Engineering in-vitro stem cell-based vascularized bone models for drug screening and predictive toxicology
Pirosa et al. Stem Cell Research & Therapy
Engineering in-vitro stem cell-based vascularized bone models for drug screening and predictive toxicology
Alessandro Pirosa 0
Riccardo Gottardi 0 1
Peter G. Alexander 0
Rocky S. Tuan 0
0 Center for Cellular and Molecular Engineering, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine , 450 Technology Drive, Pittsburgh, PA 15219 , USA
1 Ri.MED Foundation , Via Bandiera 11, Palermo 90133 , Italy
The production of veritable in-vitro models of bone tissue is essential to understand the biology of bone and its surrounding environment, to analyze the pathogenesis of bone diseases (e.g., osteoporosis, osteoarthritis, osteomyelitis, etc.), to develop effective therapeutic drug screening, and to test potential therapeutic strategies. Dysregulated interactions between vasculature and bone cells are often related to the aforementioned pathologies, underscoring the need for a bone model that contains engineered vasculature. Due to ethical restraints and limited prediction power of animal models, human stem cell-based tissue engineering has gained increasing relevance as a candidate approach to overcome the limitations of animals and to serve as preclinical models for drug testing. Since bone is a highly vascularized tissue, the concomitant development of vasculature and mineralized matrix requires a synergistic interaction between osteogenic and endothelial precursors. A number of experimental approaches have been used to achieve this goal, such as the combination of angiogenic factors and three-dimensional scaffolds, prevascularization strategies, and coculture systems. In this review, we present an overview of the current models and approaches to generate in-vitro stem cell-based vascularized bone, with emphasis on the main challenges of vasculature engineering. These challenges are related to the choice of biomaterials, scaffold fabrication techniques, and cells, as well as the type of culturing conditions required, and specifically the application of dynamic culture systems using bioreactors.
tissue engineering; vascularized bone; three-dimensional microphysiological systems; biomaterial; scaffold; preclinical model; bioreactors
Bone architecture and development
Bones are hierarchically organized over multiple length
scales. Macroscopically, bone can be divided into
compact and trabecular tissues, each with very different
mechanical strength and stiffness (Fig. 1a). The
microscopic architecture of the former is characterized by
osteons and Haversian channels containing nerves and
blood supply, whereas the latter is comprised of
interconnected trabeculae and the presence of the bone
] (Fig. 1b). At a molecular level, bone extracellular
matrix (ECM) is composed largely of the fibrous
macromolecule collagen type I and mineralized inorganic
hydroxyapatite crystals [
] (Fig. 1c). Bone is primarily
vascularized by an arterial network, but within the bone
marrow cavity and the Haversian channels the
vasculature branches into thin-walled capillaries, whose
fundamental role is the exchange of nutrients and signals
between blood and bone cells [
Bone fulfills a wide range of physiological functions. As
essential structural and load-bearing elements, bones
represent the foundation of physical locomotion and protect
our internal organs. Due to the presence of the bone
marrow, long bones serve as the tissue origin of the biological
components required for hematopoiesis. Moreover,
osseous tissues can trap potentially harmful metals (e.g., lead),
as well as maintain the homeostasis of key electrolytes via
calcium and phosphate ion storage [
]. Bone formation
can occur through two distinct pathways,
intramembranous and endochondral ossification. In both cases, the
first step is the condensation of mesenchymal cells to
produce a template for subsequent bone formation [
Intramembranous bone formation (typical of flat bones)
involves the direct differentiation of mesenchymal
progenitor cells into osteoblasts, whereas endochondral
ossification (typical of long bones) involves the initial
differentiation of the mesenchymal progenitor cells into
chondrocytes, followed by their hypertrophy, matrix
mineralization, and replacement by bone tissue [
intramembranous and endochondral ossification occur in
close proximity to vascular ingrowth. During
intramembranous ossification, capillaries invade the differentiating
mesenchymal zone, whereas in endochondral ossification,
hypertrophic chondrocytes recruit the infiltrating
vasculature. Initial vascularization is followed by invasion of
osteoclasts and osteoblasts, with coordinated resorption of
hypertrophic cartilage and subsequent mineralization of
the ECM and bone formation [
]. Hence, vascularization
occurs from the ossification centers toward the growth
plate and determines the rate of bone ossification [
]. It is
interesting to note that many of the processes that occur
during long bone formation are recapitulated during
fracture healing [
], underscoring the importance of
knowledge of bone development for the design of more
effective strategies for bone repairing [
endothelial growth factor (VEGF) is a key regulator of
angiogenesis and thus bone development [
]. For instance,
VEGF-A gene depletion was shown to attenuate the
resorption of hypertrophic chondrocytes and bone
formation, highlighting the role of VEGF-dependent
angiogenesis during bone formation [
]. VEGF-A levels
were also found to be dependent on the oxygen-sensing
hypoxia-inducible factor (HIF)-1α pathway in the
modulation of bone mass, thus confirming the role of
neoangiogenesis for the homing of osteoblast progenitor cells and
for providing bone formation/promoting factors [
fact, besides bone development, vasculature is also
essential for bone remodeling, and fracture healing is also
highly dependent on the vasculature. Dysregulated
interactions between the vasculature and bone cells are the
basis of a number of different pathologies [
]. Some of
these, such as avascular necrosis and osteoporosis, are
associated with a diminished vascular supply [
whereas others such as Gorham–Stout disease, a form of
idiopathic osteolysis caused by abnormal proliferation of
vascular structures originating in the bone , and
Klippel-Trénaunay syndrome, characterized by vessel
malformations and overgrowth of bones and soft tissues [
are caused by excessive vascularization.
Bone and related pathologies
Bone diseases can cause loss of bone strength and density,
and they may arise from nutrient deficiencies, abnormal
development, genetic disorders, impaired vasculature, and
other causes. Table 1 summarizes and compares the main
pathologies affecting bone.
Osteoporosis refers to the loss of bone density
resulting from an altered balance of the bone remodeling
process, and affects approximately 10 million US adults
50 years of age and older [
]. The most widely used
osteoporosis treatment is the administration of
bisphosphonates, which shorten the osteoclast life span and
inhibit bone resorption [
]. Although general risk factors
of osteoporosis are well documented, little is known
about the role of vasculature [
]. Some studies have
revealed a connection between low bone mineral density
and increased cardiovascular morbidity/mortality [
]. Endothelial cells (ECs) are known regulators of
vascular tone by releasing vasodilator molecules, such as
nitric oxide (NO), and they have been addressed as a
potential link between cardiovascular diseases and
osteoporosis. Studies in rats showed that the inhibition of NO
production or NO synthase (NOS) activity was followed
by marked bone loss [
], while human studies
revealed lower NOS expression resulting from estrogen
]. Since the presence of estrogen
receptors has been found in human ECs [
], it is
possible that estrogen deficiency seen in postmenopausal
women could alter the endothelial function of bone
microcirculation. Although these studies suggest that
endothelial dysfunction may play a role in the
development of osteoporosis, the exact causal relationship has
yet to be determined.
Osteoarthritis is the main cause of disability in the
], and its hallmark is the progressive
degeneration of cartilage. However, OA affects the whole joint
and all tissues play a role in the disease [
particular, the subchondral bone has been reported to be
critical in the pathogenesis of OA [
]. During movement,
there is continuous functional interaction across the
osteochondral junction. Under the diseased state, altered
mechanical loading in cartilage induces changes in bone
and vice versa [
]. The communication between the
two tissues, however, is not limited to mechanical
coupling and the associated mechanotransduction. Recent
evidence indicates that the calcified cartilage and
subchondral bone are not an impermeable barrier, and some
molecules are capable of diffusing across the
osteochondral junction [
]. Blood vessels and microchannels
have been found to reach from the subchondral bone all
the way to the uncalcified cartilage, and there is evidence
of contact between uncalcified cartilage and subchondral
bone and the marrow spaces [
]. During OA,
the osteochondral junction is significantly altered,
allowing greater transport and cellular crosstalk between
cartilage and bone [
32, 38, 42
]. Another hallmark change
of the osteochondral junction occurring during OA is
increased vascularization and neoangiogenesis [
which may further contribute to the molecular crosstalk
between cartilage and bone. Part of this signaling
involves an increase in the VEGF level in osteoarthritic
chondrocytes compared to those in healthy cartilage
], possibly contributing to the induction of vascular
invasion as part of a proregenerative mechanism. In
turn, ECs have recently been reported to enhance
chondrogenic differentiation of mesenchymal stem cells
], suggesting the potential of significant
molecular interplay between subchondral bone vasculature
and cartilage, an aspect that has not been much
investigated. Overall, increased vascularity in the subchondral
bone is associated with OA severity in cartilage and with
clinical disease activity [
Another pathogenic bone condition with devastating
consequences is osteomyelitis (OM). OM can be broadly
defined as an infection within the bone and is classified
by duration (acute or chronic), pathogenesis (trauma,
contiguous spread, hematogeneous, surgical), site,
extent, or type of patient [
]. Poor vascularity is a prime
cause for both the development of an infection and
resistance to antibiotics [
]. Acute OM can be eradicated
before osteonecrosis occurs if the infection is treated
promptly and aggressively with antibiotics and surgical
]. However, in an established/chronic
infection, fibrous tissue and chronic inflammatory cells
encapsulate the infected site, reducing vascular supply,
inhibiting an effective inflammatory response, and
limiting the action of antibiotics [
]. In addition, the
bacteria become encapsulated within a biofilm, that both
protects the bacteria from the body’s defenses and
antibiotics and serves as a chronic source of new bacterial
]. Several therapies intended to enhance
blood flow to vascular or chronically infected areas have
been tested in experimental models or clinical trials,
including hyperbaric oxygen therapy, PRP, and VEGF
]. Recently, a VEGF gene-transfected
muscle flap was shown to be effective as a treatment to
supplement systemic antibiotic treatment in the
management of experimental OM in a rodent model [
These strategies seek to enhance the body’s own defense
and the effectiveness of antibiotics by enhancing blood
flow to avascular tissue.
Osteonecrosis, or avascular necrosis (AVN), occurs
when the blood supply to the bone is disrupted,
precipitating death of the bone cells, arthritis, and
destruction of the hip joint. Common risk factors include
long-term steroid treatment, alcohol, abuse, joint
injury, arthritis, and cancer—all associated with altered
blood supply [
]. The risk greatly increases with
corticosteroid use (to treat pain and inflammation),
bisphosphonate (to prevent bone loss), and
antiangiogenic therapy (in the treatment of some cancers
or leprosy) [
]. Areas susceptible to AVN have
several common characteristics: they have limited routes
of vascular supply, they undergo relatively high rates of
bone turnover, and they may have higher than normal
exposure to bacterial infections . Evidence that
reduced vascular supply causes AVN is indicated by the
rise in AVN incidence following anti-angiogenic
treatments used in cancer therapy, such as the
VEGF-specific antibody bevacizumab, the tyrosine kinase inhibitor
sunitinib (Sutent), and the mTOR inhibitor rapamycin,
and new treatments for leprosy, such as lenalidomide.
Osteoclasts and blood vessels are closely associated
during bone remodeling, and recent studies indicate
that osteoclasts promote angiogenesis through
secretion of factors such as matrix metalloproteinase 9
(MMP9). Poor vascularization occurs in certain
inflammatory and immunosuppressive states and in the
presence of infection as well. While the pathogenesis of
AVN is largely agreed upon, the cellular and molecular
mechanisms are less understood, and thus
countertreatments to prevent the condition during
antiresorptive and anti-cancer treatments are not available.
Trauma-related injuries can lead to bone fractures,
whose healing is critically dependent upon an adequate
vascular supply. Breakage of the bone initiates the
fracture healing process, which begins with an inflammatory
reaction, followed by the processes typical of
endochondral ossification for a period of up to 28 days.
Neovascularization is critical for successful bone formation, since
vascular endothelium interruption is the first event
following trauma, which could lead to the formation of
necrotic tissue. Experimental evidence showed impaired
bone formation with administration of anti-angiogenic
], whereas VEGF treatment enhanced fracture
]. Clinical evidence showed that in the
presence of decreased vascular perfusion to the fracture, the
incidence of impaired healing (delayed union or
nonunion) increases from 10–15% to 46% [
treatment to augment bone healing and prevent delayed
union or nonunion is represented by the use of
autograft, allograft, or synthetic materials [
marrow-derived endothelial progenitor cells (EPCs)
participate in the generation of new blood vessels
(vasculogenesis) at the site of injury [
], and they have been
shown, in combination with bone marrow-derived
MSCs, to augment bone healing [
progress in improving fracture healing is hindered by the
limited knowledge of the molecular mechanisms leading
to nonunions; thus, a better understating of the
biological pathways involved in this process would certainly
benefit from the development of specific clinical
Bone also plays a role in pathologies related to ionic
homeostasis, such as calcium and phosphate [
particular, osteocytes are involved in phosphate
homeostasis through the expression of different proteins, such
as dentin matrix protein 1 (DMP1) [
], Phex, and
fibroblast growth factor 23 (FGF-23) [
]. In addition to
being key ions in systemic physiology, including muscle
functions, calcium and phosphorous homeostasis is
crucial for the development of the growth plate, which must
be mineralized to promote proper vascular invasion and
subsequent bone formation [
]. Bone may also act as a
reservoir of lead, contributing to systemic toxicity [
and it is the target tissue for the actions of a number of
teratogens (e.g., valproic acid, thalidomide, etc.) [
Studying the mechanism of how these toxins act at the
cellular and molecular levels with bone is indeed crucial
to developing new treatments.
Osteosarcoma is the most common bone malignancy
affecting predominantly adolescents and young adults, with
a 5-year survival rate of about 50–60% [
occurs mostly in the medullary cavity within the
metaphysis of long bones, an active bone growing region, and then
can propagate to the bone cortex and the surrounding soft
tissues. The molecular pathogenesis of this tumor is quite
complex and involves several elements, such as
environmental factors (e.g., UV and ionizing radiation,
methylcholanthrene and chromium salts, etc.), chromosomal
abnormalities, p53 mutations, and so forth [
]. As for
many other tumors, vasculature is a critical factor for the
survival and proliferation of cancer cells; in fact,
osteosarcoma generally involves downregulation of
antiangiogenic factors, such as thrombospondin-1 [
pigment epithelium-derived factor (PEDF) [
osteosarcoma is known as a vascular tumor, there are
contradictory data about the correlation between the
microvascular density/VEGF expression and the
formation of metastasis [
]. Emerging evidence suggests
that the blood vessels of bone may play a role in the
interactions between other tumors and the bone
microenvironment in the pathogenesis of bone metastasis . The
extravasation of tumor cells can be related to the
molecular receptors typical of a tissue/organ-specific blood vessel
]. Interestingly, blood vessels located in the
metaphysics of long bones express specific adhesion
proteins, such as P-selectin and E-selectin, which have been
shown to promote interaction and subsequent adhesion of
tumor cells [
]. One widely studied example of this kind
of interaction is the preferential spreading of breast cancer
metastasis to bone. In fact, breast cancer cells express
chemokine receptors, integrins, cadherins, and
boneremodeling factors that contribute to the successful and
preferential spread of tumor to bone [
Due to the multifactorial nature of bone diseases, the
aging population, and increased occurrence of
traumarelated injuries, bone disorders represent a big concern
and the current treatments do not provide optimal
outcomes. Furthermore, any alteration in the vascular
supply may lead to an increased susceptibility to
osteoporosis, osteonecrosis, and osteomyelitis [
models have been traditionally utilized in research to
mimic these human pathologies. Nevertheless, on
multiple occasions, animal models have been unsuccessful
and unreliable in predicting the processes and
development of human pathologies and the response to drug
candidates, because their physiology is fundamentally
different from that of humans. To overcome this
deficiency, tissue engineering may represent an alternative
platform by establishing in-vitro pathophysiological
models based on human cells to study the causes and
progression of specific diseases, and to develop and test
candidate therapeutic strategies.
In-vitro modeling of 3D vascularized bone models through tissue engineering approaches
The successful development of in-vitro engineered bone
is critically dependent on the ability to introduce a 3D
vascular network that guarantees adequate oxygenation,
mass transfer, nutrient delivery, and by-product removal
]. Here, we summarize the challenges in generating
in-vitro vascularized bone constructs and the main
materials, scaffolds, and cells required to achieve this goal,
with a focus on stem cell employment.
Challenges of in-vitro vascularized bone engineering
Bone is a highly vascularized organ and the development
of vasculature and mineralized matrix requires a
synergistic interaction between osteogenic and endothelial
]. These mechanisms have been studied in
animal models, but they are still not understood due to
the complexity of the in-vivo environment. In-vitro
scale-up of bioengineered tissues is known to be limited
by diffusion issues; therefore, the establishment of a
functional vasculature within the construct could be
essential to generate an accurate and large enough model
capable of mimicking the native tissue. Current
vascularization strategies comprise the use of angiogenic
factors combined with 3D scaffolds (Fig. 2a),
prevascularization strategies (Fig. 2b), and the use of coculture
systems (Fig. 2c) [
The first approach relies on the induction of
vascularization by endothelial precursors using
angiogenic factors such as VEGF. For instance, Braghirolli et
] demonstrated that an electrospun
poly(caprolactone) scaffold loaded with VEGF promoted the
penetration and proliferation of EPCs within the 3D matrix.
This process is primarily EC regulated and, in the
context of TE, specific interactions with the scaffold
material and other cell types are needed for optimal in-vitro
vascularization. Thus, the current research trend is
focused on the production of prevascularized constructs
in-vitro. This approach proposes the generation of stable
vasculature in-vitro using ECs and subsequent in-vivo
implantation in the target site [
]. The principal
drawback of this method, when utilized for in-vivo bone
repair, is the difficulty of generating stable in-vitro
vasculature prior to implantation. The use of coculture
systems including different cell types is more complex
and several parameters must be taken into account for
the successful outcome of a vascularized bone construct,
namely cell type (osteogenic and vasculogenic), media,
seeding methodology, culture (static or dynamic),
scaffolds, and microenvironment (e.g., oxygen tension) [
In the past decade, different attempts to generate
vascularized bone constructs using osteogenic precursor cells
and vascular progenitor cells have been made, but the
majority of these studies were conducted through
invivo models [
]. It has been difficult to create a
veritable in-vitro model containing bone tissue and
vasculature-like structures within the same construct. A
recent attempt to introduce simultaneously osteogenic
differentiation and vasculature development in-vitro was
made by Tsigkou et al. [
], which combined human
bone marrow MSCs and human umbilical vein
endothelial cells (HUVECs) seeded on a polymeric scaffold and
in a hydrogel, respectively. They observed the formation
of capillary-like structures 4–7 days after implantation in
a mouse model, and MSCs were found to be necessary
for the development of a stable vasculature. The main
outstanding issue is still the generation of accurate
invitro models, due to the difficulties of obtaining quality
mineralized bone matrix, further complicated by the
introduction of the vasculature.
Bone mineralized matrix formation is provided by
osteoblasts during embryonic development and postnatal
growth, yet about 95% of the cellular population residing
in the adult skeleton is represented by osteocytes.
Osteocytes are the terminally differentiated osteoblasts
embedded in the lacunae within the bone matrix. They
are characterized by cellular processes radially spread
toward the mineralized matrix inside tiny tunnels called
canaliculi, filled by canalicular fluid [
]. The presence
of osteocytes in an engineered bone construct is an
indication of a mature developed osseous tissue. Although
osteocytes play a secondary role in bone tissue
formation, they are functionally important in its homeostasis,
mechanosensation, and mechanotransduction [
Osteocytes sense canalicular fluid flow-derived shear
stress through their primary cilium  and specific ion
channels and mechanotransduction proteins, releasing
in response paracrine signaling molecules such as NO,
ATP, and prostaglandins, which regulate osteoblast and
osteoclast activity [
]. The lacuna–canalicular
network connects the osteocytes to the vasculature and
there is evidence linking osteocyte apoptosis and
reduced VEGF production with consequent impaired
vascularity and bone strength [
]. Taken together, these
findings highlight the importance of the presence of
mature osteocytes in an engineered bone. For this
reason, many investigations are focused on the derivation
of mature osteocytes from stem cells to better
understand their physiology and assess their potential in bone
Bone is also innervated by sensory and sympathetic
nerve fibers that, in addition to skeletal pain
transmission, play a role in bone metabolism. Bone cells have
been recently shown to express adrenoceptors, receptors
for norepinephrine (NE), calcitonin gene-related peptide
(CGRP), substance P, and vasoactive intestinal peptide
]. NE reuptake influences bone
remodeling by osteoclasts , CGRP deficiency was linked to
decreased bone formation in mice [
], while SP has
been shown to have both dose-dependent
boneresorbing and formation activity [
]. These are just a
handful of examples on how innervation has been linked
to bone production and turnover; thus, an in-vitro
engineered bone construct would certainly benefit from the
presence of nerve endings and related neurotransmitters.
However, the generation of a vascularized and
innervated bone construct clearly presents an additional level
of complexity in terms of integrating and regulating such
different tissue types [
Another parameter to consider when developing
veritable in-vitro vascularized bone is the incorporation of
osteoclast activity inside the engineered constructs.
Osteoclasts are essential for bone remodeling by
resorbing bone matrix and generating physical space for
osteoblasts and ECs, thus allowing the formation of new bone
tissue and vasculature [
]. The dissolution of the
inorganic phase of bone matrix takes place by the
secretion of hydrochloric acid, while the organic matrix is
resorbed by secreted enzymes, like cathepsin K and
metalloproteinase 9 [
]. The resorption activity as well as
the area and depth of the resorption pits can be analyzed
in-vitro using osteoclast-like cells derived from
monocytes, for instance the RAW 264.7 cell line [
however, to date, there are considerable variations in the
examined parameters. There is no specific, single
functional osteoclast cell marker; thus, different parameters
should be analyzed together, such as tartrate-resistant acid
phosphatase 5b (TRAP 5b) [
], morphological changes
from monocytes [
], and 3D volume characterization of
]. Furthermore, as different materials can affect
the response of osteoclasts, the choice of the scaffold is
critical for an optimized bone engineering strategy.
Different authors have studied the resorption ability of
osteoclasts using different scaffolds/materials, from dentin to
bone substitutes, as well as calcium phosphate and
bioactive glasses [
], and have observed specific
resorption rates depending on the material used. For
example, Keller et al.  found that natural
biomaterials were resorbed more rapidly than synthetic
ones, while Badran et al. [
] showed an inhibition
of osteoclast activity by increasing mineral density.
As highlighted by Alexander et al. in a recent
], a TE approach can be used for the
development of in-vitro preclinical models of normal/
pathological tissue function, but an effective
highthroughput assay should consist of a minimal system
with well-defined performance parameters. These
systems should model the structure and function of
human tissue, as well as the physiological response to
different stimuli, such as the interaction with adjacent
]. Bone actively interacts with cartilage,
muscles, ligaments, tendons, and many other tissues in
its physiological function within the musculoskeletal
]. An ideal in-vitro model of bone should
consider the possibility of studying its interaction with
other tissues, thus posing significant challenges in
accommodating different, tissue-specific microenvironments.
Due to their pluripotency/multipotency, stem cells
represent an exceptional tool to achieve this complex
integration between bone and other tissues. A good
model to study the interaction between bone and
cartilage was developed by Lin et al. [
], who produced a
biphasic construct mimicking the nature of the
osteochondral junction. This construct was developed using
a 3D-printed dual-chamber bioreactor that allowed the
creation of two separate yet communicating
]. Osseous and chondral phases were
generated using one stem cell type, bone
marrowderived MSCs, encapsulated in a photocrosslinked
gelatin methacrylate (gelMA) hydrogel. Osteogenic and
chondrogenic media were used to differentiate MSCs
toward bone and cartilage phenotype, respectively. The
two constructs were independently controlled and
tested by the introduction of bioactive agents or
Although different models have been created to
recapitulate the interaction between bone and other adjacent
tissues, to the authors’ knowledge, an in-vitro engineered
model coupling vascularized bone to another tissue has
not yet been developed. Such capability would be
particularly relevant when engineering the osteochondral
junction, as only the simultaneous presence of bone
matrix, vasculature, and cartilage can enhance or permit
veritable behavior of the complex. In fact, the
interactions between vasculature and cartilage are well known
during in-vivo skeletogenesis [
], and numerous
in-vitro studies have documented the molecular
exchanges driving the inhibition of chondrocyte
differentiation, including the pivotal role exerted by VEGF
secreted by ECs [
]. However, a model also
containing subchondral bone would better mimic the
complex interaction among the three tissues. For these
reasons, a triphasic scaffold was produced by the authors
using a recently developed 3D-printed
microphysiological tissue system (MPS) bioreactor that allows the
separate flow of specific media to the chondral and osseous
components, while maintaining them in contact and
allowing tissue–tissue communication  (Fig. 3). The
cartilage construct was engineered incorporating human
bone marrow-derived MSCs in a photocrosslinkable
gelMA hydrogel, while the vascularized osseous
construct was obtained by seeding MSCs and HUVECs in a
poly(ε-caprolactone) (PCL) scaffold, produced through
additive manufacturing as described by Puppi et al.
]. Results from our preliminary assessments suggest
a better differentiation of MSCs in the presence of the
HUVECs, which form interconnected tubular-like
structures in the osseous compartment.
Overall, the development of a successful in-vitro model
of vascularized bone is heavily dependent on optimal
selection of scaffold/matrix able to guide the formation of
the different tissues, and on fine control of cell signaling.
Mineralized bone and associated vasculature are
characterized by different morphological and structural
properties, thus different biomaterials are needed for the
engineering of both tissues to match their
characteristics. Different materials have been investigated for the
development of bone constructs, and evaluated both
invitro and in-vivo. Calcium phosphates (CaPs) have been
of great interest due to their osteoconductive properties.
The majority of research has been focused on two main
forms of CaPs, hydroxyapatite (HAp) and
betatricalcium phosphate (β-TCP), or the combination of
]. Other inorganic materials that have been
exploited in bone engineering are bioactive glasses [
which have been shown to improve the formation of a
hydroxycarbonate apatite layer by osteoblasts in-vitro
and to support bone formation in-vivo [
]. The most
commonly used polymeric materials for bone
engineering include many natural polymers, such as collagen,
fibrin, alginate, silk, hyaluronic acid, chitosan, and
]. These biopolymers offer
economic and environmental advantages, such as low
manufacture/disposal costs and renewability, as well as
biological advantages, such as supporting cell signaling,
adhesion, and cell-mediated degradation and remodeling
]. Synthetic polymers have also been thoroughly
investigated as engineered bone scaffolds, by virtue of their
controllable and reproducible chemical–physical and
degradation properties, which can be tailored to the
requirements of the target applications [
]. The different
classes of polymers that have received significant
attention include saturated poly(α-hydroxesters), such as
poly(glycolic acid) (PGA), poly(lactic acid) (PLA), and
PCL, and biodegradable polyurethanes, covering a wide
range of properties, including mechanical strength,
elasticity, biodegradability, hydrophilicity, and so forth
]. When considering the development of a
capillarylike network within a mineralized bone construct, the
most current models include the embedding of
endothelial-derived cells in hydrogels, such as Matrigel®,
collagen, and fibronectin [
]. However, these
materials have limitations in mechanical stability,
durability, and immunogenicity when implanted [
]. One recent example to overcome these limitations
is represented by the use of methacrylated gelatin
(gelMA), produced by conjugating methacrylate groups
to the amine-containing side groups of gelatin, which
becomes a photocrosslinkable hydrogel . This
material is characterized by the advantages of both natural
and synthetic polymers, allowing the fine-tuning of
mechanical properties, while preserving biological cues.
Different studies in the past have reported the capability
of creating vascular-like structures using gelMA and ECs
80, 89, 142
There have been a number of investigations that
explored a variety of biomaterials for the engineering of
vascularized bone; however, identifying the most suitable
biomaterials remains a difficult task, since each material
has its inherent drawbacks. Ceramics are characterized
by brittleness and the biodegradation rate not matching
the formation of new bone tissue; natural polymers
possess low mechanical, thermal, and chemical stability; and
synthetic polymers lack biological cues. A logical path to
overcome these limitations would be to combine
different materials to obtain constructs with improved
properties. An example of this paradigm is the development of
nanocomposite materials based on biopolymers and
ceramic nanofillers, in an attempt to exploit the biological
activity of natural polymers as well as the
osteoconductivity of ceramics [
All of the biomaterials already described require
multistep processing to develop 3D scaffolds/matrices capable
of inducing and supporting osteoblast proliferation and
differentiation, as well as vasculature development.
Accordingly, in addition to osteoinductivity and
osteoconductivity, an ideal scaffold should have functional
physical properties such as interconnected
macroporosity with pore size larger than 100 μm for optimal
osteoblast differentiation and formation of new blood vessels,
and biocompatible stiffness to match the mechanical
properties of native bone [
]. Substrate mechanical
properties greatly influence stem cell osteogenic
differentiation, as well as EC behavior [
]; for instance, a
scaffold Young’s modulus in the range 25–40 kPa directs
MSCs toward osteoblastic differentiation [
Current technologies do not allow the production of
scaffolds with the exact mechanical properties of bone;
however, a number of groups have explored
development-inspired precursor templates to instruct
stem cells to form a mature tissue [
such constructs do not possess the same mechanical
properties as native bone initially, they are engineered
in-vitro with sufficient stiffness to be implanted in a
load-bearing region and are able to guide stem cells to
develop mature bone. For instance, Daly et al. 
produced an MSC-laden 3D-printed hypertrophic cartilage
template, made of a reinforced alginate bioink, which
developed into functional vascularized bone when
implanted in mice.
Biologically inspired scaffolds should also harbor
signals that act to induce the simultaneous development of
bone and vasculature. Different biological scaffolds have
been used for the development of in-vitro vascularized
bone models, including decellularized bone and specific
ECM preparations. Decellularized bone has been
historically considered and applied as a biologically derived
matrix, able to induce mineralized bone production by
osteoprogenitor cells. A recent study by Correia et al.
] showed the employment of decellularized bone
plugs as scaffold for HUVECs and MSCs seeded in a
fibrin carrier. Vasculature was able to grow inside the
porosity of the scaffold both in-vitro and then in- vivo
after subcutaneous implantation in mice. The use of
decellularized ECM has been reported recently by Gao
et al., who developed a vascular patch using MSCs in a
decellularized human aortic matrix [
strategy involves the functionalization of “smart” scaffolds
with angiogenic and osteogenic factors such as VEGF,
providing a highly localized signal to control stem cell
The need to control macrostructural and
microstructural properties of a scaffold to meet key requirements
of a specific application has led to the development of
different manufacturing technologies, such as solvent
casting/particulate leaching, freeze drying, phase
separation, and combinations of these techniques [
conferring to the scaffold different properties. For
instance, using a thermally induced phase separation
(TIPS) technique, Mannella et al. [
] developed a
porous scaffold with a pore size gradient able to mimic the
porosity of the cancellous bone. Although several studies
about the production of bone scaffolds using the
aforementioned techniques have been published, these
methodologies are lacking in control over the fine
architecture of the structure, specifically in relation to
pore morphology and interconnection, fundamental
requirements for the introduction of vasculature. There
are no precise values for specific bone and/or vascular
ingrowth; pore sizes greater than 100 μm have been
shown to favor bone formation [
], while capillary
density is promoted when pore sizes are greater than
300 μm [
]. For these reasons, solid freeform
fabrication (SFF) or additive manufacturing (AM) techniques
are attracting great interest, owing to their capabilities of
producing predefined interconnected porous structures,
using natural and synthetic polymers, as well as ceramics
as starting materials [
]. AM techniques comprise the
layer-by-layer building of 3D structures, based on
computer-aided design (CAD) and computer-aided
manufacturing (CAM) processes. Depending on the
specific working principles, AM can be classified into
laser-based systems (e.g., selective laser sintering,
stereolithography), printing-based systems (e.g., 3D printing),
and nozzle/extrusion-based systems (e.g., fused
deposition modeling, computer-aided wet-spinning), each
allowing for specific scaffold macroarchitectural and
microarchitectural features [
]. AM has significantly
improved the technical ability to control key factors in
bone scaffolds, such as composition, pore geometry, size,
and interconnectivity, as well as scaffold mechanical
performance. The fine-tuning of morphological parameters
provided by AM allowed researchers to produce
scaffolds for the concomitant development of osseous and
vascular tissue. 3D printing has been thoroughly
exploited for the generation of bone scaffolds with
vascular integration, mostly using ceramics [
], but also
synthetic polymers [
], natural polymers [
]. 3D-printed scaffolds have also been
loaded with bioactive molecules, such as BMP and
VEGF, to promote bone formation and vascularization
respectively. Novel studies are exploring the
incorporation of other molecules, such as KR-34893 indene
compound that stimulates MSC differentiation and mineral
], oxygen-releasing agents like calcium
peroxide to solve O2 diffusion issues [
plateletrich fibrin to stimulate bone marrow-derived MSC
]. AM and 3D-printing technologies
have also produced great advancements in the field of
microfluidics systems and organ-tissue chips that
employ stem cells for in-vitro disease modeling and drug
]. In-vitro vascularized bone models
have steadily benefited from this technology. For
instance, Jusoh et al. [
] recently reported a
microfluidics platform based on HAp-loaded ECM to study
angiogenesis and osteogenesis in a vascularized bone
model. Furthermore, AM can be integrated with other
scaffold fabrication methods to fabricate hybrid
architectures with unique structural features, as described in
detail by Giannitelli et al. [
]. Although AM
techniques were widely studied for the development of bone
scaffolds, to date, there are few studies employing them
to develop in-vitro models of vascularized bone. A
summary of the main scaffolds and fabrication techniques
employed in bone engineering is presented in Table 2.
Another fabrication technology that is increasingly
gaining attention is bioprinting, which refers to the
predefined and precise dispense of cell-laden biomaterials
for the construction of complex “living” 3D structures
]. If the cell selection is represented by
osteoprogenitor stem cells, the resulting structure will be
composed of cells capable of producing bone matrix. The
geometry and dimension of the construct can be
controlled in an automated manner such that specific
morphologies may be fabricated based on the
application. Bioprinting, thus, represents another promising
approach for the production of in-vitro models of bone
]. Traditionally, bioprinting has been applied mainly
to the formation of soft tissues and in-vitro vasculature.
For instance, Norotte et al. [
singlelayered and double-layered vascular tubes with a
diameter ranging from 0.9 to 2.5 mm, using different vascular
cell types, such as smooth muscle cells and fibroblasts.
The identification of new stem cell sources as vascular
precursors, coupled with the advancement in bioink and
microfluidics technologies, has allowed bioprinting to
create constructs that mimic the arrangement of the
vasculature in bone for more precise organ modeling.
Numerous studies of vasculature bioprinting and chips
using stem cells have disclosed different aspects of blood
vessel biology, such as the role of transforming growth
factor beta on normal vascular function [
], the role
of MSCs in promoting vasculature formation [
regulation of the perivascular stem cell niche by MSCs
], and the role of angiopoietin in MSC transition to
mural cells in the presence of ECs [
bioprinting requires the employment of materials with better
mechanical properties, needed to mimic the stiffness of
the bone matrix. One example of mechanically
reinforced bioprinted construct has been made from alginate
and PLA nanofibers, supporting adipose-derived MSC
viability and differentiation [
]. Another approach is
the use of stem cell-laden hydrogels in combination with
thermoplastic fibers made of PCL or poly(vinyl alcohol)
]. Gao et al.  developed a
photopolymerized acrylated peptide, coprinted with
poly(ethylene glycol) (PEG) which showed robust osteogenesis of
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MSCs. MSC osteogenesis has been also stimulated by
combining low-intensity pulsed ultrasound (LIPUS), as
mechanical stimulation, and a 3D-bioprinted PEG–RGD
]. Other materials have been employed, in
various formulations, for the development of bioprinted
bone constructs, ranging from agarose and gelatin to
]. The integration of bioprinted
vasculature and mechanically stiffer substrates would greatly
improve the production of functional in-vitro
vascularized bone. One example of this paradigm featured a dual
3D bioprinting system based on fused deposition
modeling (FDM) and selective laser ablation (SLA) [
authors used alternate deposition of stiff polylactide
(PLA) fibers and MSC/HUVEC-laden gelMA hydrogel
to achieve proper mechanical strength and biological
cues of complex vascularized bone constructs. Another
approach comprised the production of a mandible
fragment using an integrated tissue-organ printer (ITOP),
which was able to bioprint Pluronic-F127 hydrogel laden
with human amniotic fluid-derived stem cells (hAFSCs),
in combination with a PCL-based mechanical backbone,
and featuring incorporated microchannels for nutrient
]. Kolesky et al. bioprinted a thick
vascularized tissue using different polymeric templates and
fugitive inks. The construct was laden with MSCs in
combination with HUVECs, as well as other
parenchymal and stromal cells, showing robust osteogenesis and
functional perfusion of the vasculature [
The great landscape of current scaffold fabrication
technologies has provided a wealth of choices for the
generation of in-vitro vascularized bone constructs.
Future attention should be focused on high-throughput
and high-resolution AM techniques that are able to
produce functional vasculature within a mature bone
construct. The key aspect is the biocompatibility of these
methods with the chosen cells of interest in order to
obtain their optimal functionality.
Osteogenic and vascular precursors
When considering the development of an in-vitro model
of vascularized bone, the selection of suitable cell
sources is crucial. A desired cell source must have little
to no limitation in terms of availability and be easy to
maintain and manipulate in-vitro. Adult MSCs are
widely employed for somatic TE by virtue of their ability
to differentiate into multiple lineages, such as cartilage,
fat, muscle, and bone [
]. MSCs can be readily
isolated from different tissue sources, such as adipose,
muscle, bone, and in particular, bone marrow, which is
the most widely used source [
]. In fact, bone
marrow-derived MSCs have been benchmarked as one
of the most appropriate cell sources for bone TE due to
their well-defined osteogenic differentiation [
By using an appropriate culture medium, MSCs can be
expanded without differentiation, and induced to
undergo specific differentiation to a stable phenotype in
]. In addition to bone marrow-derived
MSCs, adipose-derived MSCs (ASCs) are now a widely
accepted source for bone tissue engineering applications
and have been employed also in numerous preclinical
and clinical studies [
]. Induced pluripotent stem cells
(iPSCs) and embryonic stem cells (ESCs) have also been
studied as potential cell sources for bone regeneration
]. Although pluripotent ESCs are a promising
cell candidate for the development of fully functional
vascularized bone in-vitro as they can form all
specialized cell types constituting the human bone, including
its vasculature , ESCs research also raises ethical
and political controversies due to their derivation from
early human embryos [
]. For these reasons, the use
of iPSCs obtained by reprogramming of adult somatic
cells, thus avoiding the ethical problems inherent in ESC
research, has gained significant attention.
Current models of biomimetic, engineered
vascularized bone, fabricated by culturing human MSCs on 3D
scaffolds resembling the matrix of native bone [
require a vascular compartment created using other cell
sources. Various sources of stem cells, such as ESCs,
MSCs, and iPSCs, have been identified as potential
candidates for vascular engineering [
cells have also been successfully differentiated from
amniotic fluid stem cells [
]. The choice of vascular
precursor cells is crucial for functional production of a
proper vasculature within the in-vitro bone model.
Mature ECs have been traditionally used to stimulate
angiogenesis , among them HUVECs, which
represent one of the most commonly employed cells in
vascularized bone engineering. These cells naturally form
vessel-like structures when cultured in hydrogels and,
most importantly, they were demonstrated to enhance
MSC osteogenesis in-vitro [
]. Another cell
type that has gained attention are the EPCs, which were
found to be 10 times more proliferative than HUVECs
 and have been recently used, also in combination
with MSCs, to improve vasculogenesis in-vivo [
and in tissue engineering applications [
vessel development was also achieved by employing
iPSC-derived ECs .
As mentioned earlier, active osteoclasts are also a
crucial component of in-vitro bone modeling; however, the
exact optimal cell source for osteoclasts has yet to be
defined. There are several cell types that can be
differentiated into osteoclast-like cells, such as bone marrow and
peripheral blood mononuclear cells [
human mononuclear leukocytes isolated from umbilical
cord blood . Osteoclasts can be directly isolated
from native bone tissue [
]. RAW 264.7, a mouse
leukemic monocyte–macrophage cell line, has been
frequently used to study osteoclastogenesis in-vitro
]. Primary monocytes can also be differentiated into
osteoclast-like cells and used to study osteoclastogenesis
]. Furthermore, osteoclasts have been
obtained by differentiating macrophages derived from
human iPSCs [
These well-established, strong relationships among the
constituent cell types, taken together, underscore the
important and fundamental requirement of these cells in the
success for development of vascularized bone in-vitro and
the production of a veritable bone model (Fig. 4).
Based on the cells selected for the development of a
vascularized bone construct, the choice of culture media
and seeding methodology has to be carefully considered
to avoid negative, undesired effects. Different medium
formulations and supplements have been identified for
optimal osteogenic differentiation of stem cells.
Dexamethasone, β-glycerophosphate, ascorbic acid,
1,25-dihydroxyvitamin D3, and BMP-2 have significant
osteogenic inductive activity on MSCs by influencing
different aspects of their biology, such as activation of
specific genes (osteopontin, core binding factor α1,
alkaline phosphatase, osteocalcin) as well as signaling
pathways like Wnt [
]. Regarding the culture of
endothelial progenitors, VEGF, basic fibroblast growth
factor (bFGF), and epidermal growth factor (EGF) are
usually employed to stimulate endothelial progenitor
expansion and differentiation/angiogenesis [
]. The use
of individual differentiation medium, endothelial or
osseous, generally yields satisfactory vasculature and
mineralized matrix formation, albeit separately [
However, given the need for simultaneous development
of bone and vasculature, a coculture system must be set
up, and investigators have used either a combination of
the two differentiation media [
88, 206, 225–227
single type of medium [
], or osteogenic medium
with some vascular growth factors . In the case of a
coculture system, while different cell types can be seeded
simultaneously to avoid uneven distribution inside the
construct and to allow communication between them,
the choice of culture media is very important to assure
cosurvival and differentiation. In another approach,
different cell types can be expanded and differentiated
separately and then seeded together, thus avoiding the
media problem; however, some critical cell–cell
interactions will be missing and, as discussed earlier, the
generation of a veritable in-vitro model, which requires
recapitulation of the interaction between bone cells and
vasculature, may thus be compromised. Another
parameter that needs to be considered is the cell ratio.
Signaling between osteogenic and vascular precursors, as well
as their adult counterparts in the mature organ, directs
their functional integration (see Fig. 4). Altering the
balance of this crosstalk compromises the engineering of
the vascularized bone tissue. Investigators have studied
varying combinations between osteogenic and vascular
precursors, ranging from a 1:1 mix to unbalanced ratios
in favor of osteogenic or endothelial precursors.
Although a positive trend can be identified in the use of
a 1:1 ratio, the results are also heterogeneous and an
ideal ratio cannot be defined a priori [
]. Table 3
summarizes the main media and cell ratios that have been
used in the engineering of vascularized bone and their
major benefits and drawbacks.
A recently published review by Liu et al. [
different aspects of the medium combinations and
seeding methodologies used in coculture systems for
vascularized bone tissue engineering. However, due to the
heterogeneity of the experimental parameters used in
coculture studies, a clear trend is difficult to establish.
Considering the tight interactions between osteogenic
and vascular stem cells during development, and the
functional integration between bone and blood vessels in
the mature organ, the development of veritable in-vitro
models of this organ should comprise both types of cells
cultured in a mixed medium in order to not miss the
key interplay between osseous and vascular precursors/
Bioreactors are generally defined as any device that is
able to dynamically sustain biological and/or
biochemical processes under precisely monitored and controlled
experimental and operating conditions (e.g., pH,
temperature, mechanical stimuli, time, nutrient supply,
and waste removal) [
]. Different types of
experimental setups have been developed to optimize cell seeding
and mass transport, such as spinner flasks (Fig. 5a),
rotating wall vessels (RWVs) (Fig. 5b), and perfusion
bioreactors (Fig. 5c). Spinner flasks and RWVs minimize the
nutrient gradient and metabolite concentration around
the construct, while perfusion bioreactors directly
perfuse media inside the scaffold, thus assuring mass
transport within the porosity [
]. Using optimal
material–scaffold–cell systems, coupled with the higher
control over experimental parameters, has made
bioreactors ideal means for the development of 3D tissues
invitro. This is particularly true for the engineering of
biological interfaces or complex tissues, like the
osteochondral junction and vascularized bone. Bioreactors, in
combination with MSCs, have in fact been used for the
recreation of the osteochondral tissue interface, as
reported in a number of studies [
121, 233, 234
particular, culturing in flow perfusion bioreactors has
been shown to upregulate expression of osteoblastic
], and these bioreactors have been
employed for the production of engineered bone [
]. To improve the amount and quality of bone
beyond what has been produced by an in-vitro process,
various studies have reported the use of “in-vivo
bioreactor” systems to produce a clinically relevant amount of
vascularized bone (Fig. 5d). These approaches were
based on the body’s healing mechanism that supports
the formation of neotissue in different kinds of scaffolds,
ranging from calcium-alginate gel, to β-TCP, to natural
bovine bone mineral-coated titanium meshes implanted
subperiosteally in-vivo [
]. The engineered bone
produced with this approach could be harvested from
the “living bioreactor” host and subsequently
transplanted successfully at a bone defect site. While in-vivo
engineering surely ensures production of enhanced
vascularized bone, high-throughput assays require a large
number of identical samples that cannot be performed
by in-vivo systems, thus necessitating the development
of in-vitro platforms of vascularized bone engineering.
In addition to improving in-vitro osteogenic
differentiation of MSCs in the presence of ECs, several studies
have reported better matrix mineralization by osteogenic
MSCs cultured under dynamic conditions rather than
static ones [
]. However, the combination of
these two strategies using a flow perfusion bioreactor
did not significantly enhance osteogenic differentiation
O osteogenic, V vascular, Expansion expansion medium for osteogenic precursors
Pros: technically simpler; even mix distribution within
the construct; cell–cell crosstalk during differentiation
Cons: risk of suboptimal individual cell type viability and differentiation
Sequential seeding 
Pros: optimal differentiation of the first seeded cells;
Cons: uneven distribution of the two types of cells within the construct
Independent differentiation 
Pros: optimal differentiation of each cell type in their Cons: lack of cell–cell communication
of MSCs in the presence of ECs [
because shear stress affects the function of ECs [
However, another approach was used by Nishi et al.
], who cultured MSCs and ECs on a porous
poly(lactic acid) scaffold in a rotating wall vessel bioreactor.
The flow environment created by the RWV bioreactor,
coupled with the interaction between the cell types,
enhanced the distribution and differentiation of cells in the
scaffold. To date, there are only a limited number of
studies using a bioreactor in combination with bone and
vascular progenitor cells, due to the technical and
biological challenges of codifferentiating the two cell types
together. The successful construction of a physiologically
compatible bioreactor for engineered vascularized bone,
to achieve fast and easy visualization of the target cells
and their interactions, must take into account a number
of design features, including noninvasive optical access,
automation, uniform cell seeding, overall chamber
dimensions, checkpoint markers, and mechanical stimuli
In utilizing engineered vascularized bone models
invitro, instead of harvesting tissue samples and analyzing
them at designated time points, the nondestructive
tracking of cells and new matrix formation should result
in a reduction of resources and permit real-time
understanding of the processes. One example is represented
by the work reported by Bersini et al. [
developed a biological 3D in-vitro microfluidic model to study
breast cancer metastasis to bone. This microfluidics
platform is based on hydrogel-containing chambers placed
between surface-accessible microchannels, allowing
high-resolution real-time imaging of single-cell behavior,
cell–cell communication, cell–matrix interactions, and
cell population dynamics [
]. Another useful
highthroughput platform was developed by Moya et al. [
to study vasculature in real time. They produced an
invitro 3D metabolically active stroma (~ 1 mm3 volume)
containing a living and dynamically perfused human
capillary network, by combining human endothelial
colony forming cell-derived ECs (ECFC-ECs) and a
polydimethylsiloxane (PDMS) micromold. Although
diffusion of oxygen was demonstrated to not be a limiting
step in-vitro, thus allowing the development of tissue
constructs in the order of centimeters in thickness [
the major technical problem is nutrient diffusion. The
density of the ECM plays an important role in the
diffusion of nutrients and the morphogenesis of capillaries
], and this is particularly true for mineralized bone
matrix. In addition, real-time visualization of the process
occurring inside the bone matrix is also limited by the
opacity of the bone matrix itself, thus requiring the
development of specific bioreactor or in-vitro models to
overcome this limitation. X-ray microcomputed
tomography (μCT) has been extensively used in the field of
bone tissue engineering due to its ability to provide
rapid, nondestructive 3D images of bone and bone
scaffold microstructure at 1–50 μm resolution [
]. Various groups have developed μCT-compatible
], utilizing low radio-opacity
materials, such as polysulfone, with dimensions matched to
those of standard μCT chambers. Another requirement
is that such systems should allow the study of the
vasculature within in-vitro vascularized bone, which, however,
is complicated by the 3D nature and limited access to
the internal microvasculature due to the loss of optical
access. While the analysis of thin sections of engineered
vascularized bone could address this issue, it would
obviate the purpose of recapitulating the 3D in-vivo
structure. A more viable solution is the use of
contrastenhanced μCT for the investigation of microvasculature
], which was previously studied in soft tissues, such
as the kidney, heart, and liver [
]. This method is
based on the injection of a radio-opaque contrast agent
within the vasculature prior to imaging. However, again,
the opacity and density of the bone matrix put some
limitations on visualization of the vessel microstructure
when this is performed in a bone model. It is thus not
surprising that few studies have been published on the
analysis of bone microvasculature using μCT [
illustrating the future potential of contrast-enhanced
μCT for the evaluation of bone neovascularization in
Perspectives and future directions
The definition of the parameters that must be
accommodated in the design of in-vitro models of vascularized
bone represents a challenging task for researchers. The
complex interactions between blood vessels and bone
have limited our ability to develop veritable in-vitro
constructs of physiological systems representing human
bone. First, from a biological point of view, the
codifferentiation of endothelial precursors and osteogenic stem
cells is hindered by the different mechanisms and signals
to which these cells respond when recapitulating in-vivo
development. As shown in numerous studies, a
compromise between endothelial and osteogenic signals/cues
must be considered, in order to minimize undesired
differentiation of the cell types. On the other hand,
predifferentiation of vasculature or bone cells does not permit
the critical communications between the two
physiological systems during development or repair, thus
compromising the morphogenesis of the actual structure of
the tissue complex and in a manner similar to native
bone. This aspect is of high relevance when studying the
effects of drugs and/or toxicants on the structure and
development of bone, as well as screening for potential
treatments. In other words, the engineered vascularized
bone model must present advantages over the majority
of studies that have been performed in-vitro on 2D
cultures of separate endothelial and osteogenic cells or
cocultures, and through in-vivo animal models [
]. From an engineering point of view, the design of a
system which could maintain the vitality and
differentiation of different types of cells for long-term culture is
also a significant challenge. Furthermore, experimental
parameters must be controlled (i.e., system variables
must be clearly defined) and, as described earlier, the
system must be accessible for real-time imaging and
evaluation. Bioreactors have partially solved the need to
have in-vitro systems which could mimic the full
physiological structure of bone and, at the same time, have
allowed analysis of the constructs.
High-throughput screening of drugs or toxicant relies
on a minimal system defined by precise parameters that
can be stimulated by specific stimuli, such as
mechanical, biochemical, genetic, and so forth. To address this,
our group has developed a triphasic model composed of
blood vessels, bone, and cartilage to study the
interaction between these three different tissues [
3D-printed microphysiological system, in combination
with stem cells, allows for the codifferentiation of the
three different tissues, and is also responsive to the
introduction of different stimuli, including
stressinducing factors, such as IL-1β for the study of
osteoarthritis, female hormones like estrogens for the study of
], or teratogens to study their effect on
skeletal development. The capabilities of additive
manufacturing have enabled us to produce customized
scaffolds and bioreactors to meet the requirements of
vascularized bone engineering, coupled with a
highthroughput platform for drug screening and toxicology
assessment. In the future, bioprinting could represent a
suitable on-demand platform for the versatile fabrication
of specific cellular patterns at the micrometer scale for
the production of vascularized bone, but also a broad
range of other engineered tissues [
]. While still in its
early stage, bioprinting can also benefit from the wide
range of additive manufacturing techniques and
bioreactor technologies to generate veritable in-vitro models of
vascularized bone that could help understand the
physiopathology of bone, and generate valuable
treatment strategies. To become an economically viable
resource, the production of in-vitro 3D models of
vascularized bone should also consider the introduction of a
certain level of automation, to reduce human
intervention as well as product variability. An interesting and
comprehensive review about this topic was recently
published by Costa et al. [
], who reviewed the current
automated tools and strategies for the production of
In addition to applications in regenerative medicine,
tissue-engineered constructs could be used as in-vitro
preclinical models of normal or pathological tissues for
applications in drug screening and toxicity assessment.
For effective high-throughput assays, minimal systems
and accurate modeling of the structure/function of the
studied human tissue or organ are required. Bone
pathologies present significant disease burden, underscoring
the need to develop more effective treatment strategies.
The development of in-vitro high-throughput
microphysiological models that faithfully recapitulate the
physiopathology of bone is thus highly relevant, particularly
given the cost and intrinsic genetic difference of animal
models. Different biological and engineering challenges
must be overcome, including the right choice of stem
cells, regulation of codifferentiation of vasculature and
bone, control of system parameters and stimuli, access
for real-time imaging, and functional evaluation. In the
pursuit of this initiative, additive manufacturing
techniques and bioreactor technologies have increased our
ability to produce systems that integrate cells, scaffolds,
and biological/environmental stimuli to create in-vitro
models of native osseous tissue, and to study the
processes regulating its biology.
EC: Endothelial cell; EPC: Endothelial progenitor cell; ESC: Embryonic stem cell; HUVEC: Human umbilical vein endothelial cell; iPSC: Induced pluripotent stem cell; MSC: Mesenchymal stem cell; μCT: Microcomputed tomography
The authors acknowledge funding support from the sources indicated.
This work was supported by the Commonwealth of Pennsylvania, Department of Health, the Environmental Protection Agency (R835736), the US Department of Defense (W81XWH-14-1-0217 and W81XWH-14-2-0003), CASIS (GA-2016-236), the Ri.MED Foundation, and the National Institutes of Health (1UG3 TR002136).
Availability of data and materials
Not applicable to this article, as no data were generated or analyzed.
All authors participated in the writing and review of the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
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