For submission to BONE
Uncovering the Periosteum for Skeletal Regeneration: The Stem
Cell That Lies Beneath
Scott J. Roberts1,2,3#, Nick van Gastel2,4#, Geert Carmeliet2,4 and Frank P. Luyten1,2*
1Skeletal
Biology and Engineering Research Center, KU Leuven, O&N 1 Herestraat
49 bus 813, 3000 Leuven, Belgium.
2Prometheus,
Division of Skeletal Tissue Engineering, KU Leuven, O&N 1 Herestraat
49 bus 813, 3000 Leuven, Belgium.
3Institute
of Orthopaedics and Musculoskeletal Science, Division of Surgery &
Interventional Science, University College London, The Royal National Orthopaedic
Hospital, Stanmore, Middlesex, HA7 4LP, UK.
4Clinical
and Experimental Endocrinology, KU Leuven, O&N 1 Herestraat 49 bus 902,
3000 Leuven, Belgium.
# These authors contributed equally to this work
*Corresponding Author: Skeletal Biology and Engineering Research Center, O&N1
Herestraat 49 bus 813, 3000 Leuven, Belgium
Tel: +32 16 34 25 45
Fax: +32 16 34 62 00
Email: frank.luyten@uzleuven.be
Keywords: Periosteum, Stem Cells, Bone Development, Fracture Repair, Tissue
Engineering
1
Abstract
The cartilage- and bone-forming properties of the periosteum have long since been
recognized. As one of the major sources of skeletal progenitor cells, the periosteum
plays a crucial role not only in bone development and growth, but also during bone
fracture healing. Aided by the continuous expansion of tools and techniques, we are
now starting to acquire more insight into the specific role and regulation of periosteal
cells. From a therapeutic point of view, the periosteum has attracted much attention
as a cell source for bone tissue engineering purposes. This interest derives not only
from the physiological role of the periosteum during bone repair, but is supported by
the unique properties and marked bone-forming potential of expanded periosteumderived cells. We provide an overview of the current knowledge of periosteal cell
biology, focusing on the cellular composition and molecular regulation of this
remarkable tissue, as well as the application of periosteum-derived cells in
regenerative medicine approaches.
2
1. Introduction
The osteogenic potential of the periosteum, a thin vascular membrane that covers the
external surface of bone except for the articular surfaces of the long bones, was
described for the first time in 1742 by Henri-Louis Duhamel du Monceau[1]. His
findings were later confirmed by Louis Xavier Ollier, who showed that upon treatment
of bone fractures the integrity of the periosteum must be retained to achieve
successful healing[2].
Subsequently, research has revealed that the periosteum consists of two layers (Fig.
1): an outer fibrous layer containing fibroblasts dispersed in between collagen fibers,
and an inner cambium layer which contains skeletal progenitor cells and osteoblasts,
and is highly vascularized and innervated[3]. The periosteum serves as an
attachment site for tendons, ligaments and muscles. In addition, periosteal blood
vessels deliver 70-80% of the blood supply to the bone cortex[4]. Furthermore,
progenitor cells in the cambium layer continuously give rise to osteoblasts to allow
appositional bone growth as well as cortical bone modeling and remodeling in
concert with osteoclasts. The astounding potential of the periosteum is however
mainly revealed after bone fracture, when periosteal progenitor cells undergo an
impressive expansion, followed by differentiation into osteoblasts and chondrocytes,
but predominantly the latter. This process forms the cartilaginous fracture callus that
undergoes hypertrophy, followed by replacement by bone and eventual remodeling
to restore the original shape of the bone[5, 6]. This remarkable property of the
periosteum has elicited extensive research into the use of periosteum-derived cells
for regenerative approaches, and preclinical studies demonstrating the potential of
these cells in the treatment of non-healing bone fractures and large bone defects are
becoming available.
3
In this review, we summarize recent findings expanding our insight into the cellular
composition and molecular regulation of the periosteum, and provide an overview of
regenerative medicine approaches using periosteum-derived cells, as well as clinical
attempts to mimic the periosteum for improved fracture healing.
2. The periosteum during skeletal development, growth and aging
Developmental origin of the periosteum
Embryonic bone formation can proceed through either the intramembranous or the
endochondral
pathway.
During
intramembranous
ossification,
mesenchymal
stem/progenitor cells will condense and differentiate into osteoprogenitor cells
expressing runt-related transcription factor 2 (RUNX2) and osterix (OSX), which in
turn give rise to osteoblasts, the mature bone-forming cells[7]. During this process,
undifferentiated progenitor cells remain at the bone periphery, forming the
periosteum. These cells will drive further appositional growth of the bones by giving
rise to new osteoblasts[8]. The formation of an entire bone by the direct differentiation
of progenitors to osteoblasts is rather the exception and is responsible for the
formation of only a few bones, such as the flat bones of the skull. In contrast, most
bones develop through endochondral ossification, a process in which initially a
cartilage template is laid down, that is later replaced by bone[9]. More precisely,
during the development of the long bones, mesenchymal stem/progenitor cells
expressing paired-related homeobox gene 1 (PRX1) will condense and give rise to
chondrocytes, which express the transcription factor sex determining region Y box 9
(SOX9)[10, 11], in the inner part of the condensation, while at the periphery
undifferentiated cells remain to form a perichondrium. As the chondrocytes proliferate
and the cartilage anlage grows, cells in the center will stop proliferating, enlarge and
4
become hypertrophic. These hypertrophic chondrocytes will mineralize their
surrounding matrix and attract blood vessels, chondroclasts and osteoprogenitor
cells[9]. In addition, they play an important role in directing adjacent perichondrial
cells to become osteoblasts and form a mineralized bone collar, thus converting the
perichondrium into periosteum. Several molecular signals have been shown to link
the
development
of
the
cartilage
anlage
with
that
of
the
surrounding
perichondrium/periosteum, including the Indian hedgehog/parathyroid hormonerelated protein, bone morphogenetic protein (BMP) and fibroblast growth factor (FGF)
signaling pathways[9, 12-14]. The crucial role of the periosteum during further bone
development is highlighted by the fact that the periosteal layer will not only serve as
the source of osteoprogenitor cells that invade the cartilage template to form the
primary ossification center[15], but it will also contribute to the radial growth of the
long bones by continuously producing mature osteoblasts from periosteal progenitor
cells[16].
The periosteum during skeletal growth and aging
Periosteal bone apposition is a cardinal feature of skeletal growth. Long bones grow
wider as they grow taller, and there is extensive individual variation in this
process[16, 17]. One of the most striking determinants of periosteal apposition in
humans is gender. There is little difference between the sexes in the extent of
periosteal apposition until puberty, when periosteal expansion markedly accelerates,
especially in men when sexual dimorphism appears[16-18]. The skeletal gender
differences in radial bone growth are traditionally attributed to the actions of sex
steroids. In the periosteum, estrogens promote the expansion of early osteoblast
progenitors but inhibit their differentiation by osteogenic agents such as parathyroid
5
hormone (PTH) or BMP2, thus limiting periosteal bone expansion[19, 20]. Androgens
in contrast stimulate both the proliferation of early periosteal osteoprogenitors and
their differentiation to mature osteoblasts. Current evidence further indicates that in
addition to differences in sex steroid secretion, skeletal sexual dimorphism also
depends on gender-related differences in growth hormone/insulin-like growth factor 1
signaling and sensitivity to mechanical loading[18].
At the completion of longitudinal growth, periosteal apposition will decrease to no
more than a few millimeters per year and, together with continued endosteal
resorption, result in thinning of the cortical shell[16, 17]. Biomechanical analyses
suggest that these changes in bone size have important implications for the
determination of fracture risk in the elderly. In addition to many factors that affect
adult periosteal bone apposition, including mechanical forces, nutrients, endocrine
factors and lifestyle, an important gender difference remains during adulthood and
aging. Men experience a greater rate of periosteal growth with aging, leading to a
superior ability to maintain bone strength and protection against age-related fracture
risk. This might again be related to differences in sex steroid levels, as orchidectomy
reduces and ovariectomy increases periosteal bone formation in adults[21]. Since
periosteal and endosteal/trabecular osteoprogenitor cells seem to respond differently
to sex steroids as well as antiresorptive and anabolic osteoporotic drugs[3], targeting
the periosteum with anabolic agents such as PTH or androgens may prove a potent
approach for fracture risk reduction in osteoporotic patients or the elderly.
6
3. The periosteum as a central mediator of bone healing
Bone repair: the cellular picture
Bone is one of few tissues that can heal without forming a fibrous scar. The process
of fracture healing largely recapitulates bone development, although it differs slightly
due to the presence of inflammation and mechanical loading[22]. The bone healing
process comprises 4 different phases: an initial inflammatory response and
recruitment of skeletal progenitor cells, the formation of a cartilaginous callus, the
replacement of cartilage by spongy bone and finally the remodeling of the immature
bone into mature lamellar bone (Fig. 2).
Immediately following bone trauma, as a result of rupture of blood vessels, a
hematoma is generated that consists of cells from both peripheral blood and the
intramedullary hematopoietic compartment. The injury also initiates an inflammatory
response, necessary for the removal of necrotic tissue and debris from the fracture
site and for the healing to proceed. During this phase, chemokines and inflammatory
cytokines including tumor necrosis factor alpha (TNFα), interleukin 1 (IL1) and IL6
are secreted to recruit inflammatory cells while hypoxia at the fracture site will induce
the
production
of
VEGF
and
other
angiogenic
factors
to
stimulate
neoangiogenesis[23]. In addition, a myriad of growth factors, including transforming
growth factor beta 1 (TGFβ1), BMPs, FGFs, stromal-derived factor 1 alpha (SDF1α)
and platelet-derived growth factor (PDGF), are released by platelets and
inflammatory cells present in the fracture hematoma, triggering the activation and
proliferation of skeletal progenitor cells in the periosteum[23-27].
7
Depending on the biomechanics of the fracture site, periosteal cells will then
differentiate into chondrocytes and/or osteoblasts. While strictly stabilized fractures
(i.e. fractures in which the broken ends are closely opposed and mechanically
stabilized) heal through intramembranous ossification, partially stabilized or nonstabilized fractures show the presence of large amounts of cartilage and heal through
the endochondral pathway[28]. These observations might be related to the inability of
blood vessels to develop properly in an unstable environment[29]. If the fracture is
held stable, blood vessel bridging can occur, along with rapid union by periosteal and
endosteal direct bone formation. In contrast, when motion is present at the fracture
site, stable blood vessel formation is hampered and the periosteal cells near the
fracture ends will differentiate into chondrocytes. In periosteal tissue further away
from the fracture site, where less movement is present, periosteal cells can
differentiate into osteoblasts, forming woven bone. As the endochondral callus
provides new stability to the fracture site and as chondrocytes undergo hypertrophy,
blood vessels and osteoprogenitor cells are attracted from nearby periosteal regions.
The cartilage template is thus completely replaced by woven bone through
mechanisms similar to those that govern embryonic bone development. In parallel a
new cortical shell is formed by the bridging of the bony periosteal callus ends,
providing the fracture with a semi-rigid structure which allows weight bearing[22]. In
the final phase of fracture healing the woven bone is replaced by mature lamellar
bone through the cooperation of osteoclasts and osteoblasts, gradually restoring the
original bone shape including the establishment of a medullary cavity.
Throughout all of these phases, the periosteum plays a central role. Several animal
studies have shown that removal of the periosteum drastically affects bone repair[5,
6, 30], and cell tracing experiments have revealed that up to 90% of cartilage and
8
woven bone in the early fracture callus derives from the periosteum[30]. The
tendency of periosteal cells to initiate endochondral ossification upon bone injury
appears to be a distinctive periosteal characteristic, as endosteal or bone marrow
injuries heal exclusively by intramembranous bone formation[5]. This difference in
preferred bone formation pathway may furthermore represent an intrinsic difference
between periosteal and bone marrow stromal progenitor cells, as it appears to be
maintained even when cells have been expanded ex vivo[31].
Molecular signaling pathways controlling the periosteal response upon
fracture
The most studied signaling cascade with respect to bone fracture repair is the BMP
pathway. During the early stages of endochondral fracture repair BMP2, 4, 5, 6, 7
and 8, as well the receptors BMPR1A and BMPR2, can be detected in activated
periosteal cells and inflammatory cells in the granulation tissue[25, 27]. However,
while different BMP ligands are expressed, some degree of redundancy and/or
overlapping functions appear to exist, as fracture repair in mice in which Bmp4 or
Bmp7 was deleted in the limb (Prx1-Cre) progressed normally[32, 33]. In contrast, in
bones lacking Bmp2 (Prx1-Cre), the early steps of fracture healing are blocked and
skeletal progenitors at the repair site are not able to differentiate into chondrocytes or
osteoblasts[34]. Furthermore, two groups have further provided evidence that
endogenous BMP2 signaling in periosteal cells is particularly important to initiate their
osteochondrogenic differentiation and secure bone healing, although BMP2 seems
not to be required for the initial periosteal expansion[35, 36]. Additional evidence for
the importance of endogenous BMP signaling in human cells has been provided by
9
overexpressing the BMP antagonist Noggin in human periosteum-derived cells,
which completely abrogates their in vivo bone-forming capacity[37].
In addition to BMPs, the role of FGF signaling in bone healing has been extensively
studied. Expression of several FGF ligands, including Fgf2 and Fgf5, is markedly
increased during the very early stages of callus formation[26]. Moreover, progenitor
cells in the periosteum and the early periosteal callus have been shown to express
FGFR1 and respond to FGF2 and FGF9[38-40]. In line with this, several studies have
explored the use of FGF2 to promote bone healing, showing that administration of a
single dose of this factor at the fracture site can stimulate callus formation by
enhancing the proliferation of periosteal cells[41]. Recent work from our own group
has furthermore provided evidence that FGF2 not only functions as a trigger for
periosteal cell proliferation, but also primes the cells for their subsequent
osteochondrogenic differentiation and increases their production of BMP2[31].
In line with its role during bone development, expression of Indian hedgehog can be
detected in the early cartilaginous callus[42, 43]. A recent study has further
highlighted the importance of periosteal hedgehog signaling for bone healing by
deleting Smoothened, the receptor that transduces all hedgehog signals, in the
periosteum of bone grafts[44]. Disruption of hedgehog signaling markedly reduced
periosteal callus formation, suggesting that this pathway plays a significant role in
adult bone repair via enhancing differentiation of periosteal progenitors[44].
Furthermore, the use of periosteal progenitor cells overexpressing sonic hedgehog
significantly improves the healing of devitalized allogeneic bone grafts, demonstrating
the therapeutic potential of targeting this pathway in periosteum-mediated bone
repair[45].
10
In addition, several other molecular pathways are emerging as regulators of
periosteal cell function during bone healing. During the early stages of fracture
healing, inflammatory mediators such as TNFα may influence periosteal cell fate[46].
The importance of the local inflammatory environment for periosteal cell activation
and
differentiation
has further been
proven
by genetic studies
targeting
cyclooxygenase 2 (COX2), an enzyme with a central role in prostaglandin production,
showing that expression of COX2 in the injury milieu is essential for successful
periosteal progenitor cell-initiated bone healing[47, 48]. The Notch signaling pathway,
which is known to control the maintenance of stromal stem/progenitor cells in bone
marrow[49], has also been implicated in the activation of periosteal cells during the
early stages after fracture[50]. Furthermore, Wnt signaling appears to play an
important role in the osteogenic commitment of periosteal progenitor cells[37, 51, 52],
similar to its role during bone development and homeostasis[53].
Taken together, it is clear that our understanding of the signaling pathways that are
activated during fracture repair has increased substantially during the last few years.
This provides hope for the development of therapeutic strategies that target the
periosteum, or its resident progenitor cells, for the improvement of bone healing,
including the treatment of non-unions and large bone defects.
4. The periosteum as a promising cell source for bone regeneration
strategies
Isolation and characterization of skeletal progenitor cells derived from the
periosteum
To isolate periosteal tissue from the bone of patients, a periosteum elevator is
typically used. This tool, shaped like a curved chisel, is designed to maintain the
11
integrity of the periosteum by cutting the Sharpey’s fibers that anchor the periosteum
to the bone[54]. Periosteum-derived cells (PDC) are then obtained by enzymatic
digestion of the tissue or by spontaneous cell egression from the biopsy onto plastic
cell culture dishes. It is currently not known whether these alternative isolation
methods confer cells of identical functionality. For the isolation of animal PDC, either
a similar technique is used or the periosteum is isolated by simply peeling off the
periosteal layer from the bone. This method is particularly useful in young animals
where the periosteal layer is relatively thick and only loosely attached to the bone[5559]. In adult mice, the periosteum is only a few cell layers thick and thus difficult to
isolate by dissection. However, the use of murine PDC has several advantages in the
study of periosteal biology, as variability between mice is much smaller than between
human donors, and isolation of PDC from transgenic mice can provide
unprecedented insight in the molecular regulation of periosteal cells. To overcome
this limitation, we have recently developed a technique allowing the isolation of PDC
from adult mice, using an enzymatic digest directly on diaphyseal bone after
embedding the epiphyses in low-melting point agarose, which prevents the
contamination of cell cultures with cells deriving from the growth plate or articular
surface[60]. An alternative method to isolate murine PDC, proposed by Wang et al.,
employs a femoral bone grafting model[44]. The authors first performed a bone
grafting surgery and 3 days later isolated the graft, scraped off the newly formed
periosteal callus and performed an enzymatic digest to release periosteal cells. While
the number of cells which can be obtained may be higher than those isolated from
resting periosteum, this technique isolates cells which have been subjected to
hypoxia and inflammation and are already in an active state, which can make the
comparison with human PDC more difficult. However in the authors opinion, refined
12
microsurgery tools coupled to appropriate microscopy and “good” hands, should
allow microdissection of the periosteum, even in adult mice.
Regardless of differences in isolation method or species used, several features of the
obtained periosteal cell populations are highly comparable between studies. In
culture, PDC exhibit a fibroblast-like morphology, which is stably maintained over
several passages. Culture-expanded PDC are negative for telomerase, but they do
possess relatively long telomeres that may contribute to their stability in culture[61].
Furthermore, culture expansion of PDC appears to be STAT3 dependent as inhibition
of this pathway, either through interference with JAK2 or STAT3 directly, abrogates
proliferation induced by serum growth factors[62]. While PDC do not overtly express
osteogenic or chondrogenic properties during expansion, they can be induced to
differentiate along the osteogenic, chondrogenic and adipogenic lineage in vitro by
subjecting them to specific differentiation conditions, confirming their multipotent
nature[44, 57, 60, 61, 63]. Evidence for the presence of genuine skeletal stem cells in
the periosteum has been delivered by single-cell lineage analysis, showing that the
periosteum
contains
cells
with
mesenchymal
multipotency
(osteogenic,
chondrogenic, adipogenic and myogenic) at the clonal level[61]. This concept is
further underscored by the observation that expanded PDC possess the potential to
form bone, cartilage and hematopoietic marrow upon in vivo transplantation[44, 60,
61, 64]. However, it is still unclear whether the skeletal progenitor cells in the
periosteum also possess the potential to self-renew, a key feature of stem cell
identity[65].
Characterization of PDC cultures by analysis of cell surface markers has shown that
a high percentage of cells express the mesenchymal stem/progenitor cell markers
CD73, CD90, CD105, CD146 and CD166 (humans) or CD51, CD73, CD90.2, CD105
13
and Sca1 (mice), while lacking the expression of markers associated with the
hematopoietic and endothelial lineages[44, 60, 61, 64]. However, periosteal cell
cultures have also been shown to contain preosteoblasts and committed osteogenic
cells, underscoring the heterogeneous nature of the periosteum[54]. Although studies
have been conducted with sorted CD45-CD9+CD90+CD166+ PDC, it is unclear
whether this procedure of cell processing results in a pure population that represents
all biological and functional characteristics of periosteal progenitors[66]. Indeed, there
is currently a lack of specific markers for periosteal progenitors that allows their
isolation from tissue digests.
The ability of PDC to mobilize to fracture sites has prompted investigation into their
ability to respond to chemokines. Subsequently, it has been reported that PDCs
express chemokine receptors from the four chemokine subfamilies; CC, CXC,
CX(3)C, and C. Furthermore, they are responsive to the chemokines CCL2
(monocyte chemoattractant protein-1, MCP1), CCL25 (thymus expressed chemokine,
TECK), CXCL8 (interleukin 8, IL8), CXCL12 (SDF1α) and CXCL13 (B-cell-activating
chemokine 1, BCA1)[67]. In addition, PDC respond to a number of growth factors and
cytokines. Indeed, factors such as IL6, epidermal growth factor (EGF), TGFβ1 and
BMP2 have been shown to play key roles in PDC osteogenic differentiation[68, 69].
Additionally, calcium- and phosphate-enriched culture conditions promote certain
aspects of osteogenic differentiation in vitro, although this may be as a result of
enhanced BMP2 expression[70, 71].
Although in vitro studies have clearly shown that the periosteum contains multipotent
skeletal progenitor cells, their in vivo counterpart still remains elusive. However,
recent lineage-tracing studies during fracture healing in mice have shed a first light
on the markers that could be used to identify skeletal progenitor cells in the
14
periosteum in vivo. Grcevic et al. have used alpha smooth muscle actin(αSMA)CreERT2 transgenic mice to demonstrate that the majority of the cells in the fracture
callus derive from perivascular αSMA-positive cells that presumably originate from
the cambium layer of the periosteum [72]. Furthermore, when periosteal αSMApositive cells were isolated, they exhibited skeletal progenitor cell characteristics,
including the expression of several mesenchymal progenitor cell markers and the
ability to differentiate along the osteogenic, chondrogenic and adipogenic lineage[50,
72]. Following a similar approach with Prx1-CreER-GFP transgenic mice, Kawanami
et
al.
showed
that
PRX1-expressing
cells
in
the
periosteum
are
osteochondroprogenitor cells that give rise to chondrocytes and osteoblasts in the
fracture callus[73]. A recent study by Murao et al. further revealed that while no
SOX9-positive cells are detected in resting periosteum, these cells quickly arise after
bone fracture[74]. By tracing their progeny using Sox9-CreERT2 mice, the authors
showed that these cells give rise to both chondrocytes and osteoblasts in the callus.
How these different cell populations relate to one another, however, is currently
unknown.
The observation that periosteal progenitor cells express perivascular cell markers,
including αSMA, CD146 and PDGF receptor beta[44, 50, 60, 61, 72], is in
accordance with several studies that have shown a perivascular location for skeletal
stem cells in bone marrow[75-77]. As the periosteum is highly vascularized and
evidence
exists
that
periosteal
pericytes
possess
skeletal
progenitor
cell
characteristics, this model could explain the high number of progenitors found in this
tissue[39, 76, 78, 79]. The finding that PDC can enhance vasculogenesis by adapting
a pericyte-like phenotype when they are implanted together with endothelial cells
further supports this concept[60].
15
Periosteum-derived cells in (pre)clinical bone tissue engineering
approaches
The feasibility of using periosteal cells for bone TE strategies was for the first time
described by Nakahara et al. in 1991, who demonstrated in vivo bone formation by
human PDC[80]. Since then, numerous studies have used PDC together with
different scaffolds to enhance bone regeneration in critical-sized defects in a variety
of animal models[56, 81-84]. The use of PDC together with a ceramic scaffold
appears to represent an ideal combination as both natural and synthetic calcium
phosphate carriers exhibit osteoinductive effects on PDC[37, 62, 85]. While the exact
molecular mechanisms behind bone formation by PDC in TE transplants remain
unknown, endogenous BMP and Wnt signaling in PDC have been shown to be
crucial for their bone-forming potential on ceramic scaffolds, and exogenous addition
of BMP2 can further enhance periosteal cell-driven bone regeneration[37, 86].
The central role of the periosteum in bone repair and the success of periosteal cells
in preclinical animal models has also given rise to several exploratory clinical studies
using ex vivo expanded periosteal cells for bone regeneration. The first of these
studies was performed by Vacanti et al., who used culture-expanded periosteal cells
derived from the radius in combination with a porous hydroxyapatite scaffold to
replace the distal phalanx of the thumb of a 36-year-old man[87]. Although functional
restoration was obtained, histological examination after 10 months revealed that only
5% of the construct was filled with lamellar bone and ossified endochondral tissue.
Further studies using tissue engineered constructs containing expanded periosteal
cells for maxillary sinus floor augmentation have however provided more compelling
evidence that PDC can be used to regenerate substantial amounts of bone in
16
humans within 3-4 months after transplantation[88-90]. Taken together, these reports
demonstrate the clinical potential of PDC for bone regeneration therapies. New, more
extensive clinical trials will however be essential to provide irrefutable evidence for
the efficacy of periosteal cells in different clinical applications.
Periosteum-derived cells versus mesenchymal progenitor cells from
other sources
Although during postnatal fracture repair the tissue predominantly responsible for
contributing tissue-forming progenitors is the periosteum, multiple adult progenitor
cell populations can be induced into the osteogenic lineage in vitro, and hence their
applicability for skeletal regeneration is being investigated. Consequently, it is
important to define which of these cell populations are most suitable for this purpose,
both for their ease of harvesting and their ability to contribute to tissue formation in
vivo. A number of studies have attempted to directly compare mesenchymal
progenitor populations from different tissues in a range of species including mouse,
rat, pig, horse and human. The most commonly compared sources of mesenchymal
progenitor cells are PDC, synovium-derived cells (SDC), bone marrow-derived
mesenchymal stromal cells (BM-MSC), muscle-derived cells (MDC) and adipose
tissue-derived cells (ADC). From these tissues it appears that the periosteum and
synovium contain the highest quantity of stem/progenitor cells as they have the ability
to produce a higher number of colonies when plated at low density[91, 92]. However,
the large number of nucleated but non-adherent hematopoietic cells present in bone
marrow aspirates may contribute to this finding. Cell surface markers appear to be
similar in cells derived from each of the aforementioned tissues with only positivity for
VEGFR-2 (Flk-1) being higher in PDC than in ADC and MDC[93]. However, this
17
analysis was carried out in passaged cells therefore it is conceivable that these cells
have lost their tissue-specific profile during culture adaptation. When proliferative
capacity of equine mesenchymal progenitors was compared, it was found that MDC,
PDC and ADC proliferated significantly faster than BM-MSC[93]. Furthermore, in all
published comparisons, PDC and SDC appeared to have the highest proliferative
capacity when compared to BM-MSC across species, including rat[94], mouse[60]
and human[92]. A high proliferative capacity is generally a prerequisite of cells
intended for tissue engineering, as an in vitro cell expansion step usually forms part
of this strategy. For analysis of in vitro differentiation capacity it should be noted that
all mesenchymal progenitor cell populations, by definition, must have the ability to
produce adipocytes, chondrocytes and osteoblasts[95]. However it is evident from
comparative studies that cells isolated from specific tissues often have enhanced
differentiation towards one of these lineages. Indeed, human PDC, BM-MSC and
SDC display superior osteogenic differentiation when compared to MDC and ADC,
however the authors did not speculate which of these three was the most
osteogenic[92]. Indeed, studies that have specifically compared the osteogenic
capacity of PDC, SDC and BM-MSC report conflicting data. For example, Hayashi et
al. claim that rat BM-MSC have a higher osteogenic capacity when compared to
PDC[91], while Yoshimura et al. report the opposite[94]. This contradiction may be
explained by a number of technical differences between these studies including cell
passage number and osteogenic conditions used. Interestingly, SDC reproducibly
display the highest chondrogenic differentiation across species, followed by PDC[92,
94], indicating that in vitro differentiation capacity is in part reflected by the
anatomical location of the parental tissue in vivo.
18
Limited studies have compared the efficacy of different cell populations for in vivo
tissue formation. One study used an in vivo pig cranial defect model to compare the
osteogenic potential of ADC, PDC and BM-MSC. In this study all the cell populations
showed comparable osseous healing when implanted in a collagen scaffold and
compared to an empty scaffold[96]. Our laboratory has previously compared the
calcium phosphate-driven in vivo bone-forming capacity of human PDC, SDC, MDC
(mesoangioblasts)[64] and BM-MSC (unpublished data) (Fig. 3). Using these assay
conditions, including the use of specific scaffolds, only PDC and BM-MSC were able
to
form
quantifiable
bone
spicules.
Interestingly,
BM-MSC
formed
larger
hematopoietic compartments than PDC, thus providing further evidence for
progenitor populations having tissue specific memory.
5. Tissue engineering strategies mimicking the periosteum in its
absence
The importance of periosteal integrity for fracture repair has led to the proposal of
strategies to recreate this tissue for long bone repair. Indeed, this concept is currently
being used clinically through the induction of a periosteal-like membrane in a
technique proposed by Masquelet and colleagues[97]. This procedure involves the
debridement of bone before implantation of a polymethyl methacrylate cement spacer
within the fixated bone defect. The role of this spacer is to firstly provide mechanical
support for the fracture, and secondly to act as a foreign body that allows the
propagation of the periosteal like membrane on its surface. The membrane is highly
vascularized and secretes a number of growth factors that are important in skeletal
development and repair, such as BMP2, VEGF and TGFβ1[98]. This induced
membrane enhances the repair of large long bone defects when combined with
19
cancellous autografts. Interestingly, it has recently been shown that the induced
membrane contains progenitor cells which have the ability to undergo differentiation
into chondrocytes, osteoblasts and adipocytes[99]. Upon comparison to PDC, the
induced membrane-derived cells had a similar colony-forming and differentiation
capacity; however they did express higher levels SDF1α, a protein that is known to
be involved in the recruitment of progenitors to the site of injury. It is currently unclear
what the origin of these progenitors is, however, the assumption that they may come
from the periosteum is supported as induced membranes which were formed
subcutaneously have no bone-forming capacity in conjunction with a calcium
phosphate biomaterial[100].
In addition to this example of in situ tissue engineering, investigations are on-going
that aim to produce an engineered periosteum in vitro[101]. It is proposed that growth
factors and cells can be sequestered within hydrogels in a manner and at levels that
mimic the native periosteum. The investigators envisage that this system will improve
the healing and integration of allografts in vivo to levels that are seen with autografts.
Engineered artificial periosteal tissues have previously been investigated, including
the use of acellular human dermis as a substitute for periosteum for the repair of
critical sized defects[102]. When this membrane was seeded with BMP2-expressing
BM-MSC, it promoted heterotopic bone formation and could heal critical sized
mandibular defects in mice. Although interesting, it is unclear whether this construct
mimics the periosteum or simply provides a BMP2/cell delivery device. Indeed, it has
also been documented that by seeding BMP2 modified cells directly onto an allograft,
cartilaginous tissue and a new cortical shell was formed resulting in bridging of a
segmental defect. However, this did not completely mimic an autograft as slower
resorption of the graft was observed [103]. More recently it has been reported that
20
BM-MSC cell sheets can act as an engineered periosteum and promote bone callus
formation during allograft healing[104], while PDC cell sheets may have applications
in tendon-bone healing[105]. Evidently, the investigation of periosteum mimetics for
skeletal regeneration and repair is gathering pace and further investigation is eagerly
anticipated.
6. Concluding remarks and future directions
As illustrated, it is clear that the periosteum plays an essential role in bone
development, homeostasis and repair. As our insight into periosteal involvement in
each of these processes grows, so does our ability to target it for skeletal
regeneration. Indeed, during postnatal fracture repair the periosteum contributes the
vast majority of progenitors for the cartilaginous callus and bone, in contrast to the
bone marrow compartment, which appears to have only a minimal bone tissueforming potential. In cases where this repair mechanism fails, clinically described as
delayed or non-union, the ability to specifically target periosteal stem cells in vivo
would likely be a highly effective strategy. However, further characterization of the
periosteal stem cell niche, including factors regulating stem cell quiescence and
activation are required to make this possible. Furthermore, markers that robustly
identify the resident periosteal stem cells in the cambium layer and allow their
selection from the fibrous layer are urgently required. Current studies indicate that
PRX1 expression may mark a population of progenitor cells, however it is not known
whether this is the entire stem cell component or whether these cells are progeny of
the periosteal stem cell. Once these factors have been determined, in vivo targeting
of periosteal cells for skeletal repair will become a reality. In the meantime, the
investigation of ex vivo-expanded PDC for tissue engineering strategies is ongoing. It
21
is envisaged that these studies, along with advances in periosteum mimetics, will
lead to clinical translation of novel tissue engineered strategies for skeletal repair.
In conclusion, it is the authors’ belief that with this review the uncertainty regarding
which source of stem cells is most suited to skeletal regeneration has now been
addressed, and we sincerely hope that this will invigorate more investigation into the
periosteum in the future.
Acknowledgements
This work was funded by the ERC Advanced Grant REJOIND (294191-REJOIND),
the BOF-KU Leuven GOA project (3M120209) and the Stem Cell Institute of LeuvenKU Leuven (PF/10/019). The work is part of Prometheus, the Leuven Research and
Development
Division
of
Skeletal
Tissue
http://www.kuleuven.be/Prometheus.
22
Engineering
of
KU
Leuven:
References
[1] Duhamel HL. Sur le développement et la crue des os des animaux. Mem Acad Roy Sci Paris
1742:354-70.
[2] Ollier L. Traite experimentel et clinique de la regeneration des os et de la production artificielle
du tissu osseux. Paris: V. Masson; 1867.
[3] Allen MR, Hock JM, Burr DB. Periosteum: biology, regulation, and response to osteoporosis
therapies. Bone 2004;35:1003-12.
[4] Chanavaz M. Anatomy and histophysiology of the periosteum: quantification of the periosteal
blood supply to the adjacent bone with 85Sr and gamma spectrometry. J Oral Implantol
1995;21:214-9.
[5] Colnot C. Skeletal cell fate decisions within periosteum and bone marrow during bone
regeneration. J Bone Miner Res 2009;24:274-82.
[6] Ozaki A, Tsunoda M, Kinoshita S, Saura R. Role of fracture hematoma and periosteum during
fracture healing in rats: interaction of fracture hematoma and the periosteum in the initial step
of the healing process. J Orthop Sci 2000;5:64-70.
[7] Long F. Building strong bones: molecular regulation of the osteoblast lineage. Nat Rev Mol
Cell Biol 2012;13:27-38.
[8] Ochareon P, Herring SW. Cell replication in craniofacial periosteum: appositional vs.
resorptive sites. J Anat 2011;218:285-97.
[9] Kronenberg HM. Developmental regulation of the growth plate. Nature 2003;423:332-6.
[10] Akiyama H, Chaboissier MC, Martin JF, Schedl A, de CB. 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 2002;16:2813-28.
[11] Akiyama H, Kim JE, Nakashima K, Balmes G, Iwai N, Deng JM et al. Osteo-chondroprogenitor
cells are derived from Sox9 expressing precursors. Proc Natl Acad Sci U S A
2005;102:14665-70.
[12] Chung UI, Schipani E, McMahon AP, Kronenberg HM. Indian hedgehog couples
chondrogenesis to osteogenesis in endochondral bone development. J Clin Invest
2001;107:295-304.
23
[13] Ornitz DM, Marie PJ. FGF signaling pathways in endochondral and intramembranous bone
development and human genetic disease. Genes Dev 2002;16:1446-65.
[14] Pathi S, Rutenberg JB, Johnson RL, Vortkamp A. Interaction of Ihh and BMP/Noggin signaling
during cartilage differentiation. Dev Biol 1999;209:239-53.
[15] Maes C, Kobayashi T, Selig MK, Torrekens S, Roth SI, Mackem S et al. Osteoblast
precursors, but not mature osteoblasts, move into developing and fractured bones along with
invading blood vessels. Dev Cell 2010;19:329-44.
[16] Seeman E. Periosteal bone formation - a neglected determinant of bone strength. N Engl J
Med 2003;349:320-3.
[17] Orwoll ES. Toward an expanded understanding of the role of the periosteum in skeletal health.
J Bone Miner Res 2003;18:949-54.
[18] Callewaert F, Sinnesael M, Gielen E, Boonen S, Vanderschueren D. Skeletal sexual
dimorphism: relative contribution of sex steroids, GH-IGF1, and mechanical loading. J
Endocrinol 2010;207:127-34.
[19] Almeida M, Iyer S, Martin-Millan M, Bartell SM, Han L, Ambrogini E et al. Estrogen receptoralpha signaling in osteoblast progenitors stimulates cortical bone accrual. J Clin Invest
2012;123:394-404.
[20] Ogita M, Rached MT, Dworakowski E, Bilezikian JP, Kousteni S. Differentiation and
proliferation of periosteal osteoblast progenitors are differentially regulated by estrogens and
intermittent parathyroid hormone administration. Endocrinology 2008;149:5713-23.
[21] Orwoll ES. Androgens: basic biology and clinical implication. Calcif Tissue Int 2001;69:185-8.
[22] Marsell R, Einhorn TA. The biology of fracture healing. Injury 2011;42:551-5.
[23] Al-Aql ZS, Alagl AS, Graves DT, Gerstenfeld LC, Einhorn TA. Molecular mechanisms
controlling bone formation during fracture healing and distraction osteogenesis. J Dent Res
2008;87:107-18.
[24] Barnes GL, Kostenuik PJ, Gerstenfeld LC, Einhorn TA. Growth factor regulation of fracture
repair. J Bone Miner Res 1999;14:1805-15.
[25] Cho TJ, Gerstenfeld LC, Einhorn TA. Differential temporal expression of members of the
transforming growth factor beta superfamily during murine fracture healing. J Bone Miner Res
2002;17:513-20.
24
[26] Schmid GJ, Kobayashi C, Sandell LJ, Ornitz DM. Fibroblast growth factor expression during
skeletal fracture healing in mice. Dev Dyn 2009;238:766-74.
[27] Yu YY, Lieu S, Lu C, Miclau T, Marcucio RS, Colnot C. Immunolocalization of BMPs, BMP
antagonists, receptors, and effectors during fracture repair. Bone 2010;46:841-51.
[28] Thompson Z, Miclau T, Hu D, Helms JA. A model for intramembranous ossification during
fracture healing. J Orthop Res 2002;20:1091-8.
[29] Marsh DR, Li G. The biology of fracture healing: optimising outcome. Br Med Bull
1999;55:856-69.
[30] Zhang X, Xie C, Lin AS, Ito H, Awad H, Lieberman JR et al. Periosteal progenitor cell fate in
segmental cortical bone graft transplantations: implications for functional tissue engineering. J
Bone Miner Res 2005;20:2124-37.
[31] van Gastel N, Stegen S, Stockmans I, Moermans K, Schrooten J, Graf D et al. Expansion of
murine periosteal progenitor cells with FGF2 reveals an intrinsic endochondral ossification
program mediated by BMP2. Stem Cells 2014;10.1002/stem.1783 [doi].
[32] Tsuji K, Cox K, Bandyopadhyay A, Harfe BD, Tabin CJ, Rosen V. BMP4 is dispensable for
skeletogenesis and fracture-healing in the limb. J Bone Joint Surg Am 2008;90:14-8.
[33] Tsuji K, Cox K, Gamer L, Graf D, Economides A, Rosen V. Conditional deletion of BMP7 from
the limb skeleton does not affect bone formation or fracture repair. J Orthop Res 2010;28:3849.
[34] Tsuji K, Bandyopadhyay A, Harfe BD, Cox K, Kakar S, Gerstenfeld L et al. BMP2 activity,
although dispensable for bone formation, is required for the initiation of fracture healing. Nat
Genet 2006;38:1424-9.
[35] Chappuis V, Gamer L, Cox K, Lowery JW, Bosshardt DD, Rosen V. Periosteal BMP2 activity
drives bone graft healing. Bone 2012;51:800-9.
[36] Wang Q, Huang C, Xue M, Zhang X. Expression of endogenous BMP-2 in periosteal
progenitor cells is essential for bone healing. Bone 2011;48:524-32.
[37] Eyckmans J, Roberts SJ, Schrooten J, Luyten FP. A clinically relevant model of
osteoinduction: a process requiring calcium phosphate and BMP/Wnt signalling. J Cell Mol
Med 2010;14:1845-56.
25
[38] Behr B, Leucht P, Longaker MT, Quarto N. Fgf-9 is required for angiogenesis and
osteogenesis in long bone repair. Proc Natl Acad Sci U S A 2010;107:11853-8.
[39] Coutu DL, Francois M, Galipeau J. Inhibition of cellular senescence by developmentally
regulated FGF receptors in mesenchymal stem cells. Blood 2011;117:6801-12.
[40] Rundle CH, Miyakoshi N, Ramirez E, Wergedal JE, Lau KH, Baylink DJ. Expression of the
fibroblast growth factor receptor genes in fracture repair. Clin Orthop Relat Res 2002:253-63.
[41] Du X, Xie Y, Xian CJ, Chen L. Role of FGFs/FGFRs in skeletal development and bone
regeneration. J Cell Physiol 2012;227:3731-43.
[42] Le AX, Miclau T, Hu D, Helms JA. Molecular aspects of healing in stabilized and nonstabilized fractures. J Orthop Res 2001;19:78-84.
[43] Murakami S, Noda M. Expression of Indian hedgehog during fracture healing in adult rat
femora. Calcif Tissue Int 2000;66:272-6.
[44] Wang Q, Huang C, Zeng F, Xue M, Zhang X. Activation of the Hh pathway in periosteumderived mesenchymal stem cells induces bone formation in vivo: implication for postnatal
bone repair. Am J Pathol 2010;177:3100-11.
[45] Huang C, Tang M, Yehling E, Zhang X. Overexpressing Sonic Hedgehog Peptide Restores
Periosteal Bone Formation in a Murine Bone Allograft Transplantation Model. Mol Ther
2014;22:430-9.
[46] Gerstenfeld LC, Cho TJ, Kon T, Aizawa T, Tsay A, Fitch J et al. Impaired fracture healing in
the absence of TNF-alpha signaling: the role of TNF-alpha in endochondral cartilage
resorption. J Bone Miner Res 2003;18:1584-92.
[47] Xie C, Ming X, Wang Q, Schwarz EM, Guldberg RE, O'Keefe RJ et al. COX-2 from the injury
milieu is critical for the initiation of periosteal progenitor cell mediated bone healing. Bone
2008;43:1075-83.
[48] Zhang X, Schwarz EM, Young DA, Puzas JE, Rosier RN, O'Keefe RJ. Cyclooxygenase-2
regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved
in bone repair. J Clin Invest 2002;109:1405-15.
[49] Hilton MJ, Tu X, Wu X, Bai S, Zhao H, Kobayashi T et al. Notch signaling maintains bone
marrow mesenchymal progenitors by suppressing osteoblast differentiation. Nat Med
2008;14:306-14.
26
[50] Matthews BG, Grcevic D, Wang L, Hagiwara Y, Roguljic H, Joshi P et al. Analysis of
alphaSMA-Labeled Progenitor Cell Commitment Identifies Notch Signaling as an Important
Pathway in Fracture Healing. J Bone Miner Res 2013.
[51] Komatsu DE, Mary MN, Schroeder RJ, Robling AG, Turner CH, Warden SJ. Modulation of
Wnt signaling influences fracture repair. J Orthop Res 2010;28:928-36.
[52] Minear S, Leucht P, Jiang J, Liu B, Zeng A, Fuerer C et al. Wnt proteins promote bone
regeneration. Sci Transl Med 2010;2:29ra30.
[53] Baron R, Kneissel M. WNT signaling in bone homeostasis and disease: from human
mutations to treatments. Nat Med 2013;19:179-92.
[54] Chang H, Knothe Tate ML. Concise review: the periosteum: tapping into a reservoir of
clinically useful progenitor cells. Stem Cells Transl Med 2012;1:480-91.
[55] Arnsdorf EJ, Jones LM, Carter DR, Jacobs CR. The periosteum as a cellular source for
functional tissue engineering. Tissue Eng Part A 2009;15:2637-42.
[56] Breitbart AS, Grande DA, Kessler R, Ryaby JT, Fitzsimmons RJ, Grant RT. Tissue engineered
bone repair of calvarial defects using cultured periosteal cells. Plast Reconstr Surg
1998;101:567-74.
[57] Declercq HA, De Ridder LI, Cornelissen MJ. Isolation and Osteogenic Differentiation of Rat
Periosteum-derived Cells. Cytotechnology 2005;49:39-50.
[58] Eyckmans J, Luyten FP. Species specificity of ectopic bone formation using periosteumderived mesenchymal progenitor cells. Tissue Eng 2006;12:2203-13.
[59] Nakahara H, Bruder SP, Goldberg VM, Caplan AI. In vivo osteochondrogenic potential of
cultured cells derived from the periosteum. Clin Orthop Relat Res 1990;259:223-32.
[60] van Gastel N, Torrekens S, Roberts SJ, Moermans K, Schrooten J, Carmeliet P et al.
Engineering vascularized bone: osteogenic and proangiogenic potential of murine periosteal
cells. Stem Cells 2012;30:2460-71.
[61] De Bari C, Dell'Accio F, Vanlauwe J, Eyckmans J, Khan IM, Archer CW et al. Mesenchymal
multipotency of adult human periosteal cells demonstrated by single-cell lineage analysis.
Arthritis Rheum 2006;54:1209-21.
27
[62] Roberts SJ, Owen HC, Tam WL, Solie L, Van Cromphaut SJ, Van den Berghe G et al.
Humanized Culture of Periosteal Progenitors in Allogeneic Serum Enhances Osteogenic
Differentiation and In Vivo Bone Formation. Stem Cells Transl Med 2013.
[63] Nakahara H, Dennis JE, Bruder SP, Haynesworth SE, Lennon DP, Caplan AI. In vitro
differentiation of bone and hypertrophic cartilage from periosteal-derived cells. Exp Cell Res
1991;195:492-503.
[64] Roberts SJ, Geris L, Kerckhofs G, Desmet E, Schrooten J, Luyten FP. The combined bone
forming capacity of human periosteal derived cells and calcium phosphates. Biomaterials
2011;32:4393-405.
[65] Bianco P, Cao X, Frenette PS, Mao JJ, Robey PG, Simmons PJ et al. The meaning, the
sense and the significance: translating the science of mesenchymal stem cells into medicine.
Nat Med 2013;19:35-42.
[66] Lim SM, Choi YS, Shin HC, Lee CW, Kim DI. Isolation of human periosteum-derived
progenitor cells using immunophenotypes for chondrogenesis. Biotechnol Lett 2005;27:60711.
[67] Stich S, Loch A, Leinhase I, Neumann K, Kaps C, Sittinger M et al. Human periosteumderived progenitor cells express distinct chemokine receptors and migrate upon stimulation
with CCL2, CCL25, CXCL8, CXCL12, and CXCL13. Eur J Cell Biol 2008;87:365-76.
[68] Eyckmans J, Roberts SJ, Bolander J, Schrooten J, Chen CS, Luyten FP. Mapping calcium
phosphate activated gene networks as a strategy for targeted osteoinduction of human
progenitors. Biomaterials 2013;34:4612-21.
[69] Roberts SJ, Chen Y, Moesen M, Schrooten J, Luyten FP. Enhancement of osteogenic gene
expression for the differentiation of human periosteal derived cells. Stem Cell Res 2011;7:13744.
[70] Chai YC, Roberts SJ, Schrooten J, Luyten FP. Probing the osteoinductive effect of calcium
phosphate by using an in vitro biomimetic model. Tissue Eng Part A 2011;17:1083-97.
[71] Chai YC, Roberts SJ, Desmet E, Kerckhofs G, van GN, Geris L et al. Mechanisms of ectopic
bone formation by human osteoprogenitor cells on CaP biomaterial carriers. Biomaterials
2012;33:3127-42.
28
[72] Grcevic D, Pejda S, Matthews BG, Repic D, Wang L, Li H et al. In vivo fate mapping identifies
mesenchymal progenitor cells. Stem Cells 2012;30:187-96.
[73] Kawanami A, Matsushita T, Chan YY, Murakami S. Mice expressing GFP and CreER in
osteochondro progenitor cells in the periosteum. Biochem Biophys Res Commun
2009;386:477-82.
[74] Murao H, Yamamoto K, Matsuda S, Akiyama H. Periosteal cells are a major source of soft
callus in bone fracture. J Bone Miner Metab 2013;31:390-8.
[75] Mendez-Ferrer S, Michurina TV, Ferraro F, Mazloom AR, Macarthur BD, Lira SA et al.
Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature
2010;466:829-34.
[76] Park D, Spencer JA, Koh BI, Kobayashi T, Fujisaki J, Clemens TL et al. Endogenous bone
marrow MSCs are dynamic, fate-restricted participants in bone maintenance and
regeneration. Cell Stem Cell 2012;10:259-72.
[77] Sacchetti B, Funari A, Michienzi S, Di Cesare S, Piersanti S, Saggio I et al. Self-renewing
osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment.
Cell 2007;131:324-36.
[78] Diaz-Flores L, Gutierrez R, Lopez-Alonso A, Gonzalez R, Varela H. Pericytes as a
supplementary source of osteoblasts in periosteal osteogenesis. Clin Orthop Relat Res
1992:280-6.
[79] Simpson AH. The blood supply of the periosteum. J Anat 1985;140:697-704.
[80] Nakahara H, Goldberg VM, Caplan AI. Culture-expanded human periosteal-derived cells
exhibit osteochondral potential in vivo. J Orthop Res 1991;9:465-76.
[81] Bakker AD, Schrooten J, van Cleynenbreugel T, Vanlauwe J, Luyten J, Schepers E et al.
Quantitative screening of engineered implants in a long bone defect model in rabbits. Tissue
Eng Part C Methods 2008;14:251-60.
[82] Perka C, Schultz O, Spitzer RS, Lindenhayn K, Burmester GR, Sittinger M. Segmental bone
repair by tissue-engineered periosteal cell transplants with bioresorbable fleece and fibrin
scaffolds in rabbits. Biomaterials 2000;21:1145-53.
29
[83] Redlich A, Perka C, Schultz O, Spitzer R, Haupl T, Burmester GR et al. Bone engineering on
the basis of periosteal cells cultured in polymer fleeces. J Mater Sci Mater Med 1999;10:76772.
[84] Sakata Y, Ueno T, Kagawa T, Kanou M, Fujii T, Yamachika E et al. Osteogenic potential of
cultured human periosteum-derived cells - a pilot study of human cell transplantation into a rat
calvarial defect model. J Craniomaxillofac Surg 2006;34:461-5.
[85] Srouji S, Ben-David D, Funari A, Riminucci M, Bianco P. Evaluation of the osteoconductive
potential of bone substitutes embedded with schneiderian membrane- or maxillary bone
marrow-derived osteoprogenitor cells. Clin Oral Implants Res 2013;24:1288-94.
[86] Agata H, Asahina I, Yamazaki Y, Uchida M, Shinohara Y, Honda MJ et al. Effective bone
engineering with periosteum-derived cells. J Dent Res 2007;86:79-83.
[87] Vacanti CA, Bonassar LJ, Vacanti MP, Shufflebarger J. Replacement of an avulsed phalanx
with tissue-engineered bone. N Engl J Med 2001;344:1511-4.
[88] Schimming R, Schmelzeisen R. Tissue-engineered bone for maxillary sinus augmentation. J
Oral Maxillofac Surg 2004;62:724-9.
[89] Schmelzeisen R, Schimming R, Sittinger M. Making bone: implant insertion into tissueengineered
bone
for
maxillary
sinus
floor
augmentation-a
preliminary
report.
J
Craniomaxillofac Surg 2003;31:34-9.
[90] Springer IN, Nocini PF, Schlegel KA, De SD, Park J, Warnke PH et al. Two techniques for the
preparation of cell-scaffold constructs suitable for sinus augmentation: steps into clinical
application. Tissue Eng 2006;12:2649-56.
[91] Hayashi O, Katsube Y, Hirose M, Ohgushi H, Ito H. Comparison of osteogenic ability of rat
mesenchymal stem cells from bone marrow, periosteum, and adipose tissue. Calcif Tissue Int
2008;82:238-47.
[92] Sakaguchi Y, Sekiya I, Yagishita K, Muneta T. Comparison of human stem cells derived from
various mesenchymal tissues: superiority of synovium as a cell source. Arthritis Rheum
2005;52:2521-9.
[93] Radtke CL, Nino-Fong R, Esparza Gonzalez BP, Stryhn H, McDuffee LA. Characterization
and osteogenic potential of equine muscle tissue- and periosteal tissue-derived mesenchymal
30
stem cells in comparison with bone marrow- and adipose tissue-derived mesenchymal stem
cells. Am J Vet Res 2013;74:790-800.
[94] Yoshimura H, Muneta T, Nimura A, Yokoyama A, Koga H, Sekiya I. Comparison of rat
mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue,
and muscle. Cell Tissue Res 2007;327:449-62.
[95] Dominici M, Le BK, Mueller I, Slaper-Cortenbach I, Marini F, Krause D et al. Minimal criteria
for defining multipotent mesenchymal stromal cells. The International Society for Cellular
Therapy position statement. Cytotherapy 2006;8:315-7.
[96] Stockmann P, Park J, von WC, Nkenke E, Felszeghy E, Dehner JF et al. Guided bone
regeneration in pig calvarial bone defects using autologous mesenchymal stem/progenitor
cells - a comparison of different tissue sources. J Craniomaxillofac Surg 2012;40:310-20.
[97] Pelissier P, Martin D, Baudet J, Lepreux S, Masquelet AC. Behaviour of cancellous bone graft
placed in induced membranes. Br J Plast Surg 2002;55:596-8.
[98] Pelissier P, Masquelet AC, Bareille R, Pelissier SM, Amedee J. Induced membranes secrete
growth factors including vascular and osteoinductive factors and could stimulate bone
regeneration. J Orthop Res 2004;22:73-9.
[99] Cuthbert RJ, Churchman SM, Tan HB, McGonagle D, Jones E, Giannoudis PV. Induced
periosteum a complex cellular scaffold for the treatment of large bone defects. Bone
2013;57:484-92.
[100] Catros S, Zwetyenga N, Bareille R, Brouillaud B, Renard M, Amedee J et al. Subcutaneousinduced membranes have no osteoinductive effect on macroporous HA-TCP in vivo. J Orthop
Res 2009;27:155-61.
[101] Hoffman MD, Benoit DS. Emerging ideas: Engineering the periosteum: revitalizing allografts
by mimicking autograft healing. Clin Orthop Relat Res 2013;471:721-6.
[102] Schonmeyr B, Clavin N, Avraham T, Longo V, Mehrara BJ. Synthesis of a tissue-engineered
periosteum with acellular dermal matrix and cultured mesenchymal stem cells. Tissue Eng
Part A 2009;15:1833-41.
[103] Xie C, Reynolds D, Awad H, Rubery PT, Pelled G, Gazit D et al. Structural bone allograft
combined with genetically engineered mesenchymal stem cells as a novel platform for bone
tissue engineering. Tissue Eng 2007;13:435-45.
31
[104] Long T, Zhu Z, Awad HA, Schwarz EM, Hilton MJ, Dong Y. The effect of mesenchymal stem
cell sheets on structural allograft healing of critical sized femoral defects in mice. Biomaterials
2014;35:2752-9.
[105] Chang CH, Chen CH, Liu HW, Whu SW, Chen SH, Tsai CL et al. Bioengineered periosteal
progenitor cell sheets to enhance tendon-bone healing in a bone tunnel. Biomed J
2012;35:473-80.
32
Figure Legends
Figure 1. Histological analysis of the periosteum. H&E stained section of human
fibular bone showing cortical bone with periosteum. The periosteum can be divided
into an outer fibrous layer and an inner cambium layer, which is highly cellular and
contains numerous blood vessels (arrows). Scale bar = 100µm.
Figure 2. The different phases of fracture healing. Stage I: The physical
disruption of the bone, blood vessels, and surrounding soft tissue causes the
formation of a hematoma, associated with inflammation and hypoxia. Stage II:
Growth factors present in the hematoma stimulate the proliferation of skeletal
progenitor cells in the periosteum. Stage III: Progenitor cells differentiate into
chondrocytes and osteoblasts, forming a soft callus that consists mainly of cartilage
and woven bone. Stage IV: The initial woven bone is remodeled into mature lamellar
bone by the cooperative action of osteoblasts and osteoclasts, gradually restoring the
original shape of the bone.
Figure 3. In vivo bone-forming capacity of human mesenchymal progenitor
cells on calcium phosphate (CaP) scaffolds. (A-D) H&E staining of NuOss™
implants seeded with 1x106 periosteum-derived cells (PDC; A; [63]), bone marrowderived mesenchymal stromal cells (BM-MSC; B; unpublished data), muscle-derived
cells (MDC; C; [63]) and synovium-derived cells (SDC; D; [63]), retrieved eight weeks
after implantation. Bone formation (white arrows) was observed in PDC and BM-MSC
implantations. Small (unquantifiable) bone spicules were observed with MDC (white
arrow). Cell condensations (areas of increased cell density; black arrows) were
observed next to CaP granules with SDC implantation. Hematopoietic compartments
(white stars) were identifiable in both PDC and BM-MSC implants, however in larger
quantities in the latter. Scale bars = 200µm. (E) Bone quantification of implants
indicating the quantification of individual donors (black points) and mean (grey bar).
Note variability in bone formation for PDC donors indicating that factors such as age
and genetic background may play a role in the efficacy of the system.
33