: 26893264]
131. Katagiri T & Watabe T Bone morphogenetic proteins. Cold Spring Harb Perspect. Biol 10.1101/
cshperspect.a021899 (2016).
132. Salazar VS et al. Reactivation of a developmental Bmp2 signaling center is required for
therapeutic control of the murine periosteal niche. eLife 10.7554/eLife.42386 (2019).
133. Bandyopadhyay A et al. Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb
patterning and skeletogenesis. PLoS Genet. 2, 2116–2130 (2006).
Author Manuscript
134. Tsuji K et al. BMP2 activity, although dispensable for bone formation, is required for the
initiation of fracture healing. Nat. Genet 38, 1424–1429 (2006). [PubMed: 17099713] This work
demonstrates the role of reoccurring BMP signalling for limb development and fracture healing
of the limb.
135. Lim J et al. Dual function of Bmpr1a signaling in restricting preosteoblast proliferation and
stimulating osteoblast activity in mouse. Development 143, 339–347 (2016). [PubMed:
26657771]
136. Fujii M et al. Roles of bone morphogenetic protein type I receptors and Smad proteins in
osteoblast and chondroblast differentiation. Mol. Biol. Cell 10, 3801–3813 (1999). [PubMed:
10564272]
137. Singhatanadgit W & Olsen I Endogenous BMPR-IB signaling is required for early osteoblast
differentiation of human bone cells. Vitr. Cell Dev. Biol. Anim 47, 251–259 (2011).
138. Yoshida Y et al. Negative regulation of BMP/Smad signaling by Tob in osteoblasts. Cell 103,
1085–1097 (2000). [PubMed: 11163184]
139. Zhang Y et al. Loss of BMP signaling through BMPR1A in osteoblasts leads to greater collagen
Author Manuscript
cross-link maturation and material-level mechanical properties in mouse femoral trabecular

compartments. Bone 88, 74–84 (2016). [PubMed: 27113526]
140. Johnson DE & Williams LT Structural and functional diversity in the FGF receptor multigene
family. Adv. Cancer Res 60, 1–41 (1992).
141. Ornitz DM et al. Receptor specificity of the fibroblast growth factor family. J. Biol. Chem 271,
15292–15297 (1996). [PubMed: 8663044]
142. Ornitz DM FGF signaling in the developing endochondral skeleton. Cytokine Growth Factor. Rev
16, 205–213 (2005). [PubMed: 15863035]
143. Montero A et al. Disruption of the fibroblast growth factor-2 gene results in decreased bone mass
and bone formation. J. Clin. Invest 105, 1085–1093 (2000). [PubMed: 10772653]
Author Manuscript
144. Zhou M et al. Fibroblast growth factor 2 control of vascular tone. Nat. Med 4, 201–207 (1998).
[PubMed: 9461194]
145. Crossley PH, Minowada G, MacArthur CA & Martin GR Roles for FGF8 in the induction,
initiation, and maintenance of chick limb development. Cell 84, 127–136 (1996). [PubMed:
8548816]
146. Lewandoski M, Sun X & Martin GR Fgf8 signalling from the AER is essential for normal limb
development. Nat. Genet 26, 460–463 (2000). [PubMed: 11101846]
147. Martin GR The roles of FGFs in the early development of vertebrate limbs. Genes Dev. 12, 1571–
1586 (1998). [PubMed: 9620845]
148. Min H et al. Fgf-10 is required for both limb and lung development and exhibits striking
functional similarity to Drosophila branchless. Genes Dev. 12, 3156–3161 (1998). [PubMed:
9784490]
149. Ohuchi H et al. The mesenchymal factor, FGF10, initiates and maintains the outgrowth of the
chick limb bud through interaction with FGF8, an apical ectodermal factor. Development 124,
Author Manuscript
2235–2244 (1997). [PubMed: 9187149]

150. Mahmood R et al. A role for FGF-8 in the initiation and maintenance of vertebrate limb bud
outgrowth. Curr. Biol 5, 797–806 (1995). [PubMed: 7583127]
151. Heikinheimo M, Lawshé A, Shackleford GM, Wilson DB & MacArthur CA Fgf-8 expression in
the post-gastrulation mouse suggests roles in the development of the face, limbs and central
nervous system. Mech. Dev 48, 129–138 (1994). [PubMed: 7873403]
152. Lin JM et al. Actions of fibroblast growth factor-8 in bone cells in vitro. Am. J. Physiol.
Endocrinol. Metab 297, E142–E150 (2009). [PubMed: 19383871]
153. Yamaguchi TP, Conlon RA & Rossant J Expression of the fibroblast growth factor receptor
FGFR-1/flg during gastrulation and segmentation in the mouse embryo. Dev. Biol 152, 75–88
(1992). [PubMed: 1321062]
154. Deng C et al. Fibroblast growth factor receptor-1 (FGFR-1) is essential for normal neural tube and
limb development. Dev. Biol 185, 42–54 (1997). [PubMed: 9169049]
155. Jacob AL, Smith C, Partanen J & Ornitz DM Fibroblast growth factor receptor 1 signaling in the
Author Manuscript
osteo-chondrogenic cell lineage regulates sequential steps of osteoblast maturation. Dev. Biol
296, 315–328 (2006). [PubMed: 16815385]
156. Verheyden JM, Lewandoski M, Deng C, Harfe BD & Sun X Conditional inactivation of Fgfr1 in
mouse defines its role in limb bud establishment, outgrowth and digit patterning. Development
132, 4235–4245 (2005). [PubMed: 16120640]
157. Orr-Urtreger A et al. Developmental localization of the splicing alternatives of fibroblast growth
factor receptor-2 (FGFR2). Dev. Biol 158, 475–486 (1993). [PubMed: 8393815]
158. Li X et al. Fibroblast growth factor signaling and basement membrane assembly are connected
during epithelial morphogenesis of the embryoid body. J. Cell Biol 153, 811–822 (2001).
[PubMed: 11352941]
159. Arman E, Haffner-Krausz R, Chen Y, Heath JK & Lonai P Targeted disruption of fibroblast
growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian
development. Proc. Natl Acad. Sci. USA 95, 5082–5087 (1998). [PubMed: 9560232]
160. Xu X et al. Fibroblast growth factor receptor 2 (FGFR2)-mediated reciprocal regulation loop
between FGF8 and FGF10 is essential for limb induction. Development 125, 753–765 (1998).
Author Manuscript
[PubMed: 9435295]
161. Wang Y et al. Abnormalities in cartilage and bone development in the Apert syndrome FGFR2(+/
S252W) mouse. Development 132, 3537–3548 (2005). [PubMed: 15975938]
162. Claes L, Recknagel S & Ignatius A Fracture healing under healthy and inflammatory conditions.
Nat. Rev. Rheumatol 8, 133–143 (2012). [PubMed: 22293759]
163. Glynne AJ, Andrew SM, Freemont AJ & Marsh DR Inflammatory cells in normal human fracture
healing. Acta Orthop. 65, 462–466 (1994).
164. Bolander ME Regulation of fracture repair by growth factors. Exp. Biol. Med 200, 165–170
(1992).
165. Croes M et al. Proinflammatory mediators enhance the osteogenesis of human mesenchymal stem
cells after lineage commitment. PLoS ONE 10, e0132781 (2015). [PubMed: 26176237]
Author Manuscript
166. Lu LY et al. Pro-inflammatory M1 macrophages promote Osteogenesis by mesenchymal stem

cells via the COX-2-prostaglandin E2 pathway. J. Orthop. Res 35, 2378–2385 (2017). [PubMed:
28248001]
167. Bernhardsson M & Aspenberg P Osteoblast precursors and inflammatory cells arrive
simultaneously to sites of a trabecular-bone injury. Acta Orthop. 89, 457–461 (2018). [PubMed:
29865916]
168. Ono T et al. IL-17-producing γδT cells enhance bone regeneration. Nat. Commun 7, 10928
(2016). [PubMed: 26965320] This work shows that the presence of the proinflammatory cytokine
IL-17, from the niche, aided in bone regrowth after injury.
169. Goerke SM, Obermeyer J, Plaha J, Stark GB & Finkenzeller G Endothelial progenitor cells from
peripheral blood support bone regeneration by provoking an angiogenic response. Microvasc. Res
98, 40–47 (2015). [PubMed: 25497270]
170. Langen UH et al. Cell-matrix signals specify bone endothelial cells during developmental
osteogenesis. Nat. Cell Biol 19, 189–201 (2017). [PubMed: 28218908]
Author Manuscript
171. Kusumbe AP, Ramasamy SK & Adams RH Coupling of angiogenesis and osteogenesis by a
specific vessel subtype in bone. Nature. 507, 323–328 (2014). [PubMed: 24646994]
172. Ramasamy SK, Kusumbe AP, Wang L & Adams RH Endothelial Notch activity promotes
angiogenesis and osteogenesis in bone. Nature. 507, 376–380 (2014). [PubMed: 24647000]
173. Cao J et al. Sensory nerves affect bone regeneration in rabbit mandibular distraction osteogenesis.
Int. J. Med. Sci 16, 831–837 (2019). [PubMed: 31337956]
174. Jones RE et al. Skeletal stem cell-Schwann cell circuitry in Mandibular repair. Cell Rep. 28,
2757–2766.e5 (2019). [PubMed: 31509739]
175. Park BW, Kim JR, Lee JH & Byun JH Expression of nerve growth factor and vascular endothelial
growth factor in the inferior alveolar nerve after distraction osteogenesis. Int. J. Oral Maxillofac.
Surg 35, 624–630 (2006). [PubMed: 16687241]
176. Wang L et al. Locally applied nerve growth factor enhances bone consolidation in a rabbit model
of mandibular distraction osteogenesis. J. Orthop. Res 24, 2238–2245 (2006). [PubMed:
17001706]
Author Manuscript
177. Emara KM, Diab RA & Emara AK Recent biological trends in management of fracture non-
union. World J. Orthop 6, 623–628 (2015). [PubMed: 26396938]
178. Panteli M, Pountos I, Jones E & Giannoudis PV Biological and molecular profile of fracture non-
union tissue: current insights. J. Cell Mol. Med 19, 685–713 (2015). [PubMed: 25726940]
179. Jones AL et al. Recombinant human BMP-2 and allograft compared with autogenous bone graft
for reconstruction of diaphyseal tibial fractures with cortical defects: a randomized, controlled
trial. J. Bone Joint Surg. Ser. Am 88, 1431–1441 (2006). [PubMed: 16818967]
180. Kawaguchi H et al. Local application of recombinant human fibroblast growth factor-2 on bone
repair: a dose-escalation prospective trial on patients with osteotomy. J. Orthop. Res 25, 480–487
(2007). [PubMed: 17205557]
181. Babcock S & Kellam JF Hip fracture nonunions: diagnosis, treatment, and special considerations
in elderly patients. Adv. Orthop 10.1155/2018/1912762 (2018).
182. Atanelov Z & Bentley TP Greenstick fracture. StatPearls (2018).
183. Kraft CT et al. Trauma-induced heterotopic bone formation and the role of the immune system: a
review. J. Trauma. Acute Care Surg 80, 156–165 (2016). [PubMed: 26491794]
Author Manuscript
184. Huang H et al. Relationship between heterotopic ossification and traumatic brain injury: Why
severe traumatic brain injury increases the risk of heterotopic ossification. J. Orthop. Transl 12,
16–25 (2018).
185. Sorkin M et al. Regulation of heterotopic ossification by monocytes in a mouse model of aberrant
wound healing. Nat Commun. 10.1038/s41467-019-14172-4 (2020).This work determines CD47
activation as a therapeutic approach for heterotopic ossification formation during wound healing.
186. Agarwal S et al. Disruption of neutrophil extracellular traps (NETs) links mechanical strain to
post-traumatic inflammation. Front. Immunol 10, 2148 (2019). [PubMed: 31708911]
187. Torossian F et al. Macrophage-derived oncostatin M contributes to human and mouse neurogenic
heterotopic ossifications. JCI Insight 2, e96034 (2017).
Author Manuscript
188. Hwang C et al. Mesenchymal VEGFA induces aberrant differentiation in heterotopic ossification.
Bone Res. 7, 36 (2019). [PubMed: 31840004]
189. Hsieh HHS et al. Coordinating tissue regeneration through transforming growth factor-β activated
kinase 1 inactivation and reactivation. Stem Cells 37, 766–778 (2019). [PubMed: 30786091]
190. Raggatt LJ et al. Fracture healing via periosteal callus formation requires macrophages for both
initiation and progression of early endochondral ossification. Am. J. Pathol 184, 3192–3204
(2014). [PubMed: 25285719]
191. Agarwal S et al. Inhibition of Hif1α prevents both trauma-induced and genetic heterotopic
ossification. Proc. Natl Acad. Sci. USA 113, E338–E347 (2016). [PubMed: 26721400]
192. Agarwal S et al. Scleraxis-lineage cells contribute to ectopic bone formation in muscle and
tendon. Stem Cells 35, 705–710 (2017). [PubMed: 27862618]
193. Loder SJ et al. Characterizing the circulating cell populations in traumatic heterotopic
ossification. Am. J. Pathol 188, 2464–2473 (2018). [PubMed: 30142335]
194. Dey D et al. Two tissue-resident progenitor lineages drive distinct phenotypes of heterotopic
Author Manuscript
ossification. Sci. Transl Med 10.1126/scitranslmed.aaf1090 (2016).

195. Kan C et al. Gli1-labeled adult mesenchymal stem/progenitor cells and hedgehog signaling
contribute to endochondral heterotopic ossification. Bone 109, 71–79 (2018). [PubMed:
28645539]
196. Eisner C et al. Murine tissue-resident PDGFRα+ fibro-adipogenic progenitors spontaneously
acquire osteogenic phenotype in an altered inflammatory environment. J. Bone Miner. Res
(2020).
197. Agarwal S et al. Analysis of bone-cartilage-stromal progenitor populations in trauma induced and
genetic models of heterotopic ossification. Stem Cells 34, 1692–1701 (2016). [PubMed:
27068890]
198. Agarwal S et al. Strategic targeting of multiple BMP receptors prevents trauma-induced
heterotopic ossification. Mol. Ther 25, 1974–1987 (2017). [PubMed: 28716575]
199. Huber AK et al. Immobilization after injury alters extracellular matrix and stem cell fate. J. Clin.
Invest 10.1172/JCI136142 (2020).
Author Manuscript
200. Stepien DM et al. Tuning macrophage phenotype to mitigate skeletal muscle fibrosis. J. Immunol
204, 2203–2215 (2020). [PubMed: 32161098]
201. Peterson JR et al. Effects of aging on osteogenic response and heterotopic ossification following
burn injury in mice. Stem Cell Dev. 24, 205–213 (2015).
202. Ranganathan K et al. Role of gender in burn-induced heterotopic ossification and mesenchymal
cell osteogenic differentiation. Plast. Reconstr. Surg 135, 1631–1641 (2015). [PubMed:
26017598]
203. Akiyama H et al. Osteo-chondroprogenitor cells are derived from Sox9 expressing precursors.
Proc. Natl Acad. Sci. USA 102, 14665–14670 (2005). [PubMed: 16203988]
204. Maes C et al. Osteoblast precursors, but not mature osteoblasts, move into developing and
fractured bones along with invading blood vessels. Dev. Cell 19, 329–344 (2010). [PubMed:
20708594]
205. Greenbaum A et al. CXCL12 in early mesenchymal progenitors is required for haematopoietic
stem-cell maintenance. Nature. 495, 227–230 (2013). [PubMed: 23434756]
Author Manuscript
206. Xiong J et al. Osteocytes, not osteoblasts or lining cells, are the main source of the RANKL
required for osteoclast formation in remodeling bone. PLoS ONE 10.1371/journal.pone.0138189
(2015).
207. Pineault KM et al. Hox11 genes regulate postnatal longitudinal bone growth and growth plate
proliferation. Biol. Open 4, 1538–1548 (2015). [PubMed: 26500224]
208. Yu VWC et al. FIAT represses ATF4-mediated transcription to regulate bone mass in transgenic
mice. J. Cell Biol 169, 591–601 (2005). [PubMed: 15911876]
209. Ambrogini E et al. FoxO-mediated defense against oxidative stress in osteoblasts is indispensable
for skeletal homeostasis in mice. Cell Metab. 11, 136–146 (2010). [PubMed: 20142101]
210. Shimoyama A et al. Ihh/Gli2 signaling promotes osteoblast differentiation by regulating Runx2
expression and function. Mol. Biol. Cell 18, 2411–2418 (2007). [PubMed: 17442891]
Author Manuscript
211. Li J et al. Suppressor of fused restraint of hedgehog activity level is critical for osteogenic
proliferation and differentiation during calvarial bone development. J. Biol. Chem 292, 15814–
15825 (2017). [PubMed: 28794157]
212. Funato N et al. Hand2 controls osteoblast differentiation in the branchial arch by inhibiting DNA
binding of Runx2. Development 136, 615–625 (2009). [PubMed: 19144722]
213. Kanzler B, Kuschert SJ, Liu YH & Mallo M Hoxa-2 restricts the chondrogenic domain and
inhibits bone formation during development of the branchial area. Development 125, 2587–2597
(1998). [PubMed: 9636074]
214. Komori T Regulation of osteoblast differentiation by runx2. Adv. Exp. Med. Biol 658, 43–49
(2010). [PubMed: 19950014]
215. Hong JH et al. TAZ, a transcriptional modulator of mesenchymal stem cell differentiation.
Science 309, 1074–1078 (2005). [PubMed: 16099986]
216. Bialek P et al. A twist code determines the onset of osteoblast differentiation. Dev. Cell 6, 423–
435 (2004). [PubMed: 15030764]
Author Manuscript
217. Cancela L, Hsieh CL & Francke UPP Molecular structure, chromosome assignment, and
promoter organization of the human matrix Gla protein gene. J. Biol. Chem 265, 15040–15048
(1990). [PubMed: 2394711]
218. Karsenty G & Park RW Regulation of type I collagen genes expression. Int. Rev. Immunol 12,
177–185 (1995). [PubMed: 7650420]
219. Pinzone JJ et al. The role of Dickkopf-1 in bone development, homeostasis, and disease. Blood
113, 517–525 (2009). [PubMed: 18687985]
220. Kim JB et al. Reconciling the roles of FAK in osteoblast differentiation, osteoclast remodeling,
and bone regeneration. Bone 41, 39–51 (2007). [PubMed: 17459803]
221. Li X et al. Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling. J. Biol. Chem
280, 19883–19887 (2005). [PubMed: 15778503]
Author Manuscript
Author Manuscript
Osteon
Author Manuscript
A cylindrical structure consisting of a mineralized matrix and osteocytes that transports

blood through connected canaliculi.
Author Manuscript
Author Manuscript
Author Manuscript
Long bone growth plate

Author Manuscript
An area of differentiating tissue located near the ends of long bones that enables
physiological lengthening of the bones.
Author Manuscript
Author Manuscript
Author Manuscript
Axial skeleton
Author Manuscript
The portion of the skeleton consisting of the bones of the head and vertebrae.
Author Manuscript
Author Manuscript
Author Manuscript
Appendicular skeleton
Author Manuscript
The portion of the skeleton consisting of the bones of the appendages.

Author Manuscript
Author Manuscript
Author Manuscript
Cancellous bone
Author Manuscript
Mature adult bone consisting of spongy tissue meshwork typically found in the cores of
vertebral bones and the ends of long bones.
Author Manuscript
Author Manuscript
Author Manuscript
Unicortical defect
Author Manuscript
A fracture involving only the outer and/or inner cortices on one side of the bone shaft.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Fig. 1 |. Bone homeostasis.

Bone homeostasis is achieved through the activity of osteoblast lineage cells and osteoclast
lineage cells. Osteoblast lineage cells such as the osteoid (which is the unmineralized
portion of bone matrix) secrete hydroxyapatite and calcium to promote bone mineralization
and the formation of osteocytes. Osteoclast lineage cells resorb bone. The balance between
the activity of the two cell lineages results in bone homeostasis. HSC, haematopoietic stem
cell.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Fig. 2 |. Skeletal stem cell hierarchy.

Skeletal stem cells (SSCs) (and their progenitors) in mice and humans can be isolated on the
basis of distinctive immunophenotypic cell surface markers. a | Human SSC hierarchy
immunophenotypic profile, beginning with a human SSC at the apex, differentiating into a
human bone, cartilage and stromal progenitor and into its committed progenitors: human
cartilage progenitors, human osteoprogenitors and human stromal cells. b | Mouse SSC
immunophenotypic profile, beginning with a mouse SSC at the apex, differentiating into a
mouse bone, cartilage and stromal progenitor and into its committed progenitors: mouse
cartilage progenitors, mouse osteoprogenitors, mouse B lymphocyte stromal progenitors,
mouse 6C3 cells and mouse hepatic leukaemia factor-expressing cells.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Fig. 3 |. Long bone anatomy.

The metaphysis contains the epiphyseal growth plate, the site of new longitudinal bone
growth. This area can be categorized into five zones: the reserve, proliferative, hypertrophy,
Author Manuscript
calcification and ossification zones. The reserve zone contains quiescent chondrocytes found
towards the epiphyseal end of the bone. The proliferative zone contains chondrocytes that
undergo rapid proliferation. The hypertrophy zone contains chondrocytes that stop
proliferating and begin to undergo rapid growth. The calcified zone contains cells that begin
to undergo apoptosis and their matrix begins to calcify. The ossification zone contains
mature and terminally committed osteoblasts that help lay down mineralized bone.
Author Manuscript
Author Manuscript
Fig. 4 |. Developmental signalling pathways regulating osteoblast differentiation.

Various signalling pathways function in a coordinated manner to ensure appropriate bone
development and repair. a | Mesenchymal progenitors (MP) are initially marked by SOX9+
expression committing them to the osteochondroprogenitor lineage. Indian hedgehog (IHH)
binds to Smoothened homologue (SMO) to prevent the cleavage of GLI3 to GLI3 repressor
(GLI3R) and to activate GLI2 activator (GLI2A), thus leading to expression of SOX9 and
Runt-related transcription factor 2 (RUNX2) in osteochondroprogenitor cells. b | Notch
signalling is a negative regulator of osteoblast differentiation. Notch binding to Jagged
(JAG) or Delta-like protein (DLL) causes proteolytic cleavage of Notch, allowing Notch
intracellular domain (NCID) to interact with RBPJ and Mastermind-like protein 1
(MAML1) to affect the downstream targets Hairy and Enhancer of Split (HES) and HES-
related with YRPW motif (HEY), leading to the inhibition of osteoblast differentiation. c |
Author Manuscript
Canonical WNT signalling acts as a positive regulator of osteoblast differentiation. WNT

ligand binding to low-density lipoprotein receptor-related protein (LRP5) or LRP6 and
Frizzled (FZD) leads to the accumulation of β-catenin, thus allowing its translocation to the
nucleus to affect gene expression, including increasing the expression of RUNX2 and osterix
(OSX), which marks the commitment to mature osteoblasts. d | Bone morphogenetic protein
(BMP) signalling induces osteoblast differentiation. Binding of BMP2 or BMP4 leads to the
phosphorylation of SMAD1, SMAD5 or SMAD8. They form a complex with SMAD4 and
then enter the nucleus to control gene expression, allowing the transition of RUNX2+OSX+
cells to mature osteoblasts. e | Fibroblast growth factor (FGF) binds to cell surface tyrosine
kinase FGF receptors (FGFR1-FGFR4), leading to a cascade of intracellular signalling
events. FGF signalling controls preosteoblast proliferation, osteoblast differentiation and the
function of mature osteoblasts. BMPR-I, bone morphogenetic protein receptor type 1;
Author Manuscript
BMPR-II, bone morphogenetic protein receptor type 2; PKC, protein kinase C; PS,
presenilin; PTCH1, Patched homologue 1.
Author Manuscript
Author Manuscript
Fig. 5 |. Continuum of bone disorders.

Bone disorders are characterized by a gradient from ‘less bone’ to ‘too much bone’.
Beginning on the left side of the spectrum, the non-union fracture with fibrosis within the
gap indicates no bone is present. On appropriate healing, the osteotomy site is filled with a
new bone regenerate. Next, hip fracture results in a bicortical defect in the femoral head. A
greenstick fracture results in a unicortical defect on one side of the bone. In the middle is
bone homeostasis, which is represented by a physiologically normal bone. On the right end
of the spectrum is too much bone, which is represented by the pathology of heterotopic
ossification, wherein extraskeletal bone formation occurs.
Author Manuscript
Author Manuscript
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Table 1 |
Cre models applied for osteoblast lineage studies
Cre model Targeted cells Advantages Disadvantages Refs

Salhotra et al.
Sox9 SSCs, osteochondroprogenitor cells Overcomes embryonic lethality of Sox9 heterozygous mutant mice; Potential for off-target Cre-mediated recombination in 203
expressed throughout the lifetime of mice from the neonatal stage to old intestines and pancreas
age
Grem1 Osteochondroprogenitor cells, bone Expression of cells concentrated in metaphysis; distinct from Potential for off-target Cre-mediated recombination in 36
stromal cells mesenchymal stem cells intestines; low level of fibroblast CFUs (~1%) in in
vitro studies forself-renewal
Osx SSCs, osteoblast progenitor cells Expression is specific to osteoblasts; expression is associated with Unable to study precursor cells that have not 204
invading blood vessels during development committed to the osteoblast lineage fate
Prrxl SSCs Expressed throughout the lifetime of mice from the neonatal stage to Potential off-target effects around the eye 205
old age; presence of self-renewal properties for bulk cell culture in vitro
Sost Mature osteocytes Used a Sost gene within a BAC clone for better expression; no off- Potential off-target effects in haematopoietic cells and 206
target effects present in osteoblasts or myocytes osteoclasts
Pthrp (also SSCs, osteochondroprogenitor cells, Stromal contribution; expressed throughout the lifetime of mice from Involved in long-term maintenance of skeletal 42
known as bone stromal cells the neonatal stage to old age integrity; predilection for chondrogenesis over
Pthih) osteogenesis
Runx2 Mature osteoblasts Can be used to determine gene function in mature osteoblasts; robust Reporter leakage into cartilage cells 204
expression and recombination
Ctsk SSCs in the periosteum Can be used to determine the function of SSCs found in the periosteum Potential deletion of gene in germline cells 43
Hoxa11 SSCs in the periosteum Labelling of cells from development until adulthood allows cell lineage Hox11+ SSCs only mark a subset of the entire SSC 26,28,207
tracing population
BAC, bacterial artificial chromosome; CFU, colony-forming unit; Cre, Cre recombinase; SSC, skeletal stem cell.
Page 40
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Table 2 |
Transcription factors and protein-coding genes involved in osteoblast differentiation
Transcription factor or gene Effect Mode of action Refs

Salhotra et al.
Transcription factor
AP-1 Regulates osteoblast homeostasis Enhances RUNX2 expression 76
FIAT Reduces osteoblast activity Antagonizes ATF4 function 208
FOXO Increases osteoblast numbers Interacts with ATF4 209
GLI1 Stimulates osteoblast differentiation Enhances RUNX2 activity 92
GLI2 Stimulates osteoblast differentiation Enhances RUNX2 activity 210
GLI3 Suppresses osteoblast differentiation Inhibits DNA binding of RUNX2 211
HAND2 Inhibits intramembranous osteoblast differentiation in the mandible Inhibits RUNX2 212
HOX11 (also known as TLX1) Globally patterns the appendicular skeleton Enhances RUNX2 expression 28
HOXA2 Suppresses osteoblast differentiation Downregulates RUNX2 expression 213
OSX Stimulates osteoblast differentiation Interacts with MSX2 65
RUNX2 Stimulates osteoblast differentiation Acts as a scaffold regulatory factor involved in skeletal gene expression 214
TAZ Stimulates osteoblast differentiation RUNX2 co-activator 215
TWIST Inhibits osteoblast differentiation Inhibits RUNX2 216
Protein-coding gene
BGLAP Regulates bone remodelling Binds to apatite and calcium 217
COL1A1 Stimulates osteoblast differentiation Binds to calcium ion 218
DKK1 Negative regulator of bone growth Inhibits LRP5 or LRP6 interaction 219
PTK2 Stimulates osteoblast differentiation Integrin signal transduction 41,220
SOST Negative regulator of bone growth Inhibition of canonical WNT signalling 221
AP-1, activator protein 1; ATF4, activated transcription factor 4; FIAT, factor inhibiting ATF4-mediated transcription; FOXO, forkhead box protein O; LRP, low-density lipoprotein receptor-related protein;
OSX, osterix; RUNX2, Runt-related transcription factor 2.
Page 41

Nihms-1641529 Mechanisms of Bone Development and Repair

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Nihms-1641529 Mechanisms of Bone Development and Repair

Uploaded by

Copyright:

Available Formats

HHS Public Access

Published in final edited form as:

Mechanisms of bone development and repair

3Department of Surgery, University of Michigan, Ann Arbor, MI, USA.

highlighted the importance of gradients of morphogens, such as bone morphogenetic protein

development and disease. Although a certain degree of repair capacity is maintained

Cell types involved in osteogenesis

A fraction of these osteoblasts will undergo apoptosis, whereas a subset of osteoblasts

mechanical unloading, osteocytes express receptor activator of nuclear factor-κB ligand

Stem and progenitor cells

Mesenchymal stem cells

lineage specification moments during the differentiation process.

MSCs have been shown to maintain expression of genes characteristic of embryonic

Skeletal stem cells

skeletal component that heals through intramembranous ossification. During mandibular

Regulation of cell lineage specification

Embryonic origins of bones

Bone formation during embryogenesis occurs in two distinct processes: intramembranous

ossification or endochondral ossification. Intramembranous ossification begins with the

Furthermore, TABLE 2 highlights the transcription factors and protein-coding genes

SOX9.—SOX9, a member of the SRY-related high mobility group box-containing (SOX)

perturbs cartilage differentiation and inactivates chondrogenic marker expression, eventually

RUNX2.—RUNX2 is a transcription factor that drives skeletal development (TABLE 2).

2 (Ibsp) and osteocalcin 2 (Bglap2)57–59. In mice, Runx2 is strongly expressed in immature

OSX.—OSX, an osteoblast transcription factor, is required for normal skeletal development

abnormal cartilage accumulation, and osteocyte dysfunction along with a decrease in

In bones undergoing endochondral ossification, the heterogeneous population of immature

ATF4.—ATF4 is a leucine zipper-containing transcription factor and a component of the

ATF4 regulates osteoblast differentiation through post-transcriptional modification, enabling

Overexpression of FOS-related antigen 1 (FRA1), a FOS-related protein encoded by the

Signalling pathways in bone development

HH is a secreted protein that functions as a morphogen through a paracrine signalling

targets such as the GLI family of transcription factors84–86 (FIG. 4a).

In limb development, SHH functions as an important morphogen in anteroposterior

system, overexpression of Gli1 upregulated the transcription of early osteogenesis-related

Notch signalling.—Notch signalling requires direct cell-to-cell contact. Notch receptors

cell replication of the osteoblast lineage and suppresses osteoblast differentiation101,102. A

Canonical WNT signalling.—WNT ligands communicate by attaching to a cell surface

binding to the receptor Frizzled and low-density lipoprotein receptor-related protein 5

BMP signalling.—The TGFß family consists of a large group of structurally similar

Fgfr1 expression is downregulated in mouse SSCs; however, expression levels increase as

The role of the niche in bone repair

Dysregulation and clinical relevance

The opposite aspect of the spectrum is ‘excessive bone formation’, exemplified by

heterotopic ossification. Heterotopic ossification is defined as ectopic (extraskeletal) bone

Conclusions and perspectives

Understanding of the osteoblast lineage has significantly increased through identification of

2412–2424 (1999). [PubMed: 10500098]

Natl Acad. Sci. USA 112, 9920–9925 (2015). [PubMed: 26216955]

Annu. Rev. Cell Biol 4, 375–404 (1988). [PubMed: 3058162]

Tissue Res. 339, 189–195 (2010). [PubMed: 19649655]

2069 (2013). [PubMed: 23553905]

Trends Mol. Med 8, 340–345 (2002). [PubMed: 12114114]

collagen production. J. Cell Biol 190, 1093–1106 (2010). [PubMed: 20837772]

digit-patterning. Nature 452, 882–886 (2008). [PubMed: 18354396]

24898–24910 (2014). [PubMed: 25028519]

in the skeleton. Endocrinology 154, 623–634 (2013). [PubMed: 23275471]

Engl. J. Med 346, 1513–1521 (2002). [PubMed: 12015390]

cross-link maturation and material-level mechanical properties in mouse femoral trabecular

2235–2244 (1997). [PubMed: 9187149]

166. Lu LY et al. Pro-inflammatory M1 macrophages promote Osteogenesis by mesenchymal stem

ossification. Sci. Transl Med 10.1126/scitranslmed.aaf1090 (2016).

A cylindrical structure consisting of a mineralized matrix and osteocytes that transports