Masa Eli 2019

Tissue
Engineering
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© Mary Ann Liebert, Inc.

DOI: 10.1089/ten.TEB.2018.0381
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Challenges in 3D Printing of Bone Substitutes
Reza Masaeli1,*, Kavosh Zandsalimi2, Morteza Rasoulianboroujeni3, Lobat Tayebi 3,4,*
This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
1
Dental Biomaterials Department, School of Dentistry, Tehran University of Medical
Sciences, Tehran, Iran
2
Department of Life Sciences Engineering, Faculty of New Sciences and Technologies,
University of Tehran, Tehran, Iran
Downloaded by SENCKENBERG/ZEITSCHRIFTEN from www.liebertpub.com at 07/20/19. For personal use only.
3
Marquette University School of Dentistry, Milwaukee, WI 53233, USA
4
Department of Engineering Science, University of Oxford, Oxford, OX1 3PJ, UK
Challenges in 3D Printing of Bone Substitutes (DOI: 10.1089/ten.TEB.2018.0381)
* Corresponding authors:
Reza Masaeli, PhD, DDS (email: dr.rezamasaeli@yahoo.com)
Lobat Tayebi, PhD (email: lobat.tayebi@marquette.edu)
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Abstract:
It is hoped that tissue engineering will provide a solution to meet the growing needs for
bone substitutes. Among the potential solutions, 3D printing is a promising method to
fabricate functional bone substitutes especially for treatment of complex and critical‐sized
bone defects. Despite its encouraging achievements, 3D printing of bone substitutes still
faces serious challenges including mechanical strength, shape complexity, optimization of
pore parameters, and vascularization. The newer approach, i.e., 3D bioprinting, is also
confronted with challenges, which have prevented the realization of the dream of
fabricating functional patient‐specific bone substitutes. This article reviews the major
challenges toward 3D printing and bioprinting of bone substitutes and recent studies
addressing them. Potential solutions for each challenge and future directions are also
provided.
Keywords: Tissue Engineering, 3D Printing, Bioprinting, Biomaterials, Bone Substitutes
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1. Introduction:
Although bone has mechanisms for dynamic remodeling and self‐healing, large bone
defects need external interventions and/or regenerative techniques to be repaired. The
smallest size intraosseous wounds unable to self‐heal are called critical‐sized bone defects
(CSBDs) [1, 2]. A CSBD can be caused by a disease or a trauma with lifetime regeneration
rate of less than 10% [3]. Thus, external interventions are needed to repair CSBDs and
larger defects. The main treatments to restore and regenerate CSBDs are bone grafting,
bone transport, and bone tissue engineering [4‐6].
Bone grafting: Bone grafting has been a routine clinical practice in orthopedic and spine

surgeries [7, 8], reconstructive surgeries of traumatic/tumor bone lesions [9‐11], corrective
cranio‐maxillofacial surgeries [12, 13], and bone augmentation procedures in dental
implant dentistry [14, 15]. In 2012, the global number of annual bone grafting procedures
was estimated to be 2.2 million [16].
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Autografts, allografts, xenografts and alloplastic (synthetic) grafts are different classes of

bone grafts [17]. Each type of bone graft has its own advantages, disadvantages,
indications and limitations.
Autografts are usually harvested from a patient’s iliac crest or fibula and possess
osteogenic cells and osteoinductive growth factors, without the risk of immunogenicity.
The main drawbacks of autografts include potential risk of infection at the donor and graft
sites, pain and morbidity at the donor sites, and limited availability in elderly patients and
children [18, 19].
Allografts are mainly derived from donated cadaveric bones. Rigorous chemical treatments
and gamma irradiation during sterilization deteriorate osteoinductivity of allografts and
decrease their fracture toughness and bending strength [20]. The other disadvantages of
allografts are risk of infection transmission and costly laborious processes [21, 22].
Xenografts are obtained by decellularization, deproteinization, demineralization, and

freeze drying of bovine, porcine or equine bones. Xenografts suffer from low mechanical
properties [23], and the risk of the zoonotic diseases transmission [24].
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Synthetic bone grafts lack osteogenic and osteoinductive properties. Yet, due to the
precise control of structural composition and porosity, and the lack of disease transmission
and donor site morbidity, majority of the studies in the recent two decades have focused
on developing synthetic bone grafts [25‐27].
Bone transport: Bone transport technique, also known as Ilizarov surgery, is a widely
practiced method that has been used to repair bone fractures and defects. The method is
based on distraction osteogenesis, in which controlled directional mechanical stresses are
applied on the bones existed at the defect site to induce new bone formation [28, 29]. The
major applications of bone transport technique include repairing femoral and tibial
fractures, correction of limb shortening, and reconstruction of maxillofacial deformities
[30‐33]. Compared to bone grafting, bone transport is easier, safer, and cheaper.
Furthermore, it allows for simultaneous bone and soft tissue repair. However, this method
suffers from long‐lasting treatment period which results in patient discomfort and imposes
psychosocial distress over the patients and their families [34, 35].
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Bone tissue engineering: Tissue engineering has emerged as a promising solution to meet
the ever‐increasing demand for tissue substitutes. Despite the obstacles and limitations,
innovations of tissue engineering has attracted great scientific and public interests during
the last decade [36, 37].
One of the promising approaches in tissue engineering is three‐dimensional (3D) printing
of tissues and organs [38, 39]. Using an additive approach, 3D printing is developed to
position biomaterials and biomolecules precisely in a layer‐by‐layer 3D order resembling
the extracellular matrix (ECM) [40]. The ultimate goal of 3D printing is to fabricate and
assemble various tissues and organs, including skin, liver, bone, cartilage, cardiovascular
and neuronal structures [41‐48]. 3D printing is in its early days of creating functional
tissues and organs, however it has found unprecedented applications in high‐throughput
drug screening and discovery [49‐51] and fabrication of research models especially for
cancer studies [52, 53].
More than 40 different 3D printing methods have been devised, among which fused
deposition modeling (FDM), stereolithography (SLA), selective laser sintering (SLS),
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photopolymer jetting (PPJ), and powder binder jetting (PBJ) are the most widely used
modalities [54]. For 3D printing of cellular microenvironments, several biomaterials are
used as inks, including natural biomaterials (gelatin, alginate, collagen, fibrin, hyaluronic
acid and hydroxyapatite), synthetic biomaterials (polyethylene glycol (PEG),
polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic‐co‐glycolic
acid) (PLGA) and hybrid biomaterials. Recent progress had led to the emergence of
advanced inks with high printability, high strength, high viscoelasticity, high cell viability
and the ability to regulate cellular functions [55].
3D printing of bone substitutes: Since 3D printing allows control of the shape, size, and
distribution of interconnected pores, it has found many applications in the construction of
bone tissue substitutes. The most widely used materials for 3D printing of bone are
bioactive glasses [56‐58], hydroxyapatite (HA) [59‐61], brushite [62, 63], monetite [64, 65],
β‐tricalcium phosphate (β‐TCP) [66‐68], tetracalcium phosphate (TTCP) [69, 70], biphasic
calcium phosphate (BCP) [71, 72], and polycaprolactone (PCL) [73, 74] (Figure 1).
Tissue Engineering
Furthermore, the lack of osteoinductivity of synthetic bone grafts are compensated by
possible entrapment of growth factors and osteogenic drugs in 3D printed scaffolds.
Controlled sustained and localized delivery of these additives improves tissue ingrowth
and bone formation [75].
Figure 1
2. Challenges in 3D printing of bone substitutes
3D printing has created opportunities for the development of functional bone substitutes,
however, it still faces challenges. The major challenges in 3D printing of bone substitutes
include mechanical strength, shape complexity, porosity, and vascularization [76‐78].
Although the solutions proposed for these challenges have optimized printing parameters
and led to emergence of advanced powders and binders, development of 3D printing
materials and methods is still ongoing.
In recent years, many studies have been conducted to find effective solutions to the
aforementioned challenges, and the number of these studies is progressively increasing.
These studies are summarized below.
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2.1. Mechanical strength
3D printed constructs with inadequate strength are usable only in non‐load bearing
defects. Chemical composition, structural design, processing conditions and post‐

processing changes are the main factors that determine mechanical properties of the final
constructs.
Many efforts have been made to improve strength of 3D printed bone scaffolds by
modifying the chemical/structural composition and optimizing processing and post‐
processing conditions. Thermally induced densification [79], liquid phase sintering [80, 81],
microwave sintering [82, 83], monomer/polymer infiltration [84, 85] and doping [86‐89]
are the main solutions offered to improve mechanical properties of bone substitutes.
In 2014, Feng et al. studied the effects of ZnO doping on mechanical properties of β–TCP
scaffolds fabricated by selective laser sintering (SLS). Results indicated that adding 2.5 wt%
ZnO to β‐TCP powder increases hardness, stiffness, compressive strength and fracture
Tissue Engineering
toughness of β‐TCP scaffolds [90]. Another study conducted by Castilho et al. was to
develop a ceramic‐polymer composite suitable for 3D printing of TCP bone scaffolds with
improved mechanical and biological properties. Results showed that direct mixing of TCP
and 2.5% alginate powders improves mechanical properties, cytocompatibility and
proliferation rate. The authors concluded that homogeneously distributed polymers in
ceramic matrices could be promising for fabrication of bone substitutes [91].
Table 1 summarizes some of the recent studies performed to enhance the mechanical
properties of 3D printed bone substitutes.
Table 1
2.2. Shape complexity
Fabrication of patient‐specific anatomically‐shaped bone substitutes is challenging due to
the large anatomical variation of patients and variable defect size and shape. 3D printing is
a promising method to realize this goal, because it has the potential to be combined with
medical imaging techniques and acquire the anatomical data required to fabricate
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personalized bone substitutes. Furthermore, it offers high control over micro‐ and macro‐
architecture of the constructs and allows for creating complex shaped structures [92, 93].
Generally, layer‐by‐layer fabrication of complex‐shaped constructs is limited due to
excessive increase of construct’s horizontal cross section that causes deviation from the
desired shape. In industrial production methods, this problem is resolved using
structural/sacrificial layer processes in which the sacrificial layers provide mechanical
support for structural layers [94]. However, due to the lack of biocompatibility of the
materials used in these procedures, this solution is not always applicable to fabricate
tissue substitutes. This problem can be overcome through indirect 3D printing (casting the
powder into pre‐printed molds) [95, 96]. Indirect 3D printing has made it possible to
fabricate complex‐shaped large‐scale porous scaffolds with pore sizes up to 500 μm.
However, difficult demolding limits the variety of shapes manufacturable by this method,
and incomplete porogen packing reduces the uniformity of the microstructure [97, 98].
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In 2014, Castilho et al. [99] used biphasic calcium phosphates (BCPs) with different
hydroxyapatite (HA) to tricalcium phosphate (TCP) ratios and performed their setting via a
hydraulic reaction to obtain scaffolds with precisely controlled shapes. The dimensional
accuracy of scaffolds was above 96.5% with the minimum size of macropores around 300
μm. Although the resultant scaffolds lacked sufficient strength to bear loads, they could be
used as individual non‐load bearing bone implants because of their excellent shape
fidelity.
2.3. Optimization of pore parameters
Tissue responses and mechanical properties should be balanced in accordance with the
intended use for bone substitutes. One approach to achieve this is controlling pore
parameters. Minimum pore size required for infiltration and migration of bone cells is 100‐
150 μm. Larger pores (> 300 μm) enhance new bone formation and vascularization [100],
but decrease constructs’ strength [101]. Pore parameters including volume, distribution
and geometry affect tissue responses, strength and degradation kinetics of the 3D printed
constructs [102, 103].
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In 2016, Roohani‐Esfahani et al. designed a novel triphasic bioactive glass‐ceramic
consisted of a glass phase, strontium doped hardystonite (Sr‐Ca2ZnSi2O7) and submicron
crystals of gahnite (ZnAl2O4) to fabricate 3D hexagonal inter‐connected porous scaffolds
with large pores required for bone ingrowth and effective solute diffusion. Compressive
strength of the resultant scaffolds were in the range of cortical bones, implying their
possible application in load bearing bone defects [104]. In another study, Sears et al.
utilized high internal phase emulsions of propylene fumarate dimethacrylate (PFDMA)
shelled with PCL or PLA to fabricate high strength 3D porous bone scaffolds. The printed
constructs possess hierarchical macro‐pores (250 μm) and micro‐pores (5–30 μm) with
compressive strength similar to trabecular bones. The authors concluded that this dual
modality printing could be considered as a promising approach for developing complex
tissue scaffolds [105].
Table 2 lists recent studies aimed at optimization of pore parameters.
Tissue Engineering
Table 2
2.4. Vascularization
It has been well proven that a rich blood supply is required to meet nutritional needs of
bone cells and removal of their waste products. Therefore, successful function of
engineered bone substitutes in clinical conditions is dependent on the formation of
hierarchical vascular networks throughout the construct. Furthermore, vascularization is a
determining factor in integration of bone substitutes within defects [113, 114].
Many studies have focused on improving vascularization in bone substitutes (see table 3).
Growth factor delivery, co‐culturing, mechanical stimulation, utilization of angiogenic
biomaterials and microfabrication of biomimetic constructs are the most used strategies to
regeneration of vascularized bone tissues [115].
Table 3
In 2016, Cui et al. suggested an innovative dual 3D printing method consisting of SLA and
FDM to fabricate bone substitutes with improved osteogenic and angiogenic properties.
The printed constructs were composed of PLA fibers arranged in a Haversian‐like structure,
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with human umbilical vein endothelial cells (HUVEC)‐laden gelatin methacrylate hydrogels
penetrated into the hollow spaces. Furthermore, vascular endothelial growth factor (VEGF)
and bone morphogenetic protein 2 (BMP2) were regionally immobilized in constructs to
promote angiogenesis and osteogenesis, respectively. Results revealed efficient
vascularized bone regeneration of the dual printed constructs. The authors concluded that
the proposed method could be utilized in clinical translation [124].
In another study, Fahimipour et al. designed a TCP‐based scaffold containing VEGF‐loaded
PLGA microspheres to induce osteogenesis and angiogenesis simultaneously. They used
gelatin and alginate as hardening and thickening agents, respectively, and optimized the
formulation of the ink to be printable at room temperature. Results showed that the
proliferation rate of seeded HUVECs doubled due to controlled release of VEGF.
Furthermore, mechanical properties of the scaffolds were similar to cancellous bone. The
authors proposed the fabricated 3D printed porous scaffolds for bone regeneration in
critical craniofacial defects [125].
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3. 3D bioprinting of bone substitutes and its challenges:
3D bioprinting is an emerging tissue engineering approach that allows for direct printing of
cell‐laden constructs. The bioink, which is a collection of biomaterials, biological
molecules, and living cells, is printed based on the computer‐aided designed model of the
tissue [126, 127]. Compared to the conventional tissue engineering methods, 3D
bioprinting provides more control on cell distribution within the construct. Furthermore, it
makes it possible to fabricate tissue blocks and organoids, thus facilitating the construction
of tissue‐ and organ‐substitutes. The main drawbacks of 3D bioprinting include the limited
choice of bioinks and the possibility to reduce the cell viability during the printing process
[128, 129].
Among the different modalities, inkjet, microextrusion and laser‐assisted bioprinting are
the most commonly used 3D bioprinting methods [126]. Several efforts have been made to
fabricate blood vessels [130‐133], neuronal tissues [134, 135], liver [136‐138], skin [139‐
141], cartilages [142‐145], and bones [146‐151] via bioprinting.
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The main challenge of bioprinting is the design of the appropriate bio‐ink. In 3D bioprinting
of bone substitutes, the bio‐ink must meet a number of required properties including
biocompatibility, biomimicry, biodegradability, bioprintability, and mechanical integrity

[129, 152]. Many efforts have been made to find a bioink with an optimal balance of
properties to overcome the limitations of the bioprinted constructs.
One major limitation of the bio‐printed bone substitutes is their inadequate mechanical
properties. In 2015, Gao et al designed a bioink with adequate mechanical properties to
create bone and cartilage substitutes. The bioink was composed of human mesenchymal
stem cells (hMSCs) loaded in poly(ethylene glycol) dimethacrylate (PEG)‐gelatin
methacrylate (GelMA) hydrogel. During the inkjet bioprinting process, PEG and GelMA
were photo‐crosslinked to enhance the mechanical properties of the constructs. Results
obtained 21 days after printing showed the improvement of elastic modulus of hMSC‐PEG‐
GelMA constructs by 100% compared to the cell laden PEG or GelMA constructs. The
findings of the study demonstrated that photo‐cross‐linkable multimaterial hydrogels are
Tissue Engineering
promising bioinks to create bone and cartilage substitutes [146].
In another study conducted by Daly et al in 2016, cartilaginous templates were created by
printing bone marrow derived MSCs into the Arg‐Gly‐Asp‐modified alginate hydrogels. To
improve the mechanical properties of the constructs, the MSC‐laden bioink was co‐
deposited with PCL fibers. Results showed 350 fold increase in compressive modulus of
bioink/PCL templates. The constructs had the potential to be implanted as vertebral
bodies in load bearing locations [147].
In 2017, Bendtsen et al formulated a novel hydrogel with optimal printability and
biocompatibility to fabricate bone substitutes. The bioink composed of alginate, polyvinyl
alcohol (PVA), and hydroxyapatite (HA) showed excellent rheological properties and high
shape fidelity. Furthermore, its osteoconductivity made it a favorable environment for new
bone formation. The mouse calvaria 3T3‐E1 (MC3T3) cells encapsulated into the alginate‐
PVA‐HA hydrogels had viability of 95.6%. The authors concluded that the proposed
formula is a promising solution for repairing bone defects [148].
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Similar to 3D printed bone substitutes, bioprinted constructs also suffer from poor
vascularization, which could result in the failure of the constructs after implantation. This
limits the application of bioprinting constructs, especially in large bone defects. To provide
functional vasculature in large bone defects, Byambaa et al developed a novel hydrogel
that allows for co‐culturing of bone marrow derived hMSCs and human umbilical vein
endothelial cells (HUVEC). The GelMA hydrogel conjugated by vascular endothelial growth
factor (VEGF) and loaded with silicate nanoplatelets promoted simultaneous angiogenesis
and osteogenesis. 3 weeks after in vitro culture, the constructs demonstrated high cell
viability, high proliferation rate, and excellent structural stability. The results also indicated
the formation of a mature bone niche after 21 days of culture [149].
Recently, Anada et al fabricated a highly vascularized biomimetic hydrogel suitable for
bone tissue engineering applications. The dual ring bone‐mimetic construct was composed
of a GelMA/octacalcium phosphate external ring and a central ring of GelMA loaded with
HUVECs. The HUVEC spheroids promoted angiogenesis by forming capillary networks
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within the construct and the octacalcium‐containing ring stimulated bone formation [151].
4. Future directions:
In the near future, demands for bone substitutes will increase, especially in aging
populations [153,154]. 3D printing and bioprinting have the potential to fabricate complex‐
shaped patient‐specific bone substitutes.
In the next coming years, hardware and software advancements will allow for fabricating
constructs with intricate hierarchical structural details close to that of natural bones.
Moreover, multimaterial (bio)inks and multi‐head (bio)printing machines will make it
possible to create constructs with chemical composition similar to bones. Besides, pre‐
vascularized bone substitutes will facilitate host vascular ingrowth. Therefore, soon after
implanting the construct, its capillary network will undergo anastomosis with host tissue’s
microvasculature. This will dramatically reduce the failure rate of 3D (bio)printed bone
substitutes due to the occurrence of hypoxia, apoptosis, or immediate cell death at the
implantation site. In addition, advanced (bio)inks with optimal printability and
biocompatibility along with optimized fabrication processes will enable us to create bone
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substitutes with adequate mechanical properties and stability to withstand physiological
stresses at load‐bearing sites [155‐157].
3D printing and bioprinting are yet facing challenges in fabricating functional bone
substitutes. Once the existing challenges have been resolved, the fabrication processes
could be scaled up and mass production of patient‐specific functional bone substitutes will
be feasible.
5. Conclusion:
Despite its promising achievements during the last two decades, 3D printing of bone
substitutes is still challenging. Mechanical properties, shape complexity, optimization of
pore parameters, and vascularization are the main challenges in 3D printing of bone
substitutes. Many efforts have been made to resolve these challenges by optimizing
materials and methods for 3D printing of bone substitutes.
Tissue Engineering
Studies have demonstrated that thermally induced densification, liquid phase sintering,
microwave sintering, monomer/polymer infiltration and doping can improve mechanical
properties of bone substitutes. On the other hand, pore volume, distribution and
geometry should still be optimized to improve tissue responses, strength, and degradation
kinetics of 3D printed constructs. Considering the importance of angiogenesis and its
undeniable role in the formation of functional bone substitutes and their integration with
the surrounding tissues, many studies have been performed to induce vascularization in
3D printed bone grafts via growth factors delivery, co‐culturing, mechanical stimulation,
utilization of angiogenic biomaterials, and microfabrication of biomimetic constructs.
In 3D bioprinting, the main challenge is to design a bioink with optimal balance of
biocompatibility, biomimicry, biodegradability, bioprintability, and mechanical integrity.
Besides, vascularization of bioprinted constructs is still challenging. Innovations are still
needed to translate 3D (bio)printed bone substitutes from the lab to the clinic.
Funding: This research did not receive any specific grant from funding agencies in the
public, commercial, or not‐for‐profit sectors.
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Declarations of interest: none.
animals performed by any of the authors.
Ethical approval: This article does not contain any studies with human participants or
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Table 1: Solutions to enhance mechanical properties of 3D printed bone substitutes.
Material Solution Results Ref.

high‐density Thermally induced 6 and 4 times increase in 79

polyethylene densification tensile modulus and strength
(HDPE)
HA/apatite– Flexural strength increased 80
wollastonite from 1.27 MPa to 76.82 MPa.
Liquid phase sintering
β‐TCP + Bioglass 45S5 4.16 times increase in 81

as liquid phase (5 wt %) compressive strength
TCP Microwave assisted sintering 46%‐69% increase of 82

compressive strength
TCP Compressive strength 83
increased from 6.62 MPa to
Tissue Engineering
10.95 MPa.
Calcium phosphate + Significant increase in 84
collagen (1‐2 wt%) maximum flexural strength
and toughness
HA+10 wt % PCL or Improvement in elastic 85
PLGA properties:
Polymer infiltration ‐ Tensile elastic modulus:
4.3 ± 0.4 MPa (HA‐PLGA),
10.3 ± 1.3 MPa (HA‐PCL)
‐ Maximum strain:
36.1 ± 4.3% (HA‐PLGA),
61.2 ± 6.4% (HA‐PCL)
TCP + SiO2 (0.5 wt%) Addition of dopants increased 86
and ZnO (0.25 wt%) compressive strength 2.5 fold.
TCP + SrO (1 wt%) and Doping Compressive strength 87
MgO (1 wt%) increased 2 times.
Si‐doped HA Compressive strength 88
Tissue Engineering

Page 31 of 36
Mg‐doped wollastonite

increased 3 times.
Compressive strength
increased to 3.8 ± 1.1 MPa.
31
89
Page 32 of 36

32
Table 2: Findings from some studies addressed pore parameters of 3D printed bone
constructs.
Materials Pore parameters Study remarks Ref.

Structural properties of inner
Porosity ~ 75%
HA + TCP channels affects the strength of 71
Pore diameters: 150–750 μm
3D printed scaffolds.
bioactive glass + Porosity: 60% Controlling structural properties
polyvinylalcohol Mean pore size: 1000 μm of pores provides excellent 106

(PVA) binder Morphology: square or parallelogram mechanical strength.
Micro and macro pores favor
Mean pore diameter:
the internal environment of 3D
HA ‐ Macropores: 450 ‐570 μm 107
printed scaffolds for cellular
‐ Micropores: 10– 30 μm
adhesion and proliferation.
Tissue Engineering
Porosity: 88‐93%
Hierarchical porous structure: Open porous filaments enhance
Alumina (Al2O3)
‐ mm‐scale pore sizes from printing compressive strength even in 108
+ PVA
‐ μm‐scale pores form emulsion scaffolds with 90% porosity.
templating
Optimization of printing
parameters (layer thickness and
Zp150 composite Porosity: 23.6‐45%
delay time) leads to maximum 109
+ Zb63 binder Pore size: 680‐750 μm
porosity and enhanced
mechanical properties.
3D printed scaffolds with
Polybutylene
Porosity: 62‐79% complex pore structures has
terephthalate 110
Pore size: 196‐772 μm lower stiffness than their simple
(PBT)
counterparts with linear pores.
Regular porosity improves
Porosity: 92.55%
PCL + HA cellular attachment, 111
Pore size: > 600 μm
proliferation and migration.
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Page 33 of 36
glass
13‐93 bioactive

Porosity: 50%
Pore size: 300‐800 μm
printed scaffolds.
attachment of cells to 3D
and microporosity enhances
Increasing surface roughness
33
112
Page 34 of 36

34
Table 3: Solutions to induce vascularization in 3D printed bone scaffolds.
Scaffold Solution Remarks Ref.

Co‐culture of Adipose
Derived MSC (ADMSC) and
Human Umbilical Vein
Capillary‐like networks
Endothelial Cells (HUVEC)
PCL/HA generated within the 116
within hyaluronic acid and
scaffolds.
gelatin hydrogels and forming
the cell‐laden hydrogels
around 3D printed scaffolds
Sustained release of
Loading PLGA‐PEG‐PLGA
VEGF‐165/BMP‐2 with
hydrogel containing VEGF‐
HA/PCL synergistic effects led to 117
165/BMP‐2 into the porous
Tissue Engineering
favorable osteogenesis
scaffolds
and angiogenesis.
Sequential release of
Immersion of scaffolds in rhBMP‐2 and rhVEGF
gelatin and polylysine solution induced angiogenesis
Polylactic acid (PLA) 118
with sequential adsorption of and osteogenesis via a
rhBMP‐2 and rhVEGF cooperative biological
signaling system.
Multifunctional hollow
pipes facilitate
infiltration of blood
Fabrication of micro‐ vessels and bone cells
Bredigite (Ca7MgSi4O16) 119
structured hollow pipes and accelerate scaffold
degradation and release
of bioactive ions (Si, Ca
and Mg).
Gelatin methacryloyl - Incorporation of silicate Simultaneous 120
Page 35 of 36

35
(GelMA) nanoplatelets in 3d printed engineering of bone and
GelMA hydrogels vascular tissues in one
- Functionalization of construct through a
hydrogels with VEGF simple direct printing
method.
Perfusable
microchannels facilitated
Design and fabrication of
vascularized bone
PLA conjugated with nano‐ interconnected micro‐

formation due to rapid 121
HA channels within the bone
perfusion of blood
matrix
throughout the
construct.
Seeding of adipose‐derived Seeding angiogenic cells
stem cells (ASCs) with fibrin within the graft prior
Tissue Engineering
PCL 122
gel into 3D printed PCL implantation induced
scaffolds vascularization.
nHA + PLGA nanospheres Cellular adhesion,
encapsulated with TGF‐β1 + Hierarchical structural design proliferation, and
polyethylene glycol and sustained release of differentiation improved 123
diacrylate (PEG‐Da) growth factor in bio‐inspired graded
hydrogel constructs.

Page 36 of 36

36

Figure 1: (A) Examples of 3D printed bone scaffolds made of compositions of various
materials. Materials of scaffolds from left to right: PCL, Gelatin/ bioactive glass/ poly (3,4‐
ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS), PLA, PLGA, PLA, PLA, β‐
TCP/HA, PLA, PCL. (B) 3D printed β‐TCP/HA (80:20) scaffold (C) 3D printed PLA scaffold (D)
3D printed PCL scaffold (E) 3D printed PLGA/TiO2 (90:10) scaffold seeded by osteoblasts
Tissue Engineering

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Material Solution Results Ref.

high‐density Thermally induced 6 and 4 times increase in 79

β‐TCP + Bioglass 45S5 4.16 times increase in 81

TCP Microwave assisted sintering 46%‐69% increase of 82

Materials Pore parameters Study remarks Ref.

polyvinylalcohol Mean pore size: 1000 μm of pores provides excellent 106

Scaffold Solution Remarks Ref.

PLA conjugated with nano‐ interconnected micro‐

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