Biol. Rev. (2021), 96, pp. 357–375.

357
doi: 10.1111/brv.12659

New insights on the reparative cells in bone

regeneration and repair
Shuo Huang , Min Jin, Nan Su* and Lin Chen*
Department of Wound Repair and Rehabilitation Medicine, Center of Bone Metabolism and Repair, State Key Laboratory of Trauma, Burns and
Combined Injury, Trauma Center, Research Institute of Surgery, Daping Hospital, Army Medical University (Third Military Medical University),
10 Changjiang zhi Road, Yuzhong District, Chongqing, China

ABSTRACT

Bone possesses a remarkable repair capacity to regenerate completely without scar tissue formation. This unique char-
acteristic, expressed during bone development, maintenance and injury (fracture) healing, is performed by the reparative
cells including skeletal stem cells (SSCs) and their descendants. However, the identity and functional roles of SSCs remain
controversial due to technological difficulties and the heterogeneity and plasticity of SSCs. Moreover, for many years,
there has been a biased view that bone marrow is the main cell source for bone repair. Together, these limitations have
greatly hampered our understanding of these important cell populations and their potential applications in the treatment
of fractures and skeletal diseases. Here, we reanalyse and summarize current understanding of the reparative cells in bone
regeneration and repair and outline recent progress in this area, with a particular emphasis on the temporal and spatial
process of fracture healing, the sources of reparative cells, an updated definition of SSCs, and markers of skeletal stem/
progenitor cells contributing to the repair of craniofacial and long bones, as well as the debate between SSCs and peri-
cytes. Finally, we also discuss the existing problems, emerging novel technologies and future research directions in
this field.

Key words: skeletal stem cells, suture stem cells, mesenchymal stem cells, mesenchymal stromal cells, MSCs, SSCs, frac-
ture healing, bone repair, bone maintenance, craniofacial suture

CONTENTS

I.Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
II.Processes of fracture healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
III.Definition of skeletal stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
IV. Sources of cells for long bone repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
(1) Periosteum-, endosteum-, and bone marrow-derived cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .361
(2) Muscle-derived stem/progenitor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .362
(3) Cells from the circulatory system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .362
V. Sources of cells for craniofacial bone repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
VI. Sources of osteoblasts for bone maintenance and repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
VII. Markers of skeletal stem/progenitor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
(1) Prrx1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .364
(2) Ctsk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .364
(3) Gli1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .366
(4) Axin2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .367
(5) αSMA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .367
(6) Cxcl12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .367
(7) Mx1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .368

* Authors for correspondence (Tel: +0086 23 68757551; E-mail: linchen70@163.com); (Tel: +0086 23 68757553; E-
mail: sunansyd@163.com)

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358 Shuo Huang et al.

(8) Grem1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .368

(9) Sox9, Col2 and aggrecan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .368
VIII. The debate between pericytes and mscs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
IX. Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
X. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
. Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
XI. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371

I. INTRODUCTION and their descendants in skeletal development, maintenance

Bone possesses an extraordinary regeneration capacity
expressed during injury (fracture) repair, as well as during
bone development and continuous remodelling throughout
adult life (Dimitriou et al., 2011). In the clinical setting, the
most common form of bone regeneration is fracture healing.
Although most fractures can be cured through proper surgi-
cal reduction and fixation, approximately 10–15% of frac-
ture patients in the United States experience delayed union
or non-union due to various physiological (e.g. age and
smoking) and pathological conditions (e.g. concomitant
infections, osteoporosis, diabetes, and osteogenesis imper-
fecta) (Einhorn, 1995; Gruber et al., 2006; Kline et al., 2009;
Colnot, 2011; Abou-Khalil & Colnot, 2014). The success of
current therapies on such abnormal fracture healing is still
very limited (Colnot, 2011; Abou-Khalil & Colnot, 2014;
Senarath-Yapa et al., 2014). Thus, understanding the cellular
and molecular mechanisms of bone regeneration and repair
is important for the development of treatments and novel
therapeutic strategies.
Numerous studies have indicated that the processes of
bone repair are similar to those of bone development. The
two major categories of vertebral bones are the flat bones
in the skulls and the long bones that comprise most bones
in the axial and appendicular skeleton (Dirckx, Van Hul, &
Maes, 2013). All these bones are formed by two mechanisms:
long bones are formed following the formation and resorp-
tion of cartilaginous templates through a process called
endochondral ossification (EO), and the majority of craniofa-
cial bones develop through intramembranous ossification
(IO), in which stem cells differentiate directly into osteoblasts
that lay down the mineralized matrix (Senarath-Yapa
et al., 2014; Ono & Kronenberg, 2016). While bone injuries
are also repaired through EO and/or IO (Zhao et al., 2015;
Ono & Kronenberg, 2016), most pathways involved in bone
development are activated in the repair processes (Vortkamp
et al., 1998; Ferguson et al., 1999; Gerstenfeld et al., 2003),
including homeotic genes in pathways that control bone
development (Bais et al., 2009).
Bone regeneration and repair is mainly performed by the
reparative cells including skeletal stem cells (SSCs) and their
descendants in a definable spatiotemporal manner. SSCs
possess self-renewal capacity and can be activated rapidly
in response to the injury, differentiating into bone and carti-
lage (or bone only) within the bone callus that will go through
a remodelling process to be replaced by new bone tissue
(Figs 1, 2). However, the identity and functional roles of SSCs
and repair remain uncertain due to technological difficulties
and the heterogeneity and plasticity of SSCs. Moreover, for
many years, there has been a biased view that bone marrow
is the main cell source for bone repair. Such uncertainties
greatly hamper our understanding of these important cell
populations and their potential application in the treatment
of fractures and skeletal diseases.
II. PROCESSES OF FRACTURE HEALING
Craniofacial bones are derived from mesodermal and cranial
neural crest cells, whereas the appendicular skeleton is
derived from the lateral plate mesoderm and the axial skele-
ton is derived from the paraxial mesoderm (Senarath-Yapa
et al., 2014; Berendsen & Olsen, 2015). Fractures of craniofa-
cial bones heal through IO (Zhao et al., 2015; Ono &
Kronenberg, 2016) (Fig. 2). In long bones, microfractures
or mechanically stable fractures heal mainly through IO,
whereas the majority of severe fractures are repaired through
a combination of IO and EO, with the approach determined
mainly by the mechanical stability of the fracture site. For
example, after a complete transverse fracture of a long bone,
rigid fixation using fixators such as bone plates will result in
complete stability, and allow healing mainly through IO
(Bahney et al., 2015). If the fracture is not rigidly reduced to
enable some motion, such as fixation by an intramedullary
pin, or is not subjected to fixation, it heals mainly through
EO accompanied by IO in a location-specific manner
(Bahney et al., 2015) (Fig. 1). Along the periosteal surface of
the distal ends of the fractured bone (Fig. 1), where there is
no motion, periosteal cells form bones via IO, with a large
cartilaginous callus formed around the fracture gap (Fig. 1);
subsequently, bone tissue is formed through the more com-
plex process of EO (Bahney et al., 2015).
The processes of fracture healing normally include
three distinct but overlapping stages: the inflammatory stage,
the repair stage and the remodelling stage (DePalma
et al., 1972; Burchardt & Enneking, 1978) (Fig. 1). In the
inflammatory stage, upon injury, blood vessels, bone and
the surrounding soft tissues are disrupted, and a haematoma
develops at the fracture site within the first few hours to days
(Abou-Khalil & Colnot, 2014). The haematoma serves as an
important source of haematopoietic cells, neutrophils/
macrophages, and platelets that are involved in the initiation
of the inflammatory response (Mountziaris & Mikos, 2008)

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Reparative cells in bone regeneration and repair 359

Fig 1. Repair processes of an unstable long bone fracture. Unstable long bone fractures are normally repaired through both
intramembranous ossification (IO) and endochondral ossification (EO). Repair initiates after the inflammatory reaction, and
includes a soft and hard callus formation stage. In the inflammatory stage, upon injury, blood vessels, bone and the surrounding
soft tissues are disrupted, and a haematoma develops at the fracture site within the first few hours to days. The haematoma serves
as an important source of neutrophils, macrophages, and platelets that are involved in the initiation of the inflammatory response.
The outer layer of the periosteum gradually reconstitutes to contain the haematoma, while cells from the inner cambial layer
initiate and start to proliferate (periosteal reaction). The initial stage of repair begins from the periphery of the stable and well-
vascularized cortex bone at the distal ends of the fracture site, where cells from the inner layer of the lifted periosteum continually
condense, proliferate and differentiate into woven bone, resulting in the formation of a bone collar via IO. In the soft callus
formation stage, around the fracture gap, the mechanical instability of the fracture ends together with hypoxia of the local
microenvironment due to the disrupted blood supply lead to the formation of a cartilaginous mass. Chondrocytes progressively
mature into hypertrophic chondrocytes (HCs). HCs secrete collagen X in the transition zone, where cartilage will be replaced by
bone tissue and express a variety of active molecules that promote mineralization and vascular invasion into the callus from the
distal ends of the fracture site and the reconstituted periosteum at the outer margin of the callus. Along with angiogenesis,
osteoblasts derived from osteoprogenitors secrete the osteoid at the edges of cartilage tissues, leading to subsequent mineralization.
In the hard callus formation stage, the entire cartilage is continuously replaced by woven bones, and a bony framework is
eventually constructed across the fracture gap.

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360 Shuo Huang et al.

Fig 2. Maintenance and repair of craniofacial bone. Craniofacial bones are coated by the periosteum and dura/endosteum and
contain interspersed bone marrow. Sutures between craniofacial bones are composed of two osteogenic fronts with a strip of
mesenchyme between them. Specific postnatal suture stem cells (SuSCs) expressing glioma-associated oncogene 1 (Gli1), Axin2,
paired related homeobox 1 (Prrx1) and cathepsin K (Ctsk) reside within the suture mesenchyme and have been found to play a
predominant role in craniofacial bone maintenance and fracture repair in adult mice.

(Fig. 1). A variety of inflammatory cytokines, such as tumor
necrosis factor alpha (TNF-α), interleukin-1a (IL-1a), IL-1b,
IL-6, and IL-18 (Abou-Khalil & Colnot, 2014), and growth
factors, such as platelet derived growth factor (PDGF), vascu-
lar endothelial growth factor (VEGF), bone morphogenetic
proteins (BMPs), fibroblast growth factors (FGFs), transform-
ing growth factor beta (TGFβ), and insulin like growth fac-
tors (IGFs) (Ferguson et al., 1998; Gerstenfeld et al., 2003;
Schindeler et al., 2008), are released at the fracture site,
resulting in the formation of vascular tissue and the recruit-
ment of inflammatory cells and reparative cells that are
essential for bone regeneration and proper fracture healing
(Gerstenfeld et al., 2001; Kon et al., 2001; Cho, Gerstenfeld, &
Einhorn, 2002; Cho et al., 2007; Yang et al., 2007; Einhorn &
Gerstenfeld, 2015). Thereafter, the outer layer of the perios-
teum is gradually reconstituted to contain the haematoma,
while cells from the inner cambial layer initiate and start to
proliferate (periosteal reaction) (Shapiro, 2008) (Fig. 1).
In the repair stage, if the fracture heals through IO (includ-
ing craniofacial fracture), skeletal stem/osteoprogenitor cells
directly differentiate into osteoblasts and lay down the mineral-
ized matrix. In fractures healed through a combination of IO
and EO, the repair process includes the soft and hard callus for-
mation stage. The initial stage of repair begins in the periphery
of the stable and still well vascularized cortex bone at the distal
ends of the fracture site, where cells from the inner layer of the
lifted periosteum continually condense, proliferate and differ-
entiate into woven bone, which results in the formation of a
bone collar via IO (Bahney et al., 2015) (Fig. 1). In the soft callus
formation stage, around the fracture gap, the mechanical insta-
bility of the fracture ends together with hypoxia of the local
microenvironment due to a disrupted blood supply leads to
the formation of a cartilaginous mass (Fig. 1) that functions as
a ‘mechanical jack’ (Rot et al., 2014; Bahney et al., 2015). Under
the action of muscle contraction, cartilage in the callus is orga-
nized to act as a bidirectional growth plate, which in turn gen-
erates force in the opposing direction that drives the natural
reduction of the fractured bone (Rot et al., 2014). In the
cartilaginous callus, chondrocytes progressively mature into
hypertrophic chondrocytes (HCs). HCs secrete collagen X in
the transition zone (Fig. 1), where cartilage will be replaced
by bone tissue, and express a variety of active molecules,
such as VEGF (Gerber et al., 1999; Colnot & Helms, 2001;
Zelzer et al., 2004), placental growth factor (PIGF) (Maes
et al., 2006), BMP (Yu et al., 2010), matrix metalloproteinase
13 (MMP13) (Behonick et al., 2007) and alkaline phosphatase,
which promote mineralization as well as vascular invasion into
the callus from the distal ends of the fracture site and the recon-
stituted periosteum at the outer margin of the callus (Kurdy,
Weiss, & Bate, 1996; Hausman, Schaffler, & Majeska, 2001;
Bahney et al., 2015). Furthermore, HCs can also act as a
secondary source of osteoblasts (Dy et al., 2012; Yang
et al., 2014a; Zhou et al., 2014b), which will be discussed in
Section VI. In parallel with angiogenesis, osteoblasts derived
from osteoprogenitors secrete osteoid at the edges of cartilage
tissues with subsequent mineralization. In the hard callus for-
mation stage, the entirety of the cartilage is continuously
replaced by woven bones, and a bony framework is eventually
constructed across the fracture gaps (Kalfas, 2001) (Fig. 1).
In the remodelling stage, the bony callus continues to be
resorbed by osteoclasts, and secondary compact bone is laid
down by osteoblasts, which is termed coupled remodelling.
During this stage, the marrow space is re-established and
returns to its original structure as haematopoietic tissue.
The enhanced vascular flow rate gradually returns to pre-
injury levels, accompanied by extensive vascular remodelling
(Melnyk et al., 2008; Holstein et al., 2013). Finally, the healing
bone is gradually restored to its original shape, structure, and

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Reparative cells in bone regeneration and repair
mechanical strength (Einhorn & Gerstenfeld, 2015).
Although these processes take place consecutively, they over-
lap substantially and involve a continuum of changing cell
populations and signalling processes within the regenerating
tissue.
III. DEFINITION OF SKELETAL STEM CELLS
In the past, the stem cells regulating bone development, main-
tenance and repair were usually termed mesenchymal stem/
stromal cells (MSCs). MSCs are defined as multipotent stem
cells that have the potential to self-renew with maintained
properties and differentiate into a variety of specialized cell
types, such as osteoblasts, chondrocytes, adipocytes, myocytes
and neurons (Pittenger et al., 1999; Caplan & Bruder, 2001;
Sanchez-Ramos, 2002). Due to their heterogeneity, there is
no single specific marker that can be used to characterize
MSCs, and a panel of markers has been outlined by the Inter-
national Society for Cellular Therapy (ISCT) and widely
adopted by researchers. The ISCT defines MSCs as culture-
expanded cells that adhere to tissue culture plastic, retain
trilineage differentiation capability (bone, cartilage, and fat)
in vitro, express the surface markers cluster of differentiation
73 (CD73), CD90 and CD105, and lack expression of the
CD34, CD45, CD14, CD11b, CD79α/CD19 and human leu-
kocyte antigen-DR (HLA-DR) surface molecules (Dominici
et al., 2006). However, with the development of biotechnologies
such as in vivo lineage tracing and single-cell RNA sequencing
(scRNA-seq), a number of new markers have been proposed
to redefine/purify subpopulations of MSCs. Currently, it is
thought that distinct subpopulations of MSCs may have differ-
ent/overlapping phenotypes and functions. Based on scRNA-
seq data, mouse stem cells in bone tissues were redefined as
‘mouse skeletal stem cells’ (mSSCs) that possess the ability to
differentiate into osteoblasts, chondrocytes and stromal cells
and are positive for integrin alpha V but negative for CD45,
lymphocyte antigen-76 (Ly-76/Ter119), tyrosine-protein
kinase receptor TIE-2 (Tie2), CD90, Ly-51 (BP-1/6C3) and
CD105 (Chan et al., 2015; Gulati et al., 2018). Chan et al. (2018)
further redefined human skeletal stem cells (hSSCs) as multipo-
tent stem cells that can differentiate into bone, cartilage, and
stroma but not fat. hSSCs are present in fetal and adult bone
tissues and can also be obtained from induced pluripotent
stem cells or BMP2-treated human adipose stroma (Chan
et al., 2018). hSSCs are positive for podoplanin (PDPN),
CD73, and CD164 but negative for CD31, CD45, Tie2,
CD235a, and CD146 (Chan et al., 2018). These new definitions
and classification methods for SSCs have been adopted and
applied by many researchers (Debnath et al., 2018; Mizuhashi
et al., 2018; Ransom et al., 2018).
Five recent papers analysed the transcriptional landscape
of mouse non-haematopoietic bone marrow cells using
scRNA-seq technology and computationally identified many
cell clusters corresponding to distinct cell types or stages of
differentiation. Pseudotemporal cell trajectory analysis based
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361
on the known expression of key transcription factors pre-
dicted the cell state hierarchy in vivo. Data from two of these
papers supported the classic concept that MSCs/SSCs pos-
sess the ability to differentiate into osteoblasts, chondrocytes
and adipocytes via three hierarchical differentiation trajecto-
ries in vivo (Wolock et al., 2019; Baccin et al., 2020). Data from
the other three papers showed that MSCs/SSCs might only
maintain the production of osteogenic- and adipogenic-
lineage cells through bilineage differentiation pathways dur-
ing the steady-state differentiation process in vivo (Tikhonova
et al., 2019; Matsushita et al., 2020; Zhong et al., 2020). These
findings refine our understanding of the cellular architecture
of bone marrow cells, however, the inherent differentiation
potential of SSCs is still controversial, and the definition of
SSCs needs to be further refined.
IV. SOURCES OF CELLS FOR LONG BONE
REPAIR
During long bone fracture healing, reparative cells could be
recruited to the fracture site from both local tissues and the
circulatory system. Local tissues include the periosteum, end-
osteum, bone marrow, and surrounding soft tissues in close
proximity to the bone, such as tendon, fat and muscle
(Colnot, 2011). Cells obtained from systemic recruitment
include: (i) endothelial cells derived from blood vessels; (ii)
stem/progenitor cells that exist in or are mobilized to the cir-
culatory system after injury; (iii) pericytes; and (iv) monocyte/
macrophage-lineage cells. The first three types possess the
ability to differentiate/transdifferentiate into chondrocytes
and osteoblasts (Sempuku et al., 1996; Pittenger et al., 1999;
Lee et al., 2000; Zheng et al., 2006; Bi et al., 2007; Liu, Schin-
deler, & Little, 2010; Wosczyna et al., 2012; Shah et al., 2013);
thus, it is hard to distinguish which sources of cells predomi-
nantly contribute to the fracture callus.
(1) Periosteum-, endosteum-, and bone marrow-
derived cells
The periosteum is a highly vascularized connective tissue lining
the outer surface of bone. It is a bilayer membrane comprising:
(i) the outer fibrous layer, which consists of fibroblasts, collagen,
elastic fibres and microvessels (Popowics, Zhu, & Herring, 2002;
Allen, Hock, & Burr, 2004) that provide the attachment points
for tendons, ligaments and muscles and is thought to serve
mainly a structural and mechanical role (Yiannakopoulos
et al., 2008); and (ii) the inner layer, which is also known as the
cambium layer and consists of smaller and more isodiametric
fibroblasts, sympathetic nerves, osteoblasts and periosteum-
derived SSCs (Ozaki et al., 2000; Allen et al., 2004; Zhang
et al., 2005; Colnot, 2009; Colnot, Zhang, & Knothe
Tate, 2012; Matthews et al., 2014; Yukata et al., 2014).
It is generally accepted that cells from the periosteum form
the fracture callus and play the predominant role in long
bone fracture healing (Zhang et al., 2005; Xie et al., 2008;
362
Colnot, 2009; Kawanami et al., 2009; Yu et al., 2010), while
cells from the periosteum, endosteum, and bone marrow
might collaborate with each other to form the new cortex bone
connecting the graft and the host cortex (Colnot, 2009). Evi-
dence has shown that periosteum activation occurs very rap-
idly within 24–48 h in response to fracture, which closely
coincides with local cell proliferation (Iwasaki, Le, &
Helms, 1997). Mechanical disruption of the periosteum delays
healing (Utvag, Grundnes, & Reikeraos, 1996; Guichet
et al., 1998; Ozaki et al., 2000).
Although there is currently a lack of periosteum-specific
cyclization recombination enzyme (Cre) recombinants for
lineage tracing, Colnot (2009) compared in detail the distinct
contributions to fracture healing of the periosteum, endos-
teum, and bone marrow using tibia unicortical bone graft
transplantation approaches. Live bone grafts from geneti-
cally labelled mice (Rosa26) containing periosteum and/or
endosteum were transplanted into the fracture site of the
wild-type host (Colnot, 2009). The results revealed that peri-
osteal cells are the major contributors to the fracture callus,
giving rise to osteoblasts and osteocytes in a stabilized frac-
ture and to chondrocytes, osteoblasts and osteocytes in a
non-stabilized fracture. Bone marrow and endosteal cells
always give rise to osteoblasts and osteocytes at the endosteal
surface and within the bone marrow cavity (Colnot, 2009).
Moreover, periosteal cells have the potential to differentiate
into cartilage and bone in the environment of the bone mar-
row cavity, while cells from the endosteum and bone marrow
mainly differentiate into bone but do not support cartilage
formation in the environment of the periosteum during
non-stabilized fracture healing (Colnot, 2009). These data
suggest that the distinct contributions to fracture healing of
periosteum-, endosteum-, and bone marrow-derived cells
might not be related to extrinsic differences in their environ-
ment but to intrinsic cellular differences among these tissues.
(2) Muscle-derived stem/progenitor cells
Skeletal muscles and bone are closely linked and coordinated
to enable locomotion and posture control (Shah et al., 2013).
Previous studies have demonstrated that muscle-derived
stem/progenitor cells are capable of forming bone tissues
(Liu et al., 2010; Wosczyna et al., 2012; Shah et al., 2013),
and ectopic bone formation can be induced in muscle by
injection of BMPs (Tachi et al., 2011; Wosczyna
et al., 2012). However, using myogenic differentiation
(MyoD) conditional reporter mice (MyoD-Cre+:Z/AP+),
Liu et al. (2011) revealed that the intact periosteum might
act as a physical barrier to prevent the involvement of
muscle-derived stem/progenitor cells in fracture healing. In
a mouse tibial defect model or closed tibial fracture model,
which produces limited damage to the adjacent soft tissues
while leaving the periosteum largely intact, no or a minimal
number of MyoD-lineage cells were found in the fracture
callus. In a tibial open fracture model, MyoD-lineage cells
participated in all fracture healing stages; however, the pro-
portion of MyoD-positive cells was lower in areas away from
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Shuo Huang et al.
the initial fracture site and decreased with the repair process
(Liu et al., 2011). Therefore, muscle might only contribute to
bone fracture healing as an alternative source of cells when
the periosteum is disrupted.
(3) Cells from the circulatory system
It was reported that endothelial cells are able to transdiffer-
entiate into chondrocytes and osteoblasts during heterotopic
ossification (Medici et al., 2010). Endothelial cells were found
in increased numbers in the circulatory system following
fracture. However, a genetic lineage-tracing approach
proved that endothelial cells, which were represented by
Tie2-expressing cells, do not appear to transdifferentiate into
cartilage or bone within the fracture callus and make no
direct contribution to the fracture callus (Lu, Marcucio, &
Miclau, 2006; Wang et al., 2011b). Therefore, there might
be a fundamental difference between heterotopic ossification
and fracture healing (Liu et al., 2011).
Blood vessels carry cells from various sources systemically
to the fracture site. To evaluate the contribution of systemic
recruitment of skeletal stem/progenitor cells during bone
repair, Taguchi et al. (2005) systemically injected green fluo-
rescent protein (GFP)-labelled bone marrow cells into wild-
type mice, and found that GFP-expressing cells in the frac-
ture callus were not incorporated into the new bone as oste-
ocytes. Another study observed systemic mobilization and
recruitment of osteoprogenitors to the fracture site via the
peripheral circulation (Shirley et al., 2005), but these cells
do not seem to differentiate into osteocytes in new bone.
Our previous studies also showed that systemic transplanted
allogeneic MSCs are able to migrate into the fracture site,
but few were found to participate in new bone formation
(Huang et al., 2015a; Huang et al., 2015b). Therefore,
the exact contribution of systemically recruited skeletal
stem/progenitor cells remains to be determined.
Pericytes are closely associated with blood vessels. They
can either be activated locally or transported from distant
tissues to the injury site and might participate in bone repair
(da Silva Meirelles, Caplan, & Nardi, 2008; Grcevic
et al., 2012) (Fig. 1). However, the contribution of pericytes
is still controversial (see Section VIII).
Monocyte/macrophage-lineage cells are crucial cell
sources for bone repair. It was reported that macrophages
can be recruited from the circulatory system to the fracture
site, where they undergo phenotypic and functional changes
to coordinate repair (Wynn & Vannella, 2016). Depletion of
macrophages delays fracture healing (Vi et al., 2015; Alexan-
der et al., 2017). Heterochronic parabiosis experiments
revealed that macrophages from old mice slowed down the
healing process in young mice, while young macrophages
or their secretions [e.g. low-density lipoprotein receptor-
related protein 1 (Lrp1)], could rejuvenate fracture repair
in old mice (Vi et al., 2018). Evidence has also shown that
osteoclast precursors could migrate to the bone injury site
through the blood circulation and begin to participate in
Reparative cells in bone regeneration and repair
the remodelling of bone on day 7 following bone injury in
mice (Yahara et al., 2020).
Thus, systemically circulating cells recruited to the bone
injury site are mainly macrophages and osteoclast precursors
in the monocyte/macrophage lineages; systemically
recruited skeletal stem/progenitor cells are few, and their
contribution remains to be determined.
V. SOURCES OF CELLS FOR CRANIOFACIAL
BONE REPAIR
Craniofacial bones are covered by the periosteum and dura/
endosteum and contain interspersed bone marrow
(Robey, 2011; Yang, Zhang, & Gangolli, 2014b; Zhao
et al., 2015) (Fig. 2). The craniofacial periosteum was
reported to contain stem cells and osteoprogenitors (Kanou
et al., 2005; Zhang et al., 2005; De Bari et al., 2006; Brey
et al., 2007) and therefore was thought to share the same turn-
over and repair mechanisms as those of the long bone perios-
teum (Ochareon & Herring, 2011; Pagni et al., 2012).
However, recent evidence has shown that the craniofacial
periosteum might have distinct skeletal progenitors and bio-
logical characteristics compared to those of the long bone
periosteum (Leucht et al., 2008; Lin et al., 2014; Wilk
et al., 2017). Sutures between craniofacial bones might play
a predominant role in craniofacial bone development, main-
tenance and repair (Mao & Prockop, 2012; Zhao et al., 2015;
Maruyama et al., 2016; Wilk et al., 2017).
Sutures are composed of two osteogenic fronts with a strip
of mesenchyme between them (Fig. 2). Most sutures in mice
remain patent throughout their lifetime, whereas human cra-
nial sutures normally fuse in adults between 20 and 30 years
old, and facial sutures fuse after 50 years old (Badve
et al., 2013; Zhao et al., 2015). Utilizing the bone transplanta-
tion method, Zhao et al. (2015) proved that sutures rather than
craniofacial periosteum and dura possess regeneration capac-
ity. They transplanted a piece of GFP-labelled calvarial bone
containing the sagittal suture but without the periosteum and
dura into nude mice. After 1 month, the transplanted suture
vigorously generated new GFP-labelled periosteum, dura
and bones in the transplant (Zhao et al., 2015), while transplan-
tation of calvarial bone containing intact periosteum and dura
but without sutures failed to grow (Zhao et al., 2015). Further
studies have shown that the midline of the suture mesenchyme
contains a distinctive stem cell population known as suture
stem cells (SuSCs) (Fig. 2), which possess long-term self-
renewing, colony-forming and multidifferentiation abilities
in vitro and can give rise to osteogenic fronts, periosteum, dura
and bone during natural bone turnover in vivo (Ouyang
et al., 2013; Zhao et al., 2015; Maruyama et al., 2016; Wilk
et al., 2017). When a craniofacial injury occurs, SuSCs are rap-
idly activated within 24 h and migrate into the injury site to
proliferate and differentiate into osteocytes embedded in the
bone plate, directly contributing to craniofacial bone repair
(Zhao et al., 2015; Maruyama et al., 2016) (Fig. 2).
Biological Reviews 96 (2021) 357–375 © 2020 Cambridge Philosophical Society
363
These results reveal that sutural cells rather than periosteal
cells play the predominant role in craniofacial bone repair,
which might be related to differences in the populations of
skeletal stem/progenitor cells. Even so, the craniofacial
periosteum is rich in blood vessels that not only deliver sig-
nals, nutrients and oxygen but also transfer suture-derived
reparative cells to the injury site; thus, they might still play
important roles in craniofacial bone repair.
VI. SOURCES OF OSTEOBLASTS FOR BONE
MAINTENANCE AND REPAIR
Normally, osteoblasts have three possible fates: (i) to become
osteocytes embedded in mineralized bone matrix; (ii) to
undergo programmed cell death; or (iii) to transform into
quiescent bone lining cells (BLCs) that are capable of being
reactivated and contributing to mature osteoblasts during
modelling/remodelling (Hock et al., 2001; Franz-Odendaal,
Hall, & Witten, 2006) (Fig. 1). Genetic pulse-chase experi-
ments using Ocn-CreER;R26-YFP mice revealed that
mouse mature osteoblasts might be short-lived non-
replicative cells (Park et al., 2012). Mature osteoblasts labelled
by osteocalcin (Ocn) turned over rapidly and were continu-
ously replaced by other cell populations in 6- to 8-week-old
mice. The number of Ocn+ endosteal and periosteal osteo-
blasts reached a peak 14 days after induction, followed by a
significant decrease of 50% by day 30, with the number grad-
ually decreasing to the basal level by day 60 in both femurs
and calvaria. By contrast, bone-embedded Ocn-labelled
osteocytes peaked in number at day 30 after induction and
then dropped over time (Park et al., 2012). These results
suggest that the half-life of mouse mature osteoblasts is
only
30 days in vivo; thus they need to be continuously
replenished during bone maintenance and repair.
Osterix (Osx) is expressed earlier than osteocalcin in the oste-
oblast lineage, and is considered a marker of pre-osteoblasts
(osteogenic precursors) (Rodda & McMahon, 2006). Osx-
labelled cells were reported to be the origin of trabecular osteo-
blasts and osteocytes in developing bones (Maes et al., 2010).
Using a genetic pulse-chase method similar to that described
above, Park et al. (2012) found that the average number of
Osx-labelled cells gradually increased over 30 days following
induction but then decreased to the basal level after
90 days
in both femurs and calvaria, implying that Osx-labelled cells
were not durable and did not represent a self-renewing pool
of osteoprogenitors.
BLCs are post-mitotic flat osteoblast-lineage cells covering
non-remodelling (quiescent) bone surfaces and are character-
ized by their morphology and location without specific
markers (Miller et al., 1989; Matic et al., 2016) (Fig. 1). BLCs
can be induced to proliferate and/or differentiate into mature
osteoblasts by parathyroid hormone (PTH), FGF2, inhibition
of sclerostin or high doses of γ-radiation (Dobnig &
Turner, 1995; Kim et al., 2012; Turner et al., 2013; Delgado-
Calle & Bellido, 2017). BLCs display gene expression profiles
364
different from those of osteoblasts, such as increased levels of
macrophage colony-stimulating factor (M-CSF), receptor acti-
vator of nuclear factor-κ B ligand (RANKL), and MMP13,
and share some of the surface markers of MSCs, such as Ly-
6A/E (Sca1), CD51 and leptin receptor (Lepr) (Matic
et al., 2016; Matthews et al., 2016). Matic et al. (2016) discov-
ered by accident using dentin matrix protein 1 (Dmp1)
reporter mice (Dmp1-CreERT;R26-tdTomato) that Dmp1
could label BLCs, mature osteoblasts (Col2.3-GFP+) and oste-
ocytes. To remove mature osteoblasts from Dmp1+ cells, they
crossed Dmp1-CreERT;R26-tdTomato mice with Col2.3Δtk
mice, after which Col2.3-GFP+ cells could be selectively elim-
inated by 16 days of ganciclovir (GCV) treatment (Visnjic
et al., 2001). After ablation, osteoblasts were absent from the
bone surface, and calcein labelling was minimal; however,
after 21 days of recovery, rapid bone formation appeared,
and the number of Dmp1+ osteoblasts and osteocytes embed-
ded in the bone dramatically increased in both cortical and
trabecular bones, suggesting the replenishment by BLCs of
mature osteoblasts (Matic et al., 2016). Therefore, BLCs might
be an alternative to SSCs as a source of ‘predetermined’ oste-
ogenic precursors contributing to bone formation in
adult mice.
HCs are generally considered to be a postmitotic cell type,
and they undergo programmed cell death through either apo-
ptotic or autophagic mechanisms (Ahmed et al., 2007; Kroe-
mer et al., 2009; Tsang et al., 2014). However, it has recently
been reported that HCs are able to re-enter the cell cycle
and might undergo asymmetric cell division (Roach, Eren-
preisa, & Aigner, 1995; Roach & Erenpreisa, 1996), giving rise
to two daughter cells, one of which undergoes apoptosis,
whereas the other survives and subsequently transdifferenti-
ates into osteoblasts (Dy et al., 2012; Yang et al., 2014a; Zhou
et al., 2014b) or dedifferentiates into progenitor-like cells
(Song & Tuan, 2004; Bahney et al., 2014). Moreover, trans-
planted cartilage grafts can participate in bone repair
(Bahney et al., 2014), and genetically labelled HCs in donor
graft cartilage can become osteoblasts and osteocytes (Yang
et al., 2014a; Tsang, Chan, & Cheah, 2015). Therefore, HCs
might be an important source of osteoblasts during long bone
development and fracture healing through EO.
In summary, mouse mature osteoblasts are considered to
be short-lived non-replicative cells, and they can be replen-
ished by BLCs, HCs, and skeletal stem/progenitor/precur-
sor cells during bone maintenance and repair.
VII. MARKERS OF SKELETAL STEM/
PROGENITOR CELLS
The identities and functional roles of skeletal stem/
progenitor cells remain unclear or controversial due to: (i)
the multiple sources of cells that intrinsically or extrinsically
(are induced to) possess trilineage differentiation capability;
(ii) the heterogeneity of SSCs/MSCs; (iii) the fact that most
previous studies did not distinguish between bone marrow-
Biological Reviews 96 (2021) 357–375 © 2020 Cambridge Philosophical Society
Shuo Huang et al.
and periosteum-derived cells, and bone marrow cells were
usually used for cell identification in vitro; and (iv) the lack of
well-accepted cell surface markers for cell identification.
Below, we summarize some populations of skeletal stem
and/or progenitor cells involved in craniofacial and/or long
bone repair with a focus on their specific markers, spatiotem-
poral distribution, stemness, and contributions to bone main-
tenance and repair (summarized in Table 1).
(1) Prrx1
Paired related homeobox 1 (Prrx1; also known as MHox or
Prx1) is usually acknowledged as a marker of SSCs in both
craniofacial and long bones. Prrx1 expression is restricted
to the early limb bud mesenchyme during embryonic devel-
opment in mice (Ono & Kronenberg, 2016). In adult mice,
Prrx1-expressing cells are localized immediately outside of
collagen type I alpha 1 (col1a1)-expressing osteoblasts in
the cambium layer of the long bone periosteum (Ouyang
et al., 2013), and very few are localized in the endosteum
and bone marrow (Kawanami et al., 2009). Prrx1 is also
expressed in SuSCs (Ouyang et al., 2013; Wilk et al., 2017),
tendon cells (Ouyang et al., 2013) and in the precursors of
white adipocytes (Sanchez-Gurmaches et al., 2015) but not
in skeletal muscles (Logan et al., 2002; Sanchez-Gurmaches
et al., 2015). Both calvarial suture- and periosteum-derived
Prrx1+ cells could give rise to chondrocytes and osteoblasts
in vitro (Kawanami et al., 2009; Ouyang et al., 2013; Wilk
et al., 2017). Mice with deletion of Prrx1 show limb shorten-
ing, craniofacial defects, and incompletely penetrant spina
bifida (Martin & Olson, 2000). Prrx1 also inhibits adipogen-
esis by activating TGFβ signalling (Sanchez-Gurmaches
et al., 2015). During long bone fracture healing, which is
mainly exerted through EO, Prrx1+ cells derived from the
periosteum could give rise to cartilage and bone in the frac-
ture callus (Kawanami et al., 2009; Wang et al., 2017;
Duchamp de Lageneste et al., 2018), while Prrx1+ cells in
the sutures contribute to calvarial bone defect healing
through IO (Wilk et al., 2017).
(2) Ctsk
Cathepsin K (Ctsk), a lysosomal cysteine protease, was origi-
nally recognized as a marker of mature osteoclasts both in vivo
and in vitro (Lotinun et al., 2013; Bonnet et al., 2017; Panwar
et al., 2017). Ctsk plays important roles in bone resorption
and remodelling through the degradation of matrix proteins;
inactivation or deletion of Ctsk suppresses bone resorption
(Lotinun et al., 2013; Bonnet et al., 2017; Panwar
et al., 2017). Yang et al. (2013) identified a new type of Ctsk+
cells within Ranvier’s groove and termed them Ctsk+ chon-
droid progenitors (CCPs). Conditional knockout of tyrosine
phosphatase 2 (SHP2) in Ctsk+ cells resulted in multiple
osteochondromas and enchondromas, implying that Ctsk+
cells in Ranvier’s groove might possess the functional proper-
ties of skeletal stem/progenitor cells (Yang et al., 2013). Most
recently, Debnath et al. (2018) found that Ctsk not only labels
Reparative cells in bone regeneration and repair
Table 1. Markers of skeletal stem/progenitor cells in adult mice
Target cells and their locations in
Markers
adult mice
Long bones
Prrx1 SSCs in the periosteum, endosteum
and bone marrow, precursors of
white adipocytes, tendon cells, not
in skeletal muscles
Ctsk SSCs in the periosteum, endosteum,
bone marrow and Ranvier’s
groove, osteoprogenitors,
osteoblasts, osteocytes#, mature
osteoclasts
Gli1 SSCs in the periosteum, bone
marrow* and Ranvier’s groove,
cells in the chondro-osseous
junction immediately beneath the
growth plate, chondroprogenitors*
in the upper layers of the growth
plate, cells in articular cartilage,
incisors and muscles
αSMA/Acta2 SSCs in the periosteum* and bone
marrow, periodontal and sutural
cells, satellite cells, adipose stem
cells, vSMCs, pericytes
Cxcl12 Pericytes, vSMCs and CAR cells in
the bone marrow, adipogenic
precursors, not osteoblasts or
osteocytes
Mx1 SSCs in the periosteum and
endosteum, mature osteoblasts,
osteoclasts
Mx1 + αSMA SSCs in the periosteum and calvarial
sutures, nearly undetectable in
bone marrow
Grem1 Periosteal cells*, OCR stem cells in
bone marrow, not in the
perisinusoidal space
Sox9, Col2, Skeletal stem/progenitor cells in the
aggrecan periosteum and bone marrow, cells
in the growth plate and articular
cartilage
Lepr Skeletal stem/progenitor cells in the
periosteum* and bone marrow,
CAR cells, adipogenic precursors,
pericytes
Craniofacial bones
Gli1 SuSCs in the suture mesenchyme
(after P21)
Cell lineages involved in bone
maintenance and repair
Periosteal and endosteal cells,
BMSCs, osteoblasts, osteocytes,
chondrocytes (in fracture callus)
Periosteal and endosteal cells,
BMSCs, osteoblasts, osteocytes,
chondrocytes (in fracture callus),
osteoclasts
Periosteal cells, BMSCs, muscle cells,
adipocytes, osteoblasts, osteocytes,
chondrocytes
Periosteal* and endosteal* cells,
BMSCs, muscle cells*, osteoblasts,
osteocytes, chondrocytes (in
fracture callus)
BMSCs, adipocytes in the bone
marrow, osteoblasts and osteocytes
in the trabecula during bone
maintenance, osteoblasts and
osteocytes in the callus and newly
formed cortical bone during
fracture healing
Periosteal and endosteal cells,
BMSCs, osteoblasts, osteocytes,
but not chondrocytes
Periosteal and sutural cells,
osteoblasts, osteocytes and
chondrocytes* (in fracture callus)
Periosteal cells, reticular marrow
stromal cells, chondrocytes (in the
growth plate and articular
cartilage), diaphyseal osteoblasts,
osteocytes, but not adipocytes
Periosteal cells, BMSCs,
chondrocytes, osteoblasts,
osteocytes
BMSCs, adipocytes in the bone
marrow, osteoblasts and osteocytes
in the trabecula during bone
maintenance; osteoblasts,
osteocytes and chondrocytes in the
callus and osteocytes in newly
formed cortical bone during
fracture healing
Periosteal, sutural and endosteal/
dural cells, osteoblasts, osteocytes
Biological Reviews 96 (2021) 357–375 © 2020 Cambridge Philosophical Society
365
References
Kawanami et al. (2009); Ouyang
et al. (2013); Logan et al. (2002);
Sanchez-Gurmaches, Hsiao, &
Guertin (2015); Wang, Zhang, &
Bikle (2017); Duchamp de
Lageneste et al. (2018)
Lotinun et al. (2013); Panwar
et al. (2017); Bonnet et al. (2017);
Yang et al. (2013); Debnath
et al. (2018)
Shi et al. (2017); Hojo et al. (2012);
Zhao et al. (2015)
Morikawa et al. (2002); Nehls,
Denzer, & Drenckhahn (1992);
Baryawno et al. (2019); Kalajzic
et al. (2008); Grcevic et al. (2012);
Matthews et al. (2014), Matthews
et al. (2016); Roguljic et al. (2013)
Sugiyama et al. (2006); Matsushita
et al. (2020); Coutu et al. (2017);
Zhong et al. (2020)
Walkley et al. (2007); Park et al. (2012),
Park et al. (2014)
Ortinau et al. (2019)
Worthley et al. (2015); Mendez-Ferrer
et al. (2010); Zhou et al. (2014a);
Debnath et al. (2018)
He et al. (2017); Shintaku et al. (2011);
Akiyama et al. (2005); Ono
et al. (2014); Debnath et al. (2018)
Zhou et al. (2014a); Baryawno
et al. (2019); Baccin et al. (2020)
Zhao et al. (2015); Shi et al. (2017)
366 Shuo Huang et al.

Table 1. (Cont.)

Target cells and their locations in Cell lineages involved in bone

Markers
adult mice
Axin2 SuSCs in the midline of almost all
calvarial sutures,
chondroprogenitors in the resting
zone of the growth plate and the
outermost layer of the growth plate
facing Ranvier’s groove
Prrx1 SuSCs in the suture mesenchyme
Ctsk SuSCs in the suture mesenchyme;
cells in the periosteum,
endosteum/dura and bone
marrow*
References
maintenance and repair
Periosteal* and sutural cells, Maruyama et al. (2016); Usami
osteoblasts, osteocytes, et al. (2019)
chondroprogenitors and
chondrocytes (in the growth plate)
Sutural cells, osteoblasts, osteocytes Ouyang et al. (2013); Wilk et al. (2017)
Periosteal, sutural and endosteal/ Debnath et al. (2018)
dural cells, BMSCs, osteoblasts,
osteocytes, osteoclasts

Prrx1, Paired related homeobox 1; Ctsk, Cathepsin K; Gli1, Glioma-associated oncogene 1; αSMA, Alpha smooth muscle actin; Cxcl12, C-
X-C motif chemokine 12; Mx1, Myxovirus resistance 1; Grem1, Gremlin 1; Sox9, Sex-determining region Y (SRY)-box 9; Col2, collagen
type II; Lepr, leptin receptor; SSCs, skeletal stem cells; BMSCs, bone marrow mesenchymal stem/stromal cells; vSMCs, vascular smooth
muscle cells; OCR, osteochondroreticular; CAR, Cxcl12-abundant reticular; SuSCs, suture stem cells.
*Expression
#
is not mentioned in the papers referenced but can be inferred from their figures or single-cell RNA sequencing data.
Rare osteocytes express Ctsk.

osteoblasts, osteoprogenitors and osteoclasts in calvarial
sutures, periosteum, endosteum and bone marrow but also
labels SSCs in the periosteum of long bones and sutures of
the calvarium. Non-haematopoietic Ctsk+ periosteum/
suture cells contain three main subpopulations: periosteal
stem cells (PSCs: CD31− CD45− Ter119− 6C3− CD90−
CD49flow CD51low CD105− CD200+) and periosteal pro-
genitors 1 and 2 (PP1: CD31− CD45− Ter119− 6C3−
CD90− CD49flow CD51low CD105− CD200−; PP2:
CD31− CD45− Ter119− 6C3− CD90− CD49flow CD51low
CD105+ CD200variable) (Debnath et al., 2018). PSCs are at
the top of the Ctsk+ cell differentiation hierarchy and can
give rise to all the other Ctsk+ populations of periosteum/
suture cells, while PP1 and PP2 do not give rise to PSCs
(Debnath et al., 2018). In addition, PSCs are able to self-
renew and differentiate into osteoblasts and chondrocytes
in vivo and in vitro and still retain these abilities even after serial
rounds of transplantation into the kidney capsule and mam-
mary fat pad (Debnath et al., 2018). PSCs can differentiate
into bone and cartilage in the fracture callus and contribute
to long bone fracture healing (Debnath et al., 2018). Notably,
multipotent PSCs also exist in the periosteal tissue of human
femurs (Debnath et al., 2018).
(3) Gli1
Glioma-associated oncogene 1 (Gli1), a transcription factor
and an effector of the hedgehog pathway, identifies MSCs
in many tissues of adult mice, such as craniofacial bones, long
bones and incisors (Hojo et al., 2012; Zhao et al., 2015; Shi
et al., 2017). Utilizing Gli1-CreERT2;R26-tdTomato mice,
Shi et al. (2017) discovered that during embryonic long bone
development, Gli1 is predominantly expressed in the peri-
chondrium, including the layers covering the joint surface,
Ranvier’s groove, and the bone shaft where osteoblasts first
form. In postnatal mice, Gli1 is mainly expressed in five
domains of long bones: the periosteum, articular cartilage,
upper layers of the growth plates, perichondrium flanking the
growth plate, and the chondro-osseous junction immediately
beneath the growth plate; it is not expressed in mature osteo-
blasts, osteocytes and bone marrow (Shi et al., 2017). After
administrations of tamoxifen to Gli1-CreERT2;R26-tdTomato
mice at 1 month of age, lineage tracing of Gli1+ cells was carried
out over the following 1, 3, and 9 months. tdTomato (Gli1+)
could label certain periosteal cells, osteoblasts and osteocytes
of cortical bone and the remaining bone trabeculae as well as
growth plate chondrocytes. It was noted that tdTomato also
labelled many adipocytes, as shown by the identification of
perilipin- and Lepr-positive cells in bone marrow (Shi
et al., 2017). Moreover, to investigate the contribution of Gli1+
cells to long bone fracture healing, Gli1-CreERT2;
R26-tdTomato mice were injected with tamoxifen when they
were 4 weeks old, subjected to semistabilized fracture at
10 weeks old, and killed after 10 days. Gli1-lineage cells were
detected throughout the fracture callus, including in both
bone and cartilage, indicating that Gli1 might be a marker of
periosteal SSCs in long bones in postnatal mice.
Zhao et al. (2015) reported that Gli1 is expressed within the
entire craniofacial periosteum, dura and suture mesenchyme
but not in the fontanelles and osteocytes on postnatal day
0 (P0), P7, and P14. After P21, Gli1 expression is gradually
restricted to the mesenchyme of most craniofacial sutures
and to the inner surface of some bone marrow spaces (Zhao
et al., 2015). Lineage-tracing results showed that when
Gli1-CreERT2;R26-tdTomato mice were induced with
tamoxifen at 1 month of age, 2 months later, tdTomato
labelled the entire suture mesenchyme, osteogenic fronts,
periosteum, dura and parts of the calvarial osteocytes that
were located either next to the suture or on the external sur-
face of the craniofacial bones (Zhao et al., 2015). Gli1+ suture
cells normally do not undergo active division; however, they
could be rapidly activated within 24 h after craniofacial bone

Biological Reviews 96 (2021) 357–375 © 2020 Cambridge Philosophical Society

Reparative cells in bone regeneration and repair
injury (Zhao et al., 2015). One month after induction with
tamoxifen, tdTomato labelled the periosteum, dura and
many osteocytes in the repair region, indicating that Gli1+
cells contribute to injury repair in craniofacial bones (Zhao
et al., 2015). Moreover, ablation of Gli1+ cells in adult
Gli1-CreERT2;DTA mice led to skull growth arrest, osteo-
porosis, craniosynostosis and impaired craniofacial bone
defect healing (Zhao et al., 2015). Overall, Gli1+ cells in post-
natal mice contribute to maintenance and repair in both long
bones and craniofacial bones.
(4) Axin2
Axin2 inhibits canonical wingless-type MMTV integration
site (Wnt)/T cell specific transcription factor (TCF) signalling
by enhancing the formation of the beta-catenin destruction
complex. By utilizing Axin2-GFP mice, Maruyama
et al. (2016) found that Axin2 expression is restricted to the
midline of almost all calvarial sutures, including the patent
posterior frontal suture, and is colocalized with slow-cycling
cells that retained 5-ethynyl-20 -deoxyuridine (Edu). This
highly specific pattern of expression was first detected at P9
and was maintained in adult mice. By contrast, Gli1+ cells
in craniofacial bones might target a larger area within the
sutures and be retained in the periosteum and dura at an
early stage (Zhao et al., 2015; Maruyama et al., 2016). For
lineage-tracing analysis, Axin2-Cre-Dox;R26RLacZ mice
were administered doxycycline for 3 days starting at P28
and analysed after 1, 3, and 12 months. The results showed
that Axin2+ labelled all patent sutures and calvarial bones
but was not detectable in the osteoblasts and osteocytes in
long bones, suggesting that there was very little contribution
of Axin2+ cells to the homeostasis of long bones (Maruyama
et al., 2016). Further analysis showed that Axin2+ suture cells
possess long-term self-renewal, clonal expansion and differ-
entiation abilities in vitro (Maruyama et al., 2016). Moreover,
in response to calvarial injury, Axin2+ suture cells could
move into the damage site and then differentiate into osteo-
cytes embedded in the bone plate, directly contributing to
calvarial injury healing (Maruyama et al., 2016). By contrast,
there was a very limited contribution of Axin2+ cells to the
long bone fracture repair (Maruyama et al., 2016).
Recently, using Axin2-CreERT2;R26-ZsGreen mice,
Usami et al. (2019) identified two new Axin2+ cell populations
as chondroprogenitors: one population in the resting zone of
the central part of the growth plate in postnatal mice, and the
other localized to the outermost layer of the growth plate fac-
ing Ranvier’s groove (the perichondrium adjacent to the
growth plate). Lineage-tracing results showed that the first
population of Axin2+ cells contributed to the formation of
the central part of the growth plate columns in the growth
stage, and ablation of β-catenin in these Axin2+ cells strongly
inhibited their expansion and induced ectopic chondrogen-
esis in the growth plate (Usami et al., 2019). The second pop-
ulation could give rise to the outer growth plate during the
neonatal stage, but their growth was paused when the growth
plate stopped growing, and these cells were thought to be
Biological Reviews 96 (2021) 357–375 © 2020 Cambridge Philosophical Society
367
involved in the lateral growth of the growth plate (Usami
et al., 2019). In summary, these findings suggested that Axin2
might be an ideal marker of SuSCs and chondroprogenitors
in the growth plate.
(5) αSMA
Alpha smooth muscle actin (αSMA, also known as Acta2) was
reported to label 0.02–0.03% of bone marrow cells, approx-
imately 1% of scraped periosteal cells (Grcevic et al., 2012),
SuSCs, satellite cells in muscles, and MSCs in adipose tissue
and periodontium, and these cells possess self-renewing and
dual/multiple-lineage differentiation potential (Kalajzic
et al., 2008; Grcevic et al., 2012; Roguljic et al., 2013; Mat-
thews et al., 2014; Matthews et al., 2016). αSMA is not only
expressed in pericytes localized to venules (Nehls
et al., 1992; Morikawa et al., 2002; Baryawno et al., 2019),
but also present in vascular smooth muscle cells (vSMCs) of
larger blood vessels (Kalajzic et al., 2008; Baryawno
et al., 2019). Grcevic et al. (2012) found that αSMA+ bone
marrow cells have the ability to differentiate into mature
osteoblasts, chondrocytes and adipocytes in vitro. However,
most of these cells express the haematopoietic lineage
markers CD11b, CD45 and Ter119; αSMA+ (CD11b−
CD45− Ter119−) bone marrow cells are mostly negative
for the mesenchymal lineage markers Sca1, CD51, CD90,
CD106, CD140b and CD146 (Grcevic et al., 2012). By con-
trast, the majority of αSMA+ periosteal cells are negative
for CD11b, CD45, and Ter119, but αSMA+ (CD11b−
CD45− Ter119−) periosteal cells express higher levels of
Sca1, CD51, CD90, and CD140b than bone marrow cells
(Grcevic et al., 2012). Using αSMA-CreERT2;
R26-tdTomato mice for lineage tracing, results showed that
during tibia fracture healing, αSMA+ cells are able to expand
and differentiate towards chondrogenic and osteogenic line-
ages (Grcevic et al., 2012; Matthews et al., 2014; Wang
et al., 2017) and contribute to new cortical bone formation
6 weeks following the fracture (Grcevic et al., 2012; Matthews
et al., 2016), which indicates that αSMA may label periosteal
SSCs of long bones in postnatal mice.
(6) Cxcl12
C–X–C motif chemokine 12 (Cxcl12) is also known as stro-
mal cell-derived factor 1 (SDF1). Cxcl12-abundant reticular
(CAR) cells are the major producers of Cxcl12 in mouse bone
marrow (Sugiyama et al., 2006). CAR cells can be found in
the periosteum and endosteum and are widely scattered in
adult bone marrow surrounding the sinusoids but Cxcl12
are barely detected in osteoblasts and osteocytes (Tokoyoda
et al., 2004; Sugiyama et al., 2006; Matsushita et al., 2020).
CAR cells usually colocalize with haematopoietic stem cells
(HSCs) and play important roles in the maintenance of the
HSC pool (Sugiyama et al., 2006). Recently, using
Cxcl12-CreERT;R26-tdTomato mice carrying either
Col12.3-GFP or Cxcl12-GFP reporters, Matsushita
et al. (2020) identified a population of quiescent perisinusoidal
368
Cxcl12-CreERT+ bone marrow MSCs (BMSCs). Although
these cells displayed little colony-forming activity, they pos-
sessed trilineage differentiation potential in vitro and could
contribute to the formation of trabecular bone and fat in
the bone marrow cavity but did not participate in cortical
bone formation during bone maintenance in vivo
(Matsushita et al., 2020). However, these cells partly contrib-
uted to newly formed bone in the callus and cortical bone in
both the cortical injury model and the tibial complete frac-
ture model. Matsushita et al. (2020) suggested that these cells
might transform into osteoblast precursor cells in a manner
mediated by canonical Wnt signalling in response to bone
injury. We found that Lepr+ cells might display similar char-
acteristics according to fate-mapping data from Lepr-cre;
R26-tdTomato;Col2.3-GFP mice (Zhou et al., 2014a).
Therefore, Cxcl12 might be a marker of particular popula-
tions of skeletal stem/progenitor cells in some circumstances.
(7) Mx1
Myxovirus resistance 1 (Mx1) was originally reported to be
expressed in bone marrow stromal cells, cranial stem/pro-
genitor cells, a fraction of osteocalcin (Ocn)-positive mature
osteoblasts, haematopoietic-derived osteoclasts, and perios-
teal (inner cambial) and endosteal cells in growing and adult
mice (Walkley et al., 2007; Park et al., 2012; Park et al., 2014).
Chondrocytes, adipocytes, skeletal muscle cells, fibroblasts,
osteocytes and blood vessels were not labelled by Mx1
(Park et al., 2012). In vivo pulse-chase results showed that
Mx1+ cells could differentiate into osteoblasts, osteocytes,
bone marrow cells, and periosteal and endosteal cells but
not chondrocytes (Park et al., 2012). Mx1+ cells could also
respond to tissue stress and migrate to fracture sites, supply-
ing new bone tissue during microfracture healing of the cal-
varium and metaphysis. Therefore, Mx1 was identified as a
marker of osteogenic progenitor cells.
Recent findings show that Mx1 and αSMA in combination
can selectively label postnatal periosteal stem cells (P-SSCs)
and calvarial SuSCs but are nearly undetectable in bone
marrow (Ortinau et al., 2019). Mx1+aSMA+ periosteal cells
possess SSC characteristics in vitro and are able to form colo-
nies with chondrogenic, osteogenic and adipogenic differen-
tiation abilities at the single-cell level (Ortinau et al., 2019).
Mx1+αSMA+ P-SSCs/SuSCs could respond rapidly and
migrate towards the injury site. Mx1+αSMA+ P-SSCs repo-
pulated the outer layer of the callus, generated new perios-
teum, and formed the majority of callus by supplying
osteoblasts and chondrocytes following tibial transverse frac-
ture (Ortinau et al., 2019). Mx1+αSMA+ SuSCs at the top
surface of the injury maintained their undifferentiated status
and formed a new periosteum, while Mx1+αSMA+ cells in
the middle of the callus differentiated into Mx1+aSMA−
osteoblasts and osteocytes embedded in newly formed bone
plates after 21 days during calvarial defect healing (Ortinau
et al., 2019). Therefore, Mx1 and αSMA in combination
might label SSCs in both long bones and craniofacial bones.
Biological Reviews 96 (2021) 357–375 © 2020 Cambridge Philosophical Society
Shuo Huang et al.
(8) Grem1
Gremlin 1 (Grem1), a secreted antagonist of BMP2, -4, and
-7 and a VEGFR2 agonist (Merino et al., 1999; Khokha
et al., 2003; Canalis, Parker, & Zanotti, 2012), is critical for
osteogenesis. Grem1 null mice displayed decreased mass
and body fat and a shortened femoral length (Canalis
et al., 2012). They also exhibited developmental skeletal
abnormalities with incomplete formation of the forelimbs,
hindlimbs and metatarsal bones (Canalis et al., 2012). Worth-
ley et al. (2015) reported that Grem1 could label a population
of osteochondroreticular (OCR) stem cells in the embryonic
and postnatal bone marrow. To trace Grem1 lineages
in vivo, Grem1-CreERT;R26-tdTomato mice were orally
administered tamoxifen at 6–8 weeks of age. After approxi-
mately 12 months of induction, Grem1+ cells differentiated
into columns of chondrocytes in the growth plate, articular
cartilage, periosteal cells, reticular marrow stromal cells,
diaphyseal osteoblasts, and osteocytes but not in adipocytes
in either the vertebral bodies or the femur (Worthley
et al., 2015). Grem1+ cells also have the ability to self-renew
in vivo and in vitro and are tripotent, differentiating into bone,
cartilage, and reticular marrow stromal cells in both embry-
onic and postnatal mice (Worthley et al., 2015). Gene-
expression microarray analysis of Lin−(CD31− CD45−
Ter119−)Grem1+ and Lin−Grem1− mesenchymal cells
revealed that Lin−Grem1+ cells were significantly more
active in BMP signalling as well as extracellular matrix
(ECM)-receptor interactions, phosphatidylinositol 3-kinase
(PI3K)-serine/threonine kinase Akt (Akt) signalling, and
focal adhesion pathways, which might be correlated with
the osteochondral lineage potential of Lin−Grem1+ cells
(Worthley et al., 2015). To investigate the contribution of
Grem1+ cells to fracture healing, adult Grem1-creERT;
R26-TdTomato;2.3ColGFP mice were administered tamox-
ifen with a 1-week washout period. Mice were killed at 7 days
following unilateral femoral fracture. The results showed that
Grem1+ cells could generate osteoblasts and chondrocytes in
the callus during fracture healing through EO (Worthley
et al., 2015). These data indicate that Grem1 might label
SSCs in long bones.
(9) Sox9, Col2 and aggrecan
Sex-determining region Y (SRY)-box 9 (Sox9), collagen type
II (Col2) and aggrecan (Agc) are markers of chondrocytes.
However, recent lineage-tracing experiments using a
tamoxifen-inducible form of Cre (CreERT) system revealed
that early postnatal cells at P3 labelled by Sox9-CreERT,
Col2-CreERT and Agc-CreERT progressively contributed
to the formation of chondrocytes, osteoblasts, osteocytes
and bone marrow stromal cells for at least 6 months in mice
(Ono et al., 2014). In addition, Col2-CreERT-labelled cells
could become adipocytes in the metaphyseal bone marrow
(Ono et al., 2014). Conditional gene manipulation using
Col2-Cre affected both cartilage and bone (Ford-Hutchinson
et al., 2007; Wang et al., 2011a). Another study reported that
Reparative cells in bone regeneration and repair
Sox9 was expressed in the primary spongiosa, periosteum
and endosteum of adult long bones (He et al., 2017). In a fem-
oral fracture model, Sox9-CreERT;R26-tdTomato mice
were given three consecutive doses of tamoxifen 2 weeks
before fracture, and lineage-tracing results showed that at
9 days postfracture, Sox9+-lineage cells differentiated into
large numbers of chondrocytes, osteoblasts and osteocytes
in the callus (He et al., 2017). However, using the same
Sox9-CreERT mice but with different tamoxifen induction
schedules for lineage tracing, Kuwahara et al. (2019)
observed different results. When Sox9-CreERT;
R26-tdTomato mice were injected with tamoxifen for three
consecutive days starting 7 days before large-scale rib re-
section surgery, at 10 days post resection, only approxi-
mately 22 ± 1.3% of tdTomato-labelled cells contributed
to both cartilage and bone in the callus (Kuwahara
et al., 2019). However, when these mice were induced for
4 consecutive days starting the day before the surgery, at
10 days post resection, a much greater portion of the callus
was labeled by tdTomato (85.5 ± 2.8%), including the newly
formed cartilage, bone and periosteum surrounding the cal-
lus (Kuwahara et al., 2019). Deletion of the obligate hedgehog
co-receptor, Smoothened, in Sox9+ cells before surgery
resulted in a near-complete loss of rib bone regeneration abil-
ity (Kuwahara et al., 2019). The latter authors speculated that
Sox9-expressing periosteal cells (termed Sox9+ messenger
cells) might play a major role in orchestrating large-scale
bone regeneration by stimulating neighbouring cells to dif-
ferentiate through an unknown signal into osteochondropro-
genitors. We suggest that Sox9+ periosteal cells might
represent primed SSCs/transit-amplifying cells/osteochon-
droprogenitors. In summary, the above results imply that
Col2, aggrecan and Sox9 might be considered markers of
skeletal stem/progenitor cells in long bones in some
circumstances.
VIII. THE DEBATE BETWEEN PERICYTES
AND MSCS
Pericytes are perivascular multipotent cells that show hetero-
geneity in different organs or even within the same tissue
(Nirwane, Gautam, & Yao, 2017). Pericytes do not have a
precisely defined phenotype; instead, they are recognized
by their phenotype, distribution and origin (Armulik, Gen-
ove, & Betsholtz, 2011). Pericytes reside on the abluminal
surface of smaller calibre vessels of every vascularized con-
nective tissue, whereas vSMCs are found around larger ves-
sels (Friedenstein et al., 1974). It is generally accepted that
pericytes and vSMCs belong to the same lineage and cate-
gory, and there are no specific markers to distinguish peri-
cytes from vSMCs (Armulik et al., 2011). Pericytes are
believed to function as MSCs with the capacity to differenti-
ate into osteoblasts, chondrocytes, fibroblasts, adipocytes
(Nehls & Drenckhahn, 1993) and myogenic cells in vitro
(Crisan et al., 2008). In addition, pericytes exist in the
Biological Reviews 96 (2021) 357–375 © 2020 Cambridge Philosophical Society
369
periosteum and bone marrow, and were reported to contrib-
ute to callus formation during bone repair (Diaz-Flores
et al., 1992). In bone marrow, there are two types of pericytes
expressing two different surface markers, Lepr and nerve/
glial antigen 2 (NG2/Cspg4), which are generally associated
with sinusoids and arterioles, respectively (Kunisaki
et al., 2013; Coutu et al., 2017; Kunisaki, 2019; Liu, Li, &
Tan, 2019a). Both Lepr+ and NG2+ pericytes express stem
cell factor (SCF/Kitl), Cxcl12 and Nestin (Kunisaki
et al., 2013; Asada et al., 2017; Coutu et al., 2017), all of which
were reported as markers of bone marrow MSCs (Ding &
Morrison, 2013; Greenbaum et al., 2013; Kunisaki, 2019).
However, contradictory data hinted that pericytes might
not or may only partly possess the characteristics of MSCs/
SSCs in vivo. Zhou et al. (2014a) fate-mapped Lepr+ cells in vivo
using Lepr-Cre;R26-tdTomato;Col2.3-GFP mice and found
that Lepr+ cells made little contribution to stromal cells and
bone in early postnatal bone marrow, but they increased in
number over time and became the source of osteoblasts
and adipocytes in adult bone marrow. However, as discussed
in Section VII-(6), both Cxcl12+ and Lepr+ cells only partly
contribute to cortical bone formation during bone repair,
but do not participate in cortical bone maintenance. Utiliz-
ing Nes-CreERT;R26-EGFP mice, Mendez-Ferrer
et al. (2010) found that Nestin+ cells in bone marrow exhib-
ited spontaneous multilineage differentiation ability in cul-
ture and robust self-renewal ability after serial
transplantation in vivo. Lineage-tracing results showed that
Nestin+ cells did not give rise to bone-forming cells after a
1-month chase but differentiated into chondrocytes in the
growth plate, osteoblasts, and osteocytes after an 8-month
chase. However, Ono et al. (2014) reported that when Nes-
CreERT;R26-tdTomato mice received tamoxifen at P3,
Nestin+ cells predominantly contributed to the formation of
endothelial cells in bone marrow after a 1-month chase, only
a small number of Nestin+ cells became osteoblasts and oste-
ocytes in the cortical bone and very infrequently became
chondrocytes in the growth plate, but they never differenti-
ated into adipocytes in bone marrow. Zhou et al. (2014a)
revealed that Nestin+ cells from different Nestin transgenic
mice displayed different characteristics in bone marrow.
Nestin+ cells from Nestin-GFP mice possessed colony-
forming capacity in vitro, but Nestin+ cells from Nestin-
CreERT;R26-EYFP mice did not, and a lineage-tracing
experiment also confirmed that Nestin-CreERT lineages
rarely contributed to the formation of osteoblasts and bone
in vivo after 9 months of tamoxifen induction (Zhou
et al., 2014a). Guimaraes-Camboa et al. (2017) found that
T-box transcription factor 18 (Tbx18) could selectively label
pericytes and vSMCs in multiple organs of adult mice.
Fluorescence-activated cell sorting (FACS)-purified
Tbx18-expressing cells behaved as MSCs in vitro. However,
lineage-tracing experiments using an inducible
Tbx18-CreERT2 cell line indicated that pericytes and
vSMCs remained identical in various pathological settings
and during ageing but did not contribute significantly to
other cell lineages (Guimaraes-Camboa et al., 2017).
370
Recently, two studies used scRNA-seq technology to ana-
lyse and compare MSCs and pericytes in periosteal, endos-
teal and bone marrow stromal cells of mouse limb bones,
but resulted in almost diametrically opposed conclusions.
Based on the ‘consensus’ that Lepr is a marker of adult bone
marrow MSCs (Zhou et al., 2014a), Baryawno et al. (2019)
annotated one cell population as Lepr+ mesenchymal stromal
cells (referred to as Lepr-MSCs) with adipogenic and osteo-
genic features and another distinct population of perivascular
mesenchymal stromal cells and pericytes (referred to as peri-
cytes) according to their gene expression profiles. Lepr-MSCs
highly expressed Lepr and adiponectin (Adipoq), which is
highly expressed in preadipocytes (Lara-Castro et al., 2007),
and the key HSC niche factors SCF, Cxcl12 and
angiopoietin-1 (Angpt1) (Baryawno et al., 2019). They also
expressed Grem1 and Runx2, which is the master regulator
transcription factor controlling the commitment of MSCs to
osteolineages, as well as the classical MSC markers CD73,
CD105 and CD106 (Vcam1), but they did not express
CD90, Sca1, NG2, Nestin and αSMA (Baryawno
et al., 2019). The annotated pericyte population expressed
the classical pericyte markers NG2, Nestin, αSMA, myosin
heavy chain 11 (Myh11), and melanoma cell adhesion mole-
cule (Mcam) (Armulik et al., 2011) but only expressed very
low levels of Lepr, in contrast to Lepr-MSCs (Baryawno
et al., 2019).
In the second paper, Baccin et al. (2020) annotated Ng2-
and Nestin-expressing mesenchymal cells in Lin− CD45−
CD71− periosteal, endosteal and bone marrow stromal cells
of mouse limb bones as Ng2+ MSCs, which showed tran-
scriptomic similarity to previously described Ng2+Nestin+
MSCs [and were annotated as the ‘pericyte’ population by
Baryawno et al., 2019]. Moreover, they annotated another
two populations with similar overall transcriptomic profiles
as Adipo-CAR and Osteo-CAR cells that expressed adipo-
cyte and osteolineage genes, respectively (Baccin
et al., 2020). Most Adipo-CAR cells localized abundantly to
sinusoidal endothelia, and Osteo-CAR cells localized to
non-vascular regions or covered arteriolar endothelia
(Baccin et al., 2020). Both CAR cell populations produced
high levels of cytokines and growth factors involved in HSC
maintenance and differentiation (Baccin et al., 2020). Inter-
estingly, the Adipo-CAR cells expressed Lepr and Adipoq
at high levels and showed strong transcriptomic similarity
to Lepr-Cre cells (Baccin et al., 2020) [annotated as ‘Lepr-
MSCs’ in Baryawno et al., 2019]. Both Adipo-CAR cells
and Ng2+ MSCs express NG2, Nestin, Cxcl12 and SCF;
however, Adipo-CAR cells express higher levels of Cxcl12
and SCF and lower levels of Nestin than Ng2+ MSCs but
do not express Grem1 (Baccin et al., 2020). Furthermore,
pseudo-time analysis results showed that Ng2+ MSCs are at
the apex of a differentiation hierarchy with CAR cells, fibro-
blasts, chondrocytes and osteoblasts downstream (Baccin
et al., 2020).
It is interesting that three other studies used the scRNA-
seq technique to identify a similar cell cluster expressing high
levels of Lepr, Cxcl12 and many adipocyte markers in mouse
Biological Reviews 96 (2021) 357–375 © 2020 Cambridge Philosophical Society
Shuo Huang et al.
bone marrow cells, and this cluster was annotated as MSCs
(Wolock et al., 2019; Matsushita et al., 2020; Zhong
et al., 2020). It is still hard to distinguish which cell popula-
tions are true MSCs/pericytes based on these opposing
views, however, these results indicate that MSCs and peri-
cytes might represent different cell types. Moreover, both
populations are still heterogeneous according to their gene
expression profiles, and the homogeneous subsets need to
be further extracted and identified.
IX. FUTURE PROSPECTS
Current knowledge reveals the unique spatiotemporal distri-
bution of SSCs/MSCs in the periosteum, bone marrow, and
suture mesenchyme as well as the growth plates and Ran-
vier’s groove in the embryonic and postnatal period and
the lineage-specific contributions of these cells during bone
development, maintenance and fracture repair. However,
much remains to be confirmed due to inherent biases associ-
ated with experimental assays and conceptual controversy.
New technologies will help us to refine our understanding
and overcome the limitations of the technologies currently
in use. The mineral composition, macro- and microarchitec-
ture, remodelling capacity and biomechanics of bone in large
animals such as sheep, pigs and monkeys are similar to those
of humans, and the availability of clustered regularly inter-
spaced short palindromic repeat (CRISPR)-mediated
genome editing in these large animals will provide us with
better opportunities to study the cellular and molecular
mechanisms of bone development, diseases, and injury/
regeneration that are more similar to those in humans
(Truong et al., 2019; Sparks et al., 2020). The dual-recombi-
nase-activated lineage-tracing platform (DeaLT) that com-
bines the D6 site-specific DNA recombinase-rox
recombination site (Dre-rox) and Cre recombinase-locus of
crossover in P1 (Cre-loxP) recombination system could partly
overcome the limitations of Cre-mediated lineage tracing,
such as the specificity of Cre and off-target labelling
(He et al., 2018; Li et al., 2018). The cellular barcoding tech-
nique, in which individual cells are labelled with unique
nucleic acid sequences (termed barcodes), could be used for
fate mapping, lineage tracing and high-throughput screening
of millions of cells in parallel and thus is an efficient approach
for investigating heterogeneous populations of SSCs
(Kebschull & Zador, 2018). The stochastic multicolour label-
ling technique (Brainbow) that labels individual cells with
multiple fluorescent proteins (Livet et al., 2007; Cai
et al., 2013; Sakaguchi, Leiwe, & Imai, 2018) could also be
used for investigating the heterogeneity of SSCs. If such a
technique is combined with tissue-clearing techniques such
as CUBIC (clear, unobstructed brain/body imaging cock-
tails and computational analysis) (Susaki et al., 2015) and
SHANEL (small micelle-mediated human organ efficient
clearing and labeling) (Zhao et al., 2020), researchers could
comprehensively map cells in the whole body of mice or large
Reparative cells in bone regeneration and repair
intact human organs at the cellular level. Intravital bone-
imaging technology using multiphoton microscopy is a novel
and powerful tool that will enable us to visualize the spatio-
temporal cellular behaviours of SSCs and their offspring in
their natural tissue microenvironment (Liu, Yuan, &
Zhang, 2019b). scRNA-seq will further deepen our aware-
ness of distinct SSC populations and their niches, interrela-
tionships and cellular and molecular mechanisms
(Baryawno et al., 2019; Tikhonova et al., 2019; Wolock
et al., 2019; Baccin et al., 2020; Matsushita et al., 2020). The
utilization of these new technologies in future studies will
help us increase our understanding of the mechanisms under-
lying the development, maintenance, degeneration, injury
and regeneration of the skeleton, leading to the development
of novel therapies to treat bone diseases and promote bone
repair.
X. CONCLUSIONS
(1) We summarize in detail the temporal and spatial pro-
cesses of injury (fracture) repair in both craniofacial
and long bones.
(2) We discuss the definition of MSCs and SSCs, as well as
their inherent differentiation potential.
(3) We describe newly discovered populations of skeletal
stem/progenitor cells involved in craniofacial and/or
long bone repair with a specific focus on their markers,
spatiotemporal distribution, stemness, and contribu-
tions to bone maintenance and repair.
(4) We compare the contributions of reparative cells from
multiple sources to bone maintenance and repair, and
also summarize the debate between the roles of
MSCs/SSCs and pericytes.
(5) We discuss the existing problems, emerging novel tech-
nologies and future research directions in this field.
ACKNOWLEDGEMENTS
This work was supported by research funding from the
National Key Research and Development Program of
China (2018YFA0800802), the National Natural Science
Foundation of China (No. 81530071, No. 81772359,
No. 81601906) and the Chongqing Postdoctoral Researchers
Research Project (Xm2016084).
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