876286

review-article2019
JAO0010.1177/0391398819876286The International Journal of Artificial OrgansPerić Kačarević et al.

IJAO The International

Journal of Artificial
Organs
Review

The International Journal of Artificial

An introduction to bone tissue engineering

Organs
1­–18
© The Author(s) 2019
Article reuse guidelines:
sagepub.com/journals-permissions
https://doi.org/10.1177/0391398819876286
DOI: 10.1177/0391398819876286
journals.sagepub.com/home/jao
Željka Perić Kačarević1, Patrick Rider2 , Said Alkildani3,
Sujith Retnasingh4, Marija Pejakić5, Reinhard Schnettler6,7,
Martin Gosau6,7, Ralf Smeets6,7, Ole Jung7,*
and Mike Barbeck2,6,8,*

Abstract
Bone tissue has the capability to regenerate itself; however, defects of a critical size prevent the bone from regenerating
and require additional support. To aid regeneration, bone scaffolds created out of autologous or allograft bone can be
used, yet these produce problems such as fast degradation rates, reduced bioactivity, donor site morbidity or the risk of
pathogen transmission. The development of bone tissue engineering has been used to create functional alternatives to
regenerate bone. This can be achieved by producing bone tissue scaffolds that induce osteoconduction and integration,
provide mechanical stability, and either integrate into the bone structure or degrade and are excreted by the body. A
range of different biomaterials have been used to this end, each with their own advantages and disadvantages. This review
will introduce the requirements of bone tissue engineering, beginning with the regeneration process of bone before
exploring the requirements of bone tissue scaffolds. Aspects covered include the manufacturing process as well as the
different materials used and the incorporation of bioactive molecules, growth factors and cells.

Keywords
Bone tissue engineering, osteoinductive, osteoconductive, bone remodelling, macrophages

Date received: 3 April 2019; accepted: 26 July 2019

Introduction 100–230 MPa, while trabecular or cancellous bone has a

Bone tissue
Bone is a composite of organic and inorganic phases.1
The organic components are mainly collagen type I
(COL-I) (~30%) and inorganic components are mostly
calcium phosphates (~70%) in the form of hydroxyapa-
tite (HA). The nanoscale organization of the organic and
inorganic phases is responsible for producing the
mechanical strength and durability that characterizes
higher porosity between 30% and 90% and a lower com-
pressive strength of 2–12 MPa.2
1Department of Anatomy Histology, Embryology, Pathology Anatomy
and Pathology Histology, Faculty of Dental Medicine and Health, Josip
Juraj Strossmayer University of Osijek, Osijek, Croatia
2Research and Development, botiss biomaterials GmbH, Berlin, Germany
3Department of Biomedical Engineering, School of Applied Medical
Sciences, German Jordanian University, Amman, Jordan
4Institute for Environmental Toxicology, Martin-Luther-Universität

bone. Microfibrils of collagen, synthesized by osteo- Halle-Wittenberg, Halle, Germany

blasts, aggregate laterally and longitudinally; forming 5Department of Dental Medicine, Faculty of Dental Medicine and

fibres that are then called an osteoid. The osteoblasts
deposit HA crystals into the voids between the collagen
fibrils during a process called biomineralization. The
Health, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia
6Department of Oral and Maxillofacial Surgery, University Hospital
Hamburg-Eppendorf, Hamburg, Germany
7Department of Oral and Maxillofacial Surgery, Division of

degree of biomineralization determines the mechanical Regenerative Orofacial Medicine, University Medical Center Hamburg-
properties of the bone tissue; a higher degree of mineral- Eppendorf, Hamburg, Germany
8BerlinAnalytix GmbH, Berlin, Germany
ized bone matrix provides a stiffer structure, however,
one that is more susceptible to fracture. *The authors contributed equally.
The two main types of bone are trabecular and cortical.
Corresponding author:
The bone types differ from each other by their structure. Patrick Rider, Research and Development, botiss biomaterials GmbH,
Cortical, also known as compact bone, has a porosity Hauptstaße 28, 15806 Zossen, Germany.
between 5% and 30% and a compressive strength of Email: patrick.rider@botiss.com
2 The International Journal of Artificial Organs 00(0)

Figure 1.  Demonstration of the tissue reaction to the biphasic bone substitute granules 2 months postimplantation and the bony
integration of the biphasic granules into new-built bone tissue within the peripheral regions of the implant beds neighboured to the
defect borders. The detection of (a) M1 and (b) M2 positive macrophages into the implantation beds. Representative biomaterial;
synthetic bone graft substitute (maxresorb, botiss biomaterials GmbH, Germany). Immunohistochemical detection of macrophages
and their M1 and M2 subforms by means of antibodies against the pro-inflammatory and anti-inflammatory molecules, that is,
haemoglobin scavenger receptor (CD163) and mannose receptor (MR; also known as CD206): (a) 400× magnification and (b)
100× magnification. *: Synthetic bone graft substitute; →: macrophages.

Bone regeneration substitute material.4 Once a material is implanted, the

Before designing a scaffold for bone tissue engineering
(BTE), it is important to understand the fundamentals of
bone regeneration, which occurs after injury or trauma
such as after the implantation of a BTE scaffold.
The fundamental process of bone formation, otherwise
known as osteogenesis, can happen via two routes: endo-
chondral ossification or intramembranous ossification.3
Both processes begin with the condensation of specialized
mesenchymal stem cells (MSCs) (discussed in more detail
below) and are dependent on the differentiation of this
condensate. In endochondral ossification, the MSCs dif-
ferentiate into chondrocytes that deposit cartilage tissue
which is then mineralized by osteoblasts. The chondro-
cytes and cartilage matrix reach a terminal and hyper-
trophic state, and are then engulfed and resorbed by
preosteoblasts, osteoblasts and blood vessels. This struc-
ture is eventually replaced by bone marrow. Therefore, the
endochondral ossification process is limited to the forma-
tion of marrow-containing bone types in the body.
Intramembranous ossification involves direct osteogenic
differentiation of the MSCs condensate; hence, does not
involve and intermediate chondrogenic phase. This pro-
cess is used in the formation of facial and cranial bones.
These osteogenic processes are effective at repairing and
restoring bone after trauma and injury. However, should
the size of the defect reach a critical size, the regeneration
process requires external support.
Current evidence around the definition of a critical sized
bone defect ranges from 2.5–3 cm or greater. The regenera-
tive process is unable to repair the bone without external
intervention, often in the form of an implanted bone
blood/biomaterial interaction begins immediately from the
moment the implant is inserted in to the body.5 During
implantation, the body triggers a tissue response involving
inflammatory and wound healing responses. Immediately,
after the implantation in the injury site, changes in blood
flow results in the discharging of fluids, cells and the pro-
teins from the surrounding tissues. These adsorb onto the
surface of the implanted materials and induce thrombus
formation. The thrombus is composed of platelets, fibrin
fibres, as well as red and white blood cells. The combina-
tion of these components enables the thrombus to form a
provisional blood-based matrix that is rich with growth fac-
tors, cytokines and chemo-attractants that recruit and medi-
ate the innate immune cells residing in the bone marrow.
When stimulated, the innate immune cells enter the
blood circulation and travel towards the injured site. The
arrival of these cells orchestrates the healing process. First
to arrive are neutrophils that initiate an acute inflammatory
reaction. Neutrophils begin to debride the wound, kill path-
ogens and release more soluble factors for the further
recruitment of immune cells. Once their function is com-
plete, the neutrophils undergo apoptosis. Along with the
neutrophils, monocytes are also recruited to the site of injury
via chemotaxis. These enter the blood circulation and dif-
ferentiate en route into macrophages. The macrophages
remove apoptosed neutrophils via phagocytosis and secrete
cytokines and growth factors. Macrophages change their
phenotype and the cytokines they secrete depend on molec-
ular signalling and their cellular environment that are influ-
enced via their interaction with the biomaterial.
Macrophages have several different phenotypes, two of
them being: M1 and M2. Each phenotype is not terminally
Perić Kačarević et al. 3

differentiated, but are instead reversible, responding to

external cues. Pro-inflammatory mediators from a cell-
mediated immune response will activate macrophages into
an M1 phenotype, commonly referred to as ‘classically
activated macrophages’. These cells take on a defensive
role when interacting with a biomaterial, performing
phagocytotic functions to remove pathogens and debris
from the injury site, as well as performing pro-inflamma-
tory functions by secreting tumour necrosis factor-alpha
(TNF-α) and interleukin-6 (IL-6) cytokines.6 M2 mac-
rophages, commonly referred to as ‘alternatively activated
macrophages’, act to instigate repair functions. Figure 1
contains two histological images of chemo-stained M1 and
M2 macrophages in implanted bone grafts.
M2 macrophages are the phenotype of resident tissue
macrophages, and are activated by IL-4 and IL-13 as well
as the phagocytosis of apoptosed neutrophils.6 These cells
perform anti-inflammatory functions by secreting
cytokines such as IL-10,6 as well as increase the produc-
tion of extracellular matrix (ECM). The M2 phenotype can
be further split into four types: M2a, M2b, M2c and M2d;
however, the precise function of each group has yet to be
precisely defined. Macrophages residing on the implant
bed have been demonstrated to promote vascularization,
bone cell differentiation and overall bone growth via the
expression of vascular endothelial growth factor (VEGF).7 Figure 2.  A depiction of the cyclic process of bone
remodelling in healthy bone, along with the influence of
Some studies have shown a correlation of increased vascu-
the different phenotypes of macrophages during healing.
larization with higher numbers of macrophages.8 In soft Osteoblasts, ascending of mesenchymal stem cells (MSCs)
tissue it has been shown that without the presence of M2 lineage, deposit and organize both organic and inorganic
macrophages during mid-stage healing, there was a lack a phases during regeneration. Osteoblasts activity is increased by
vascularization of the wound bed.6 Figure 2 depicts the anti-inflammatory cytokines secreted from M2 macrophages.
remodelling process of bone and the role of macrophages Osteoclasts are multinucleated cells with monocytic origin that
in healing. breakdown bone tissue during resorption. Osteoclast activity
is increased by pro-inflammatory cytokines secreted from M1
Macrophages can remain mononucleated individual macrophages. Regeneration and resorption occur in tandem in
cells, or fuse into multinucleated giant cells (MGCs) healthy bone as a lifelong ongoing process of bone remodelling.
depending upon their interaction with the biomaterial. If MSCs: mesenchymal stem cells; HSCs: haematopoietic stem cells.
the biomaterial is too large to phagocytose, the MGCs can *Secretion of pro-inflammatory cytokines.
**Secretion of anti-inflammatory cytokines.
fuse together, enabling them to surround the biomaterial.
Yet as they increase in size, their ability to perform phago-
cytosis decreases. Instead, as the multinucleated cell size surrounding tissue matrix. Due to their perceived similarity
increases, its capability for secreting extracellular degra- to osteoclasts, the MGCs were described as foreign body
dative enzymes improves. If the particle remains too large giant cells (FBGCs). However, studies have found MGCs
to be phagocytosed, the MGCs remain at the interface residing next to bone substitute materials years after
between the biomaterial and tissue for the duration of time implantation, surrounded by a flourishing bony matrix,
the implanted device remains in situ. thus contradicting this theory. This indicates that some
For biomaterials with a slow rate of degradation, MGCs MGCs have an inability to resorb biomaterial and there-
are observed to persist on the biomaterial surface and dem- fore, there may be differences between MGCs and the func-
onstrate similar histological features to that of osteoclasts. tions they perform.10 The presence of MGCs around bone
However, unlike osteoclasts, the MGCs do not show evi- grafts over prolonged period of time has shown evidence of
dence of particle resorption over prolonged periods.9 inducing vascularization and bone formation.8,9,11
Osteoclasts are bone-absorbing MGCs that work in tandem
with osteoblasts in natural process called bone remodula-
Growth factors in BTE
tion. It had previously been assumed that these MGCs
residing on the surface of biomaterials were similar to oste- Bone is a dynamic tissue that undergoes remodelling
oclasts and would breakdown the foreign body and throughout life. Growth factors are important for regulating
4 The International Journal of Artificial Organs 00(0)

bone formation because their regulatory functions include

cell adhesion, proliferation and differentiation. Bone
remodelling is executed by a functional and anatomic struc-
ture known as basic multicellular units (BMUs), which
includes four major bone cells: bone-lining cells, osteo-
cytes, osteoclasts and osteoblasts. The surface of the bone
is covered by a monolayer of bone-lining cells. Osteocytes
are the most abundant bone cells and play a vital role in the
initiation of bone remodelling, which is important for
maintaining the strength of the bone matrix. Osteoclasts are
MGCs that have differentiated from mononuclear cells by
two different growth factors: the monocyte/macrophage
colony stimulating factor (M-CSF) and the receptor activa-
tor of nuclear factor kappa-Β ligand (RANKL).12 Various
concentration levels of RANKL throughout several organs
reconfirm the importance of RANKL in tissue growth (par-
ticularly bone growth) and immune functions within the Figure 3.  Tissue reaction to biphasic bone substitute granules
body. Osteoblasts produce bone and are derived from 6 months postimplantation. Bony integration of the biphasic
MSCs. Osteoblasts are responsible for the deposition of granules into new-built bone tissue within the peripheral
collagen fibres and HA and the activation of osteoclast by regions of the implant beds neighboured to the defect borders.
expressing RANKL and M-CSF growth factors. Osteoclasts The granules were optimally integrated into bone tissue up
to their pores without any histological signs of exaggerated
secrete different growth factors including bone morpho- inflammatory tissue reactions. Representative biomaterial: *
genic proteins (BMPs) that influence the formation of new synthetic bone graft substitute (botiss biomaterials GmbH,
bone.13 However, many skeletal defects or extensive bone Germany), + new bone, and  soft tissue (haematoxylin and
loss through deterioration, may result in failure of bone eosin (H&E)-staining, 100× magnification).
healing.
The essential growth factors for BTE include VEGF,
been shown to be successful in bone regeneration in vitro,
fibroblast growth factors (FGFs) and BMPs.12,14,15 VEGF is
larger pore sizes, such as those similar to cancellous bone
an angiogenic protein which regulates the endothelial cell
(between 200 and 500 µm), are found to provide optimal
proliferation. BMPs induce osteogenesis through MSC dif-
tissue penetration and enable vascularization to occur in
ferentiation to osteoblasts. The combined delivery of VEGF
vivo.3,12,13
and BMPs could provide a consorted effect to induce angi-
The material used to fabricate the scaffold will also
ogenesis and osteogenesis, as the VEGF has the potential to
impact the success of the implant. Non-resorbable implants
enhance BMP-induced bone formation.12 FGFs are essen-
are useful for immediate load-bearing applications and are
tial for angiogenesis as FGFs induce endothelial and osteo-
also important for patients with a low bone metabolism,
blast cell proliferation, crucial for normal vascular
such as patients suffering from cancer or osteoporosis.10 In
developmental stage. In humans, 22 different FGFs (FGF1–
healthy bone, a biodegradable implant is ideal as it is
FGF23, although there is no human FGF15) have been
identified. FGF2, FGF9 and FGF18 are based on geneti- replaced by the patient’s native bone. Figure 3 is a histo-
cally modified temporal expression and have been identi- logical image of new bone regeneration during bone graft
fied as the best candidates for bone formation.14 resorption.
The rate of biodegradation should relate to the rate of
new bone formation, so that the biomaterial is replaced by
Scaffold properties new bone in a process called creeping substitution.10 A
For successful integration and support of the regeneration BTE scaffold must endure mechanical stresses that are
of bone, scaffolds for BTE should provide characteristics physiologically relevant to the surrounding bone.16
similar to the native bone structure. Important characteris- Overall, the mechanical properties need to be taken into
tics to be considered for bone scaffolds include surface consideration when designing scaffold architecture, as the
roughness, porosity and pore size, interconnectivity, deg- mechanical strength can be compromised by increased
radation, mechanical properties and biocompatibility.16 porosity and degradation rates.
The microarchitecture of the scaffold, which includes The inflammatory reaction caused by a biocompatible
porosity, pore size and interconnectivity between pores, is biomaterial should resolve within 2 weeks. Chronic
essential for proper mass transfer of nutrients and waste inflammation could be an indication of an infection, or a
products, as well as vascularization and tissue infiltration. response to the degradative by-products of the biomateri-
Although smaller pore sizes between 75 and 100 µm have als used. Biomaterials in BTE must have an osteoinductive
Perić Kačarević et al. 5
capacity, whereby they should instigate MSCs to differen-
tiate into bone-forming cells called osteoblasts. However,
many biomaterials do not possess this property and are
therefore incorporated with bioactive inclusions; for exam-
ple, demineralized bone matrix, bioceramics or BMPs.17
Biomaterials
Due to the highly regenerative properties of bone, small
defects are able to spontaneously begin the healing process
without intervention. Large-scale defects caused by trauma
or genetic disorders require surgical intervention to implant
materials that guide and accelerate the healing process.
Biomaterials provide an essential component for bone
regeneration. Their overall characteristics such as their
surface and bulk properties will ultimately affect the suc-
cess of the scaffold. An idealized biomaterial must fulfil a
specific criteria: be biocompatible, be replaced by the host
bone at a rate matching new bone formation, degrade into
non-toxic components and not to elicit a chronic immune
response. Bio-incompatibility results in an immune system
activation, tumour formation and inflammation.
Biodegradability of the material refers to the chemical or
enzymatic breakdown of the material over time when
introduced into living organisms. The material should
degrade into non-toxic components that are either recycled
or excreted by the body.18
Bone grafting materials can be divided into autolo-
gous, allogeneic, xenogeneic and alloplastic. The gold
standard material for repairing bone tissue is to use autol-
ogous bone. Autologous bone is bone harvested from the
same individual receiving the treatment. Although this
can produce excellent healing results, the necessity of a
secondary surgical site increases patient trauma and the
risk of infection.19 Allografts are bone tissue harvested
from one individual and transplanted between genetically
non-identical individuals of the same species.20 Allografts
provide an alternative to autografts, however, can suffer
from poor quality as the material is usually sourced from
cadavers or from bone removed during surgical opera-
tions. However, when the quality of the sourced material
is high, allografts can provide results similar to that of
autologous grafts.21 Xenografts are tissue transferred from
one species to another. Xenografts are readily available
and also provide excellent bone tissue regeneration in
comparison to autologous bone,11 but risk disease trans-
mission and can be unsuitable for patients from certain
religious groups.22 Alloplastic grafts are of a synthetic ori-
gin; this provides the benefit of preventing disease trans-
mission and improving availability. One of the most
commonly used types of alloplastic materials in BTE are
calcium phosphates, the properties of which can be tai-
lored by their composition.23
Ultimately the bone grafts should provide specific
characteristics: osteointegration, osteoinduction and
osteoconduction. Osteoinduction is a regular phenomenon
seen in any type of bone healing processes and is the stim-
ulation of immature cells (MSCs) to develop into preosteo-
blasts. Bone growth on an implant surface depends on the
differentiation of bone cells, recruited from pre-existing
preosteoblasts/osteoblasts, that are activated by primitive
mesenchymal cells via osteoinduction. Grafts should also
be osteoconductive, whereby they easily integrate with the
adjacent bone. In the case of implants, osteoconduction is
dependent on the biomaterials used and its morphology.24
Osteointegration is the direct structural and functional
integration between the host bone cells and the implants.
Natural polymers
Natural polymers benefit from a low immunological
potential and possible bioactive behaviour.25 Natural poly-
mers that have been investigated for BTE include poly-
mers naturally occurring in humans; collagen, fibrin and
elastin, as well as other natural polymers such as silk, chi-
tosan and alginate.
Collagen is the main component of the ECM, a network
of macromolecules that provides structural and mechani-
cal support to cells within tissues. Collagen can be
extracted from animal sources for use in BTE. It can be
removed from animal tissue either by decellularization or
demineralization of an organ, or via the process of extrac-
tion, purification and polymerization.26 The extracted col-
lagen is typically ground down to be further processed into
hydrogels, foams and fibres that are used to mimic the
native ECM. As one of the most commonly used scaffold
materials, collagen and its denatured form gelatine are
favoured due to their fast biodegradability, weak anti-
genicity, as well as excellent biocompatibility and cell
adhesion.27 Collagen scaffolds have also reported the
potential to induce fast vascularization.28 However, colla-
gen has a poor elasticity and processed collagen can suffer
from poor mechanical strength and requires methods such
as crosslinking or blending with other polymers for
improving the mechanical properties.29,30 Another alterna-
tive is to produce a composite material by mixing the col-
lagen with a ceramic such as beta-tricalcium phosphate
(β-TCP). The combined effects of increased mechanical
strength and the osteogenic characteristics of β-TCP were
shown to improve bone regeneration in rabbit femoral
defects, when compared to a pure collagen scaffold.31
Silk has had a long history of use as a biomaterial.32 It
is highly utilized in the field of biomaterials as it has the
ability to absorb and release water easily, demonstrates
excellent mechanical strength, and has a resistance to
ultraviolet (UV) and oxidation, thereby providing a versa-
tility for its sterilization.33 Silk is also highly processable,
and has been used to fabricate sponges, films, hydrogels
and mats.34,35 Many of its characteristics can be adapted,
such as its mechanical strength and degradation rate,
6 The International Journal of Artificial Organs 00(0)
controlled via its crystallinity.36–38 As part of the silkworm
cocoon, silk has a tensile strength of 27.6 MPa;39 however,
in its regenerated form, this can drop down to 3.45 MPa.40
The ability to domesticate silkworms means that silk is
widely available. The silkworms must be treated under a
controlled environment as the properties of silk can be
drastically influenced by the silkworms’ diet. The structure
of silk fibres spun by the silkworm includes an outer coat
of a protein called sericin, which acts as a glue to hold the
silk fibres together when spinning the silkworm cocoon.
Sericin is generally considered to be a negative component
of silk fibres as it triggers immunological responses
(although with some conflicting reports)41,42 and therefore
it must be removed in a process called ‘degumming’, a
process well established in the textile industry.34,35
The polysaccharide chitosan is a good candidate in the
field of biomaterials due to its highly antibacterial/anti-
fungal, biocompatible and biodegradable properties.
While it is not naturally formed, it is extracted from the
natural polymer chitin, commonly found in invertebrates,
and it is therefore abundantly available and easy to source.
Mushrooms can also be a source of chitin and offer a con-
sistent source all year round, avoiding inconsistent physi-
ochemical characteristics. Biodegradation rate of chitosan
correlates to the deacetylation degree (DD)43 which is
related to its crystallinity. DD can be controlled by alka-
line/acid conditions as well as by chitin deacetylases.
Crystallinity reaches a maximum when DD is 0% or 100%
and decreases with intermediate DD. As polymer crystal-
linity is inversely related to degradation kinetics, when
the DD of chitosan decreases to an intermediate point,
there is an in increase in the rate of biodegradation.
Chitosan and chitin are mechanically weak and are there-
fore usually blended with other more mechanically stable
polymers.44,45
Alginate is a natural polysaccharide sourced from
brown seaweed. It has a good biocompatibility and low
toxicity when processed to a high purity. When high-
purity alginate is implanted, it causes only a minimal
foreign body reaction.46 Alginate can be gelated into a
three-dimensional (3D) structure, most commonly via
ionic crosslinking using divalent cations.47 Although
alginate is non-degradable in mammals as they do not
possess the correct enzyme (alginase), ionically
crosslinked gels can break down by the release of the
divalent cation. Increasing the molecular weight of algi-
nate will improve its mechanical properties; however,
this will increase its gelatinous viscosity, which can
cause problems with its processing. An alternative
method to controlling alginate mechanical properties is
via its level of methacrylation in photocrosslinkable alg-
inate. By increasing the level of methacrylation from
~7% to ~25%, the elastic modulus can be increased from
34.29 ± 9.22 kPa to 174.77 ± 14.88 kPa, as well as
reduce its degradation rate.48
Synthetic polymers
Synthetic polymers are more predictable and reproducible
in their mechanical and physical properties as they are syn-
thesized under controlled conditions. As they are synthetic,
they do not pose any immunological risks. Aliphatic poly-
esters are the most commonly used synthetic polymers for
BTE and include poly(ε-caprolactone) (PCL), polylactide
(PLA), polyglycolic acid (PGA) and their copolymers.49
Polyesters degrade through the hydrolysis of ester groups
along their molecular backbone. By controlling the poly-
mer molecular weight, crystallinity, molecular side groups
as well as by their blending or co-polymerization, the
required mechanical and degradative rates are adapted.
The degradative products of polyesters cause a reduction
in pH, which if left to accumulate in the surrounding envi-
ronment can have cytotoxic effects, increase the inflam-
matory reaction, as well as increase the degradation rate.25
PCL is a semi-crystalline, biodegradable and non-toxic
polyesters that can be used for the fabrication of scaffolds
for BTE. It is highly processable, as it has a low melting
point (55°C–60°C) and glass transition temperature
(60°C), is soluble in a wide range of organic solvents.50
PCL is a ductile polymer with a high elongation to break
(>700%).50 Its main disadvantages are its hydrophobicity
and its slow degradation, as PCL can remain in situ for up
to 24 months.51 These problems can be addressed by blend-
ing with different polymers or producing a composite
material. It was reported that PCL/calcium silicate com-
posites, coated with decellularized ECM reduced the
hydrophobic nature of PCL and provided excellent bio-
compatibility. The incorporation of calcium silicate and
ECM effectively enhanced cellular adhesion, proliferation
and differentiation in both in vitro and in vivo.52
PLA is highly biocompatible and can be made from
renewable sources such as through the fermentation of
sugars.53 PLAs are composed of a chiral molecule that
exists as two enantiomers: l- and d-lactic acid, and has
several stereoisomers such as poly(l-lactide) (PLLA) and
poly(d-lactide) (PDLA). It generally has a slow degrada-
tion rate (3–5 years).53 Porous PLA scaffold showed excel-
lent biocompatibility with homogeneous distribution of
cells in vitro.54 PLA scaffolds with a pore size of 750 µm
has been demonstrated to enhance osteoblast proliferation
and metabolic activity.55 PLA can be combined with differ-
ent calcium phosphates to improve the hydrophilic nature
and osteoblast activity.56 Combining PLA with HA has
also been shown to improve its mechanical properties,
such as its flexural strength.57
Bioceramics
Bioceramics have long been applied as bone grafting
materials due to their promising biocompatibility, bioac-
tivity, osteoconductivity and mechanical strength.
Perić Kačarević et al. 7
Bioceramics enhance the osteo-potential of scaffolds and
promote new bone formation.58 The most commonly used
bioceramics include calcium phosphates, calcium sul-
phates and bioactive glasses (BGs). The most extensively
used bioceramics are calcium phosphates, due to their
similarity to the mineral phase of bone, HA. The majority
of calcium phosphates used are HA, tricalcium phosphates
(β-TCP/α-TCP) and biphasic calcium phosphates (BCPs).
HA is the main mineral component of natural bone
which represents approximately 60% of the weight of the
bone tissue.59 Although HA is already found within the
bone structure, it has very slow resorption and remodelling
rates, meaning that its properties might not be ideal for all
BTE applications. Due to its long remodelling and resorp-
tion times, nano-hydroxyapatite (nHA) has also shown to
offer some of the best properties for BTE, as nHA more
closely resembles the mineral size and structure found in
natural bone and is therefore more easily incorporated into
a regenerating bone structure.60 As HA lacks osteoinduc-
tivity, using an adsorption method, Zhou et al. incorpo-
rated BMP-2 onto the surface of the nHA. Their method
provided a sustained release of growth factor over a 35-day
period, enhanced the expression of osteogenic markers and
showed new ectopic bone formation in mice.61
β-TCP/α-TCP offers faster resorption times than HA.
α-TCP has a much higher biocompatibility than β-TCP,
however, is also more soluble, hence degrades faster in
vivo.62 β-TCP, although demonstrating a lower biocom-
patibility to α-TCP, has a more stable degradation rate and
can be used as an alternative to HA.63 TCP can be com-
bined with HA to form BCP to provide the ideal resorption
rates and biocompatibility. Many studies have shown that
a ratio of 60:40 HA to TCP will provide the best ratio for
use in BTE.23
BG is a silicate-based glass that elicits biological
response upon contact with bodily fluids, forming bonds
with both hard and soft tissues via the formation of amor-
phous calcium phosphates or crystalline HA on the surface
of the glass.64 The first and most famous BG composition
belongs to Hench,65 registered as 45S5 Bioglass®. 45S5 BG
is composed of 45% SiO2 and 55% of sodium, calcium and
phosphorous oxides. The inclusion of the phosphorous
oxides on the surface of the glass is highly reactive and
results in strong bonding with host tissue. A main advantage
of BG is its controllable degradation rate that can be matched
with the regular rate of remodelling of native bone.66 During
degradation, released Na, Ca, Si and P ions increase the
induction of osteogenesis and angiogenesis. Some of the
additional elements like fluorine, magnesium, strontium and
zinc can reinforce the function of BG.67 However, the major
downfall of BG is the low mechanical competence in BTE
due to its brittleness. An extensive review of mechanical
properties on the different compositions of BG was done by
Fu et al.,66 with some compositions reaching the compres-
sive strength range of cortical bone.59
Metallic scaffolds
Although polymer and bioceramics scaffolds have
received the most attention as bone tissue regeneration
materials, they suffer from poor mechanical properties,
either in strength or ductility. Certain metals have demon-
strated excellent biocompatibility, also known as biomet-
als and are well established as a bone replacement
material. Metals have a long history of use as for the
repair of bones, such as stainless steel, titanium-alloy,
cobalt–chromium-based, aluminium, lead and silver.
These materials were used because of their excellent
mechanical strength and fatigue resistance and ability to
be rehabilitate load-bearing bone defects. However, these
metals are non-degradable, and either require a secondary
surgery for their removal, or remain in situ for the lifespan
of the metallic implant.
There are several biometals that are also biodegradable,
such as magnesium (Mg) and iron (Fe) that are gaining
increasing interest for bone tissue scaffolds due to their
similar mechanical properties to bone.68 The release of
metallic ions could also be beneficial for the regeneration
of bone as many metallic ions are important for the proper
function of the body.69
Fe has excellent mechanical properties (similar to alu-
minium), however, has a very slow rate of degradation in
vivo that can cause it to induce a similar tissue reaction to
that of a permanent scaffold.70 In contrast, Mg has more
ductile mechanical properties, similar to bone, but has a
fast degradation rate.70 Alloying each metal with ions such
as strontium or calcium can be useful for adapting the
mechanical and corrosive properties, as well as be benefi-
cial to bone regeneration.69,71 Open porous degradable
magnesium scaffolds fabricated with pore sizes of 250 and
400 µm have demonstrated mechanical properties within
the range of human cancellous bone. In vivo experiments
demonstrated a high degree of vascularization and bone
formation.72
Fabrication techniques
Fabricating materials with satisfactory mechanical and
bioactive properties are one of the great challenges in
attempting to design bone or cartilage scaffolds, as the
implanted structure should be equivalent to the anatomical
site. Many materials with good mechanical properties have
shown great potential in vitro, but failed when implanted
in vivo due to an imbalance between the mechanical prop-
erties and porosity of the structure, as well as by its vascu-
larization,22 all properties influenced by the fabrication
process.
Conventional methods
Solvent casting and particulate leaching.  In solvent casting
and particulate leaching (SCPL), a solvent is used to
8 The International Journal of Artificial Organs 00(0)
Figure 4.  Depiction of solvent casting and particulate leaching.
Particulates are dispersed in the polymer solution, solvent
evaporates, and the particulates leach out, eroding the polymer
and creating porous structures.
dissolve a polymer with salt/polymer particles. After the
solvent evaporates, the matrix of polymer and salt/polymer
particles is submerged in water, causing the particulates to
leach out; creating a porous structure.73 This process is
depicted in Figure 4. SCPL is a simple cost-effective pro-
cedure, which has controllability over porosity and pore
size. Achievable porosity and pore sizes are 20%–50% and
30–300 µm, respectively.74 Disadvantages of this tech-
nique are cytotoxic residual of the solvent and/or particu-
late and low controllability over spatial geometry, as well
as low-pore interconnectivity.
Cao and Kuboyama75 fabricated composite scaffolds
made of PGA/β-TCP with different weight ratios (1:1 and
1:3 PGA:β-TCP) via SCPL. The solution was mixed with
NaCl particles, with a particle size of 425–500 µm.
Porosities of the 1:1 and 1:3 scaffolds were 93.6% ±
2.04% and 88.4% ± 0.7%, respectively. The scaffolds
were implanted and assessed in femoral defects in rats
over 3 months. New bone formation was the highest in 1:3
composite scaffolds, compared to 1:1 scaffold. After 90
days, the wound site had healed by >96% and all the
PGA/β-TCP scaffolds had degraded. A higher content of
β-TCP improved the osteoconductivity of the scaffolds.
Instead of mixing the porogen with the polymer in a
solution composed of a polymer and an organic solvent,
the polymers can be melted and mixed with the porogen.
PLA was combined with demineralized bone particles
(DBPs) in SCPL.76 The scaffolds proved to be osteoinduc-
tive; however, an increase in the DBP decreased porosity
of the resultant scaffolds. In SCPL, bioglass powders have
been mixed with the base polymers.77 The addition of bio-
glass enhanced hydrophilicity, degradability and bioactiv-
ity; however, increased content of bioglass decreased
porosity.
SCPL has proved to be a successful technique in fabri-
cating BTE scaffolds with many different bone-inductive
additives;73 however, addition of these particles could
Figure 5.  Depiction of freeze-drying. An emulsion of polymer
solution and water is prepared, frozen and vacuum dried to
create porous structures.
compromise the porosity. Moreover, it is limited to a thick-
ness between 3 and 4 mm to ensure efficient leaching, hin-
dering its use in defects with thicker bone.
Freeze-drying.  In freeze-drying, or lyophilization, an emul-
sion of polymer solution and water is cooled down below
its freezing temperature and subjected to vacuum drying
that evaporates the water and solvent via sublimation. As
the water leaves the frozen polymer it creates intercon-
nected porosities.3 The freeze-drying process is depicted in
Figure 5. Freeze-drying is a type of thermally induced
phase separation (TIPS) process, and is discussed further
in section ‘TIPS’.
This technique does not require high temperatures,
which allows for the incorporation of natural polymers and
biomolecules. Problems with the technique include the
potential for solvent residuals to remain within the struc-
ture as well as an irregular pore size. Achievable porosity
and pore size are reported to be >90% and 20–400 µm.78
Li et al.79 freeze-dried blends of poly(lactide-co-gly-
colide) (PLGA), PCL and HA. The scaffolds had a poros-
ity of 75.74% ± 1.21% and a pore size of 179.07 ± 0.75
µm. Compressive strength was 47 ± 2 MPa. The scaffolds
promoted human MSCs to differentiate into osteoblasts
after 21 days in culture. For in vivo assessment, the scaf-
folds were implanted into rabbit calvarial defects. After 12
weeks, the PLGA/PCL/HA scaffolds exhibited higher vol-
umes of new bone ingrowth compared to PLGA/HA, PCL/
HA and a negative control. The PLGA/PCL/HA scaffolds
were sufficiently degradable, biocompatible and induced
bone tissue regeneration.
Due to the fact that this technique does not require high
temperatures during fabrication, natural polymers can be
used80 as well as biomolecules.81 Research scholars have
successfully combined the mechanical properties and pro-
cessability of silk with the osteogenic capacity of HA to
fabricate scaffolds via freeze-drying. By combining silk
and HA resulted in scaffolds that have mechanical proper-
ties with similar load-bearing properties to bone, while also
mimicking its native ECM. Sangkert et al.80 fabricated
scaffolds with silk fibroin via freeze-drying, however
incorporated different biological components to improve
its biological response. The scaffolds were modified with
collagen, decellularized pulp and fibronectin coating. The
silk provided a mechanical support structure, while the
Perić Kačarević et al. 9
collagen improved cellular adhesion and proliferation. The
human decellularized tooth pulp was used to stimulate a
higher biofunctionality of the scaffolds, while the fibronec-
tin induced mineralization. The coating provided the sam-
ples with an osteoinductive bioactivity; exhibiting a higher
alkaline phosphatase (ALP) expression after 21 days in cul-
ture with MG-63 osteoblasts when compared to non-modi-
fied samples.
Using the freeze-drying method, it is possible to create
scaffolds that gradually release bioactive compounds.
Menon et al.81 fabricated bone tissue scaffolds from cellu-
lose, chitosan, nHA and chrysin via freeze-drying. Chrysin
is a flavonoid that promotes osteogenic growth.82 The scaf-
folds were seeded with mouse MSCs and incubated for 7
days. The sustained release of chrysin upregulated the
expression of osteogenic differentiation marker genes.
Freeze-drying has great potential in BTE with the pos-
sibility of using natural biomaterials and biomolecules.
Despite its promising outcomes in tissue engineering and
drug delivery, this technique is time-consuming and has
high energy-consumption, as well as providing only a lim-
ited control of scaffold architecture.
TIPS.  In TIPS, a homogeneous polymer solution is sepa-
rated into polymer and solvent phases by the change of
thermal energies.83 This liquid–liquid phase separation pro-
cess results in polymer-rich and solvent-rich phases. After
using thermal energy to separate the solution, freeze-drying
is performed to evaporate the solvent, resulting in a
microporous structure. Although this technique can pro-
duce large volumes of pores with controllable size and
morphology, it requires the use of polymers with low melt-
ing temperatures and organic solvents. Achievable porosity
and pore sizes are <97% and <200 µm, respectively.74
Carfi Pavia et al.84 fabricated PLLA/HA scaffolds with
ratios of 95:5, 90:10, 70:30, 50:50 and 34:66. HA was sus-
pended in PLLA solution and the scaffolds were fabricated
via TIPS and freeze-dried. Porosity decreased with an
increase in the HA content, although the results were statis-
tically insignificant, and all ratios produced porosities
>90%. Compressive moduli increased with the increase in
the HA content until a concentration of 30% HA was
reached. It is thought that higher concentrations caused a
less homogeneous distribution of HA, resulting in a decrease
of the modulus. MC3T3-E1 preosteoblasts were seeded on
PLLA/HA (95:5) and neat PLLA scaffolds. After 21 days,
the PLLA/HA scaffolds exhibited higher ALP activity.
Chen et al.85 fabricated PLLA scaffolds via TIPS. The
scaffolds had a porosity of >94%, pore size of 300 µm and
compressive strength of 1 MPa. To improve the cellular
attachment and degradation kinetics of PLLA, the scaf-
folds were modified with chitosan coating to increase their
hydrophilicity. The TIPS-fabricated scaffolds were sub-
merged in high concentration of chitosan and freeze-dried.
Mouse bone marrow stromal cells (mBMSCs) were seeded
Figure 6.  Depiction of gas foaming. Polymer discs are put
under a high-pressure inert gas, creating a foam. A drop of
pressure will create a phase separation, resulting in a porous
structure.
on the scaffolds for in vitro assessment. After 14 days, the
modified scaffolds exhibited a higher proliferation, cell
number and osteogenic gene expressions than pure PLLA
scaffolds. The scaffolds were implanted in mouse calvarial
critical-size defects. Groups with modified PLLA scaf-
folds exhibited 90% regeneration of the defects, after 12
days.
TIPS appear to be a suitable technique for the fabrica-
tion of BTE, a wide span of pore sizes and morphologies
are achievable. Due to the relatively higher temperatures,
however, natural biomaterials and biomolecules cannot be
incorporated during the fabrication process but must be
added in a post-fabrication modification. It has also been
observed that the incorporation of bone substitutes during
the fabrication process decreased porosities and resulted in
smaller irregular pore sizes.
Gas foaming.  Polymer discs are subjected to high-pressure
inert gases like CO2, N2 or He, to saturate and create foams
of the polymer and gas.86 A drop of pressure to ambient
levels will cause the gas phase to separate, instigating the
nucleation of bubbles. The gas then diffuses through the
polymer, resulting in the growth of pores. Figure 6 demon-
strates the gas foaming technique. Alternative chemical-
based gas foaming uses foaming agents, polymer and
binding agents. The semi-solidified mixture is then sub-
merged in chemicals that react with the foaming agents,
creating gas bubbles that diffuse through the polymer and
have an erosion effect that creates a porous structure. This
technique is fast and simple; however, the resulting porous
structure contains closed pores that lack interconnectivity.
The interconnectivity can be improved through combining
the process with salt/polymer leaching or freeze-drying.3
Complex structures require negative scaffolds as moulds,
which is not cost-effective. Achievable porosity and pore
sizes of >93% and 40–800 µm have been reported with
this technique.3,87
Duarte et al.88 fabricated PCL scaffolds loaded with β-
TCP and dexamethasone. A concentration of 10 wt% β-
TCP was and 5 or 10 wt% dexamethasone was used. The
fabrication was carried out with subcritical CO2 that
resulted in milder processing conditions. Resultant poros-
ity ranged between 73% and 99% and pore sizes between
164 and 882 µm. Young’s modulus ranged between 1.76
10 The International Journal of Artificial Organs 00(0)
Figure 7.  Depiction of electrospinning. Polymer is fed to the
capillary tube and a high voltage is used to introduce a polarity
that accelerates filaments of the polymer from the syringe tip
towards the collector.
and 2.92 MPa, decreasing with increasing β-TCP content.
The addition of the osteoinductive and osteoconductive
additives did not affect internal morphology of the scaf-
folds and the dexamethasone provided a sustained release
over 35 days.
Catanzano et al.89 fabricated porous scaffolds via inter-
nal gelation, followed by gas foaming and freeze-drying.
Alginate was crosslinked with a mixture of strontium (Sr)
and calcium (Ca) ions. Sr2+ and Ca2+ have osteogenic
benefits.69 Scaffold porosities were around 50.74 ± 4.57
and had a pore size between 100 and 400 µm. Calcium
ions were released quicker than the strontium ions. The
scaffolds were seeded with human MSCs and after 7 days,
the scaffolds demonstrated a high level of cellular attach-
ment. Osteocalcin (OCN) expression was significantly
higher with scaffolds crosslinked with Sr2+ and Ca2+ after
21 days, compared to cells incubated on tissue culture
plates.
Electrospinning. Electrospinning creates fine fibres using
electrostatic forces that accumulate in a mat on a collection
plate. The precursor material is either a polymer solution
or a molten polymer. Thicknesses of the fibres can range
from micrometres to nanometres. An electrospinning pro-
cess requires three essential components: (1) high voltage,
(2) feeding capillary tube and (3) metal collector. The pol-
ymer liquid is fed to the capillary tube and a high voltage
introduces a polarity that accelerates strings of the polymer
from the tubes towards the collector. The continuous jet of
polymer is received on the collector that is either neutral or
has the opposite polarity of the polymer. The process of
electrospinning is depicted in Figure 7. The process is
affected by parameters such as polymer type, solution con-
centration, polymer feed rate, distance between collector
and capillary tube, temperature and humidity. Due to the
latter two, the process should happen under a well-con-
trolled atmosphere. Electrospinning produces structures
with a high surface area that mimic natural tissue ECM
and has the potential of incorporating natural biomaterials/
biomolecules as it is a low-temperature process. Although
this technique is versatile, simple and inexpensive, the
resulted structures lack mechanical competence.90
Sharifi et al.91 fabricated nanofibrous scaffolds via elec-
trospinning, with fibre diameters of 356 nm. The scaffolds
were based on PCL and carboxylmethyl chitosan.
Carboxylmethylation of the amine and hydroxyl groups of
chitosan enhances its solubility in water and surface hydro-
philicity. Scaffolds were seeded with MG-63 osteoblasts
and after 3 days, the PCL/carboxylmethyl chitosan exhib-
ited higher proliferation rate compared to the PCL and
PCL/chitosan scaffolds.
Aragón et al. incorporated BMP-2-loaded PLGA parti-
cles with electrospun PCL/HA fibres via electrospraying.
The fibres had PCL shells and PCL/HA cores, with an
overall fibre diameter of 380 ± 108 nm. Core-shell nanofi-
bres can be fabricated using coaxial electrospinning, a
developed method to created coated fibres.92 BMP-2 con-
tent ranged between 1.2 and 1.7 µg/g and HA nanoparti-
cles between 8.8 and 12.6 wt%. The scaffolds loaded with
PLGA/BMP-2 particles exhibited higher ALP, OCN and
osteopontin expression compared to PCL/HA. Suspending
the HA nanoparticles in the polymer solution did not affect
the diameter of the fibres.
Cheng et al.93 employed coaxial electrospinning to fab-
ricate platelet-rich plasma (PRP)-filled PVA nanofibres.
Fibres had a diameter of 471.6 ± 210.8 nm. The fibres
exhibited a sustainable release of the PRP over a 30-day
period. In vitro, the growth factors from the PRP enhanced
proliferation and induced mineralization. Chitosan/β-TCP
scaffolds were seeded with MSCs and incubated for 1
week. After that, the scaffold was covered with electro-
spun PVA/PRP nanofibres and implanted subcutaneously
in nude mice. After 30 days, the scaffolds exhibited a
higher collagen II expression and accumulated more cells
when compared to a sham surgery.
Ao et al.94 fabricated nanofibres from cellulose and HA.
HA nanoparticles were incorporated with the polymer
solution at different weight percentages: 3, 5 and 10 wt%.
Diameter of fibres increased with increasing HA content.
For the highest HA content (10 wt%), the fibre diameters
ranged from 100 to 500 µm. Neat cellulose nanofibre
diameters ranged between 100 and 200 µm. The tensile
strength and modulus reached a maximum at 5 wt% HA
with 70.6 MPa and 3.12 GPa, respectively. However, at 10
wt% HA, the mechanical properties deteriorated as a result
of increased agglomerates. After 3 days seeded with
human dental follicle cells, all scaffolds did not exhibit any
cytotoxicity and increasing HA content increased cell via-
bility and proliferation rates.
Perić Kačarević et al. 11
Figure 8.  A flowchart of the different additive manufacturing techniques employed in tissue engineering indicating which methods
are suitable for cell printing.
SLA: stereolithography; LAB: laser-assisted bioprinting; SLS: selective laser sintering.
Electrospinning has great possibilities in BTE.95 The
fibrous structures produced closely mimic the fibrous
structure of natural tissue ECM, where collagen fibres
have diameters ranging from 50 to 500 nm. The ability to
incorporate bioceramics particles at low concentrations
within the electrospun fibres improves scaffold mechani-
cal properties as well as its bioactivity, however, can affect
fibre diameters and uniformity. Coaxial electrospinning
and electrospraying have shown great success in fabricat-
ing bone tissue scaffolds that sustain growth factor deliv-
ery, enhancing the bone regeneration process as whole.
Additive manufacturing
Additive manufacturing (AM), or 3D printing, is a fabrica-
tion process that builds structures from the bottom-up.16 It
has been successfully employed in regenerative medicine,
especially in the fields of tissue engineering and drug
delivery/screening. AM techniques fabricate structures or
patterns in a pre-defined fashion via computer-aided
designs (CADs). The CADs can be geometrically tailored
for individual patient specifications and have macro- and
microarchitecture that corresponds to the anatomical prop-
erties of the to-be-replaced tissue. Figure 8 provides a
flowchart of the most common 3D printing techniques
used in tissue engineering.
Inkjet printing. Inkjet printing is a low-cost, simple and
non-contact fabrication technique. Inkjet printing builds
up structures using droplets as individual building blocks.
The ejection of each droplet is driven either by piezoelec-
tric or thermal–based processes. In thermal inkjet printing,
an air pocket is nucleated within the printhead via a ther-
mal element, which causes a pressure increase within the
printhead leading to droplet ejection. The heating element
can reach temperatures ranging between 100°C and 300°C.
Although such high temperatures could degrade natural
polymers or biological molecules, studies have shown that
during the duration of heating, the heat is very localized
and does not affect the quality or integrity of the biological
materials.16 Piezoelectric printers use acoustic waves to
print. The process enables a larger variety of materials to
be printed compared with thermal inkjet printing, how-
ever, is limited by the viscosity of the ink, as high viscosi-
ties will dampen out the acoustic waves before a droplet
can be ejected. The main advantages of inkjet printing
include its high speed (up to 10,000 droplets per second)
and high cell viability (>90%). The main disadvantage of
inkjet printing is the low concentration of the inks, which
can influence the integrity of the printed structure, as well
as increase fabrication times.
Ge et al.96 fabricated PLGA scaffolds using a binder of
ethanol and acetone that had a pore size of 1 mm, a poros-
ity of 50%, a compressive strength of 7.8 ± 3.1 MPa, and
a Young’s modulus of 77.2 ± 10.8 MPa. The scaffolds
were compared in vitro to a commercially available open
pore poly-lactic acid scaffold (OPLA®, Becton-Dickinson
(BD) Inc., Murray, UT, USA) and a collagen scaffold
(Becton-Dickinson (BD) Inc.). The mechanical properties
of the printed scaffold were 40 times higher than the
OPLA® scaffold and 1800 times higher than the BD col-
lagen scaffold. Cell viability of human foetal osteoblasts
seeded onto the scaffold was 95% after 24 h. The results
demonstrated that the 3D-printed PLGA scaffolds support
proliferation and differentiation of osteoblasts, compara-
ble to commercially available scaffolds.
Extrusion printing.  Extrusion bioprinting is the most com-
monly used printing technique for the extrusion of poly-
mers or hydrogels through a micro-nozzle. Extrusion is
controlled via pressure-based mechanisms, such as via a
piston- or pneumatic-based.16 The main advantage of
12 The International Journal of Artificial Organs 00(0)
extrusion bioprinting is its fast fabrication times, however
its resolution of around of 200 μm is considerably lower
compared to other AM techniques. Pneumatic systems
generally use compressed gas, which work better for
highly viscous molten polymers but lack the same preci-
sion as piston-driven mechanisms that offer more direct
control over the flow of the ink from the nozzle.97 Overall,
this technique provides excellent structural integrity due to
the continuous deposition and high loading of the ink.
Boga et al.98 demonstrated bone regeneration in vitro
with different ratios of TCP and alginic acid (AA) (60:40,
70:30, 80:20). AA was crosslinked by immersing the scaf-
folds in a solution of CaCl2. The printed scaffolds had a
high mechanical strength and elasticity and also shown
bone-like structure. High loading of the scaffold with TCP
deteriorated the mechanical properties. To improve the
mechanical properties further, graphene oxide was incor-
porated into the structure. By including a 0.5% (w/w) con-
centration of graphene oxide, Young’s modulus of scaffolds
with a 60:40 TCP:AA ratio increased from 154 ± 8.7 MPa
to 188.3 ± 18.5 MPa.
To test the effect of scaffold architecture on the mechan-
ical properties of bone tissue scaffolds, Roohani-Esfahani
et al.99 fabricated BG ceramics, Sr-HT Gahnite scaffolds
with hexagonal, rectangular, curved and zig-zag architec-
tures. Scaffold porosities tested were: 50%, 55%, 60% and
70%. The scaffolds with hexagonal patterns had the high-
est compressive strengths of 122 ± 12 MPa, which is in
the range of human cortical bone (100–150 MPa). The
compressive strength from 90 MPa at 70% porosity to 180
MPa at 50% porosity, the result shows the application of
Sr-HT Gahnite–based scaffolds for load-bearing bone
defects.
Vozzi et al.100 demonstrated the fabrication of PLLA
combined with multiwalled carbon nanotubes (MWCNTs)
to enhance the mechanical properties. The scaffold was
fabricated with an octagonal architecture and a porosity
between 65% and 70%. In comparison to pure PLLA scaf-
folds, the inclusion of MWCNTs improved cell viability
when seeded with human foetal osteoblasts. The inclusion
of a small concentration (6.25 mg/mL) of MWCNTs
within the ink, increased the elastic modulus and osteo-
blastic proliferation rates.
Altogether, extrusion printing is considered to have
great potential as it allows the fabrication of complex
structures at clinically relevant sizes. It provides versatility
in the printing of multiple biomaterials and biomolecules.
Of the different fabrication techniques, it has the capability
of printing scaffolds with the most physiologically rele-
vant mechanical properties.
Stereolithography. Stereolithography (SLA) is an additive
technique based on the photopolymerization of layers in
vat of photosensitive resin.16 Crosslinking occurs in a
layer-by-layer fashion on a platform that moves
downwards after each subsequent layer. This technique
offers a limited range of materials, as the materials are
required to be photosensitive. To improve photosensitivity,
the materials can be mixed with photoinitiators (PIs). PIs
are chemical compounds that form free radicals upon
exposure to light energy, UV or visible.97 These radicals
initiate a chain polymer reaction between the monomers of
the material, hardening the liquid resin into predesigned
structures. The type of PI and concentration are essential in
determining the stiffness of the structure, fabrication time
as well as the cellular reaction with the 3D structure.101,102
For UV-based SLA, the most common and suitable PIs
used are Irgacure 2959 and Irgacure 184 that polymerize at
wavelengths 257–276 nm and 246, 288 or 333 nm, respec-
tively.101 For visible light-based SLA, eosin Y and LAP PIs
are the most commonly used, photocured at 514 and 405
nm, respectively.103,104
Visible light-based SLA was developed in order to ena-
ble the incorporation of cells during fabrication and has
shown advantageous results over UV; as it rules out
mutagenesis of cells, uses less cytotoxic PIs and produces
higher mechanical integrity of the prints. This technique
can produce scaffolds in a short time, regardless of their
complexity as the fabrication time is instead dependent
upon the height of the design. The main disadvantage of
this technique is the limited choice of materials and bio-
compatibility issues caused by the PIs.
Lan et al.105 fabricated BTE scaffolds from
poly(propylene fumarate) (PPF) via SLA. PPF was mixed
with diethyl fumarate (DEF) and 1 wt% bis-acyl phos-
phine oxide (BAPO) to prepare a photocurable resin. The
fabricated scaffolds had a lattice design with 65% porosity
and 250 µm pore size. The scaffolds were further modified
post-fabrication to enhance their osteo-potential. The scaf-
folds were coated subsequently with HA and arginine–
glycine–aspartic acid (RGD). These coatings improve
spreading, proliferation and osteogenic differentiation.
The coatings did not affect the bulk structure of the scaf-
folds. After 14 days of seeding MC3T3-E1 preosteoblasts,
the scaffolds with both RGD and HA coatings and only HA
coating had the same cell number.
Lee et al.106 incorporated BMP-2-loaded PLGA spheres
with PPF/DEF/BAPO liquid resin to fabricate osteoinduc-
tive scaffolds and compared them to conventionally fabri-
cated scaffolds. The scaffolds had 69.6% porosity. The
3D-printed scaffolds sustained the release of BMP-2 for 4
weeks, while conventional scaffolds only lasted up to 14
days. In vitro MC3 T3-E1 proliferation was higher on SLA
scaffolds compared to conventional scaffolds, after 14
days. Most importantly, the SLA scaffolds had signifi-
cantly higher expression of ALP, OCN and COL-I. The
SLA scaffolds were implanted in rat cranial defects and
after 11 weeks, had developed new bone that made up
90.90% ± 5.09% volume compared to 32.85% ± 8.27%
in sham surgeries.
Perić Kačarević et al. 13
Ronca et al.107 prepared a photocurable resin from syn-
thesized dimethacrylate poly(d,l-lactic acid) (PDLLA)
and 4 wt% of Lucirin 2,4,6-Trimethylbenzoyldi-
Phenylphosphinate (TPO-L) (an PI). HA particles (<200
µm) were dispersed in the liquid resin at 10 and 20 wt%.
The scaffolds had porosities ~80% and pore sizes ~745 µm
and demonstrated a high degree of similarity to the CAD.
The incorporation of HA increased the mechanical strength
from 0.61 ± 0.2 MPa to 1.23 ± 0.2 MPa for neat PDLLA
scaffolds to PDLLA/20 wt% HA scaffolds, respectively. In
vitro assessment was carried out by seeding human bone
marrow–derived stromal cells (hBMSCs) on scaffolds
without HA, scaffolds with 10% HA and scaffolds with 20
wt% HA. Scaffolds with 10 wt% HA exhibited the highest
proliferation rate and osseous differentiation while a 20
wt% HA content hindered the migration and attachment of
the BMSCs.
SLA has a great potential for the fabrication of tissue
engineering scaffolds; however, the mechanical properties
of the resulted structures might not be adequate for BTE
applications, more specifically at load-bearing sites. This
technique can be combined with other AM techniques to
create biphasic structures. A rigid phase could provide the
mechanical strength to support the structure, and a soft
phase that could facilitate vascularization and cellular
recruitment.108
Selective laser sintering.  Selective laser sintering (SLS) is a
process where compacted powder setting on a piston plat-
form is exposed to a laser beam that melts the powder into
a pre-defined shape.16 Printing is performed in a layer-by-
layer sequence, whereby for every layer; the powder is sin-
tered, the piston platform moves downwards and a
cylindrical roller replenishes powder into the powder bed.
Morphology and the size of the powder are crucial param-
eters in this process and impact flowability of the powder
into the powder bed, as well as the quality of the print-
ing.109 Powder bed is usually heated just below the melting
point of the material, speeding up the process. However,
SLS suffers from a limited choice of materials and warp-
ing of the structure. Biomolecules and natural polymers
cannot be employed in this process as the process requires
high thermal energies.
Feng et al.110 reinforced β-TCP-based SLS-
manufactured scaffolds with zinc oxide (ZnO). ZnO has
shown to encourage angiogenesis and osteogensis,111
which are both required and equally essential in BTE. The
scaffolds with 2.5 wt% ZnO had porosity of 56.8% and
compressive strength increased from 3.01 to 17.89 MPa.
Increased content of ZnO slowed down the degradation
rate and enhanced cellular response of MG-63 osteoblasts.
Deng et al.112 reinforced forsterite-based scaffolds with 20
wt% nano-58S BG. Compressive strength increased from
28.6 to 43.9 MPa. An increase in the BG enhanced cellular
adhesion of MG-63 cells.
Xia et al.113 fabricated PCL/HA scaffolds using an SLS
apparatus. The scaffolds had porosity of 70.31% and pore size
between 600 and 800 µm. At 15 wt% of HA, compressive
strength was 3.17 MPa while pure PCL scaffolds had a com-
pressive strength of only 1.38 MPa. The PCL/HA and PCL
scaffolds were seeded with hBMSCs for 21 days. Attachment,
proliferation, mineralization and ALP expression increased
with increasing HA content. For in vivo assessment, the PCL
and PCL/HA scaffolds were implanted in rabbit femur
defects. After 9 weeks, PCL with 15 wt% HA has fully
degraded and replaced by new tissue, while defects implanted
with pure PCL scaffolds still had remnants of the implanted
graft. MGCs were present at the surface of the implants,
which were associated to the remodelling process at the site.
Liao et al.114 fabricated PCL and PCL/β-TCP scaffolds
by SLS. The β-TCP content was 30 wt%, increasing the
compressive strength from 6.77 ± 0.19 MPa to 13.66 ±
0.19 MPa. The porosity of the scaffolds was between 75%
and 77% and had a pore size between 300 and 500 µm. The
scaffolds were coated with collagen I. The coating did not
impact the structure nor the mechanical strength. In vitro,
human adipose-derived stem cells (hASCs) expressed
highest ALP, OCN and mineralization on collagen-coated
PCL/β-TCP scaffolds after 28 days. hASCs were seeded
on PCL/β-TCP/COL-I and PCL scaffolds for 21 days and
subsequently implanted the gluteal muscle pockets in nude
mice. The PCL/β-TCP/COL-I formed woven bone con-
taining a vascular structure and PCL scaffolds formed a
fibrous granulated tissue, all after 4 months.
SLS can produce scaffolds with architectures and
mechanical strengths that are relevant to cortical bone.
However, SLS is limited by the choice of materials, and
via this technique, drug delivery methods are not possible
due to the high thermal energies involved.16 With complex
structures, powder could be entrapped within the structure
which is problematic as well.
Bioprinting.  Bioprinting is the process of creating 3D struc-
tures from cell-laden precursor materials (bioinks). Bio-
printing is commonly carried out by one or a combination
of the four hydrogel-based printing techniques, which are
SLA, thermal or piezoelectric inkjet, mechanical or pneu-
matic pressure-based extrusion and laser-assisted bioprint-
ing (LAB).115,116 Bioprinting is advantageous over the
manual seeding of cells in terms of cell viability and
distribution.
In SLA, cells are suspended in a photosensitive hydrogel
positioned in a vat.97 However, the use of UV radiation is
known to induce mutagenesis in mammalian cells, and
therefore, visible light-based SLA has been adapted to avoid
the adverse effects of UV. Since PIs respond to certain
wavelengths, visible light-sensitive PIs, which respond to
400–700 nm wavelengths, have been used. The same
parameters of SLA apply on SLA with cell-laden hydrogels;
however, encapsulation of the cells during the gelation
14 The International Journal of Artificial Organs 00(0)
process. A technical challenge of SLA is oxygen inhibition.
O2 molecules can scavenge the free radicals produced by the
light exposure of PIs, hindering the crosslinking. This nega-
tively impacts the print quality and could result in print col-
lapse. Oxygen inhibition can be reduced by increasing light
intensity, concentration of PIs or carrying the process under
inert conditions such as N2 or CO2; however, all of these
possibilities will negatively impact cell viability in case of
SLA printing using bioinks. Visible light-based SLA over-
comes issue as the increase in the light intensity or PI con-
centration will not impact the cells as much, enhancing the
printing quality and integrity.117,118
Lim et al.117 demonstrated a novel SLA technique that
produced enhanced print qualities using a visible light–sen-
sitive PI with high intensities, the PI was ruthenium (Ru)/
sodium persulfate. Immediate cell viability using this sys-
tem was higher when UV-sensitive Irgacure 2959 was used,
which is the least toxic PI among the UV-sensitive options.
Inkjet bioprinting employs localized heat or acoustic pie-
zoelectric waves to propel bioink from a micro-nozzle.116
This process is very similar to conventional inkjet printing;
in fact, inkjet bioprinting was the first AM technique to be
utilized for bioprinting by filling an ink cartridge of a con-
ventional table-top printer with a bioink.115 However, this
technique requires that cell suspensions/bioinks have a low
viscosity, and therefore a low concentration of materials and
cells. Low viscosity also affects the quality of the print, espe-
cially in vertical configurations and structures with over-
hangs. To overcome issues accompanied with overhanging
structures, Christensen et al.119 synthesized inkjet-printed
structures in a vat of calcium chloride solution. The CaCl2
acted as a mid-fabrication crosslinking agent and provided
buoyancy to support the print. Inkjet bioprinting has achieved
accuracies of singular cells per droplet,120 enabling cell posi-
tioning that can be utilized in many applications in tissue
engineering such as prevascularization and innervation.
In extrusion-based bioprinting, mechanical or pneumatic
pressures are applied to eject continuous strand-like bioink
through a micro-nozzle in a predesigned layer-by-layer fash-
ion.116 Mechanical-based processes are either piston- or
screw-driven but screw-driven forces will produce deadly
shear stresses on the cells; and therefore demonstrate the
lowest cell viabilities of all the bioprinting techniques.
Extrusion bioprinting offers versatile options (in terms of
material choice) and enables the use of high cell-density
bioinks. Studying rheology of the printing bioink is very
important in extrusion bioprinting as the more viscous the
bioink, the higher the induced shear stress is during printing,
resulting in higher cell apoptotic activity. Moreover, this
technique has been developed for in situ applications and
used for osteochondral repair with high success.121
Conclusion
Bone regeneration is a complex and challenging appli-
cation of tissue engineering. The balance between
mechanical competence and macro- and microarchitec-
ture is critical to produce scaffolds relevance to the
nature of native bone tissue. Critical size bone defects,
as a result of trauma or disease, can be regenerated
using a bone tissue scaffold. Currently, autografts are
the gold standard for BTE; however, extraction of auto-
grafts requires a second surgical site, hence increased
morbidity and potential for infection.
Bone tissue scaffolds can provide an alternative to
autologous bone grafts, and can offer highly tailorable
characteristics. Many variables must be considered when
deciding upon the materials and fabrication technique
used that is most suited for the end application of the
bone tissue scaffold. The main consideration should be
the positioning of the implant, as the requirements for
load-bearing and non-load-bearing scaffolds can be sig-
nificantly different. The aim of scaffolding material is to
preserve the existing bone and stimulate osteogenesis for
the regeneration of bone and functional restoration. For
this purpose, it is important to consider the material to be
used: polymeric scaffolds are more suited to non-load-
bearing sites, provide excellent ductility but have low
strength; ceramics can maintain space for long periods of
time and exhibit excellent osteoconductivity, but suffer
from brittleness; biometals are ductile and strong, yet can
be expensive and difficult to process. For this purpose,
investigating novel biomaterials or combinations of bio-
materials is essential for determining a suitable BTE
approach.
The manufacturing process used for the scaffold is also
an important consideration as this influences the structural
properties such as porosity, pore size and interconnectivity
of pores. Conventional manufacturing techniques offer an
established, low-cost and accessible method for manufac-
turing scaffolds. They have demonstrated a high degree of
success in vivo and clinically; however, the more recent
development of 3D printing has led to improvements in
scaffold design and repeatability. 3D printing has an
advantage over conventional techniques as it can provide
control over spatial geometry and microarchitecture, as
well as reproducibility between prints, yet usually requires
a high setup-cost. Overall, the techniques discussed in this
review have demonstrated excellent potential for the appli-
cation of BTE.
Author contributions
S.A., S.R. and M.P. conducted a literature review to provide the
information for this review article; Z.P.K., P.R. and M.B. wrote
the article; and R.S. and O.J. proof read the manuscript and
helped with the final editing.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with
respect to the research, authorship and/or publication of this
article.
Perić Kačarević et al. 15
Ethical statement
There are no animal or human experiments carried out for this
article.
Funding
The author(s) received no financial support for the research,
authorship and/or publication of this article.
ORCID iDs
Patrick Rider https://orcid.org/0000-0002-3620-7199
Mike Barbeck https://orcid.org/0000-0002-3001-1347
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