J Mater Sci (2020) 55:3729–3749

Review
REVIEW

A review of 3D bio-printing for bone and skin tissue

engineering: a commercial approach
Nima Beheshtizadeh1 , Nasrin Lotfibakhshaiesh1,* , Zahra Pazhouhnia1 ,
Mahdieh Hoseinpour1 , and Masoud Nafari2

1
Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of
Medical Sciences, Tehran, Iran
2
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and
Technology, ACECR, Tehran, Iran

Received: 10 July 2019 ABSTRACT

Accepted: 27 November 2019 The ultimate prospect of tissue engineering is to create autologous tissue grafts
Published online: for future replacement therapies through utilization of cells and biomaterials
4 December 2019 simultaneously. Bio-printing is a novel technique, a growing field that is leading
to the global revolution in medical sciences that has gained significant attention.
Ó Springer Science+Business Bio-printing has the potential to be used in producing human engineered tissues
Media, LLC, part of Springer like bone and skin which then ultimately can be used in the clinics. In this paper,
Nature 2019 the 3D bio-printing applications of the engineered human tissues that are
available (skin and bone) are reviewed. It is evident that various tissue engi-
neering techniques have been applied in the fabrication of skin tissue; therefore,
it leads to introduce tissue substitutes such as complementary, split-thickness
skin graft, allografts, acellular dermal substitutes and cellularized graft-like
commercial products, i.e., Dermagraft and Apligraf. Also, some bone scaffolds
based on hydroxyapatite and biphasic calcium phosphate are available in the
market. The technology of bio-printing has got validated for bone and skin
tissue fabrication, and it is hoped that other tissues could be produced by this
technique.

Introduction implantation, substitution and restoration. Graft

implantation has comprehensively extensive lists of
Regenerative medicine holds the promise to restitute anticipants all around the world, for example, organ
the normal function of cells, tissues or organs lost due transplant waiting list updated every 15 in the USA
to disease or damage via replacing or regenerating [2]. The ultimate prospect of tissue engineering is to
[1]. In fact, there are three solutions for patients with create autologous tissue grafts for future replacement
organ impairment, based upon the condition and therapies through utilization of cells and biomaterials
severity of the destruction, they are: graft [3, 4]. Besides, tissue engineering has been seen as an

Address correspondence to E-mail: [email protected]

https://doi.org/10.1007/s10853-019-04259-0
3730 J Mater Sci (2020) 55:3729–3749

efficient method to assist in rescuing lives and for fabrication that asserts micrometric cell patterning
improving the quality of life. There are many meth- for extensive biomedical engineering applications
ods to produce an appropriate scaffold that cells [21]. Generating the printing paths, selecting appro-
could seed on, like foam casting, electrospinning, priate bio-inks, printing control and performing
phase separation, decellularizing, etc. quality control after printing are major steps that
Recently human donor organs, such as kidney assure passable print [22].
[5–7], lung [8–10], heart [11–13] and liver [14–16], Generating the printing pathways and verifying its
have been experimented to obtain decellularized feasibility by designers is the first step of the bio-
extracellular matrix scaffolds (dECM) in order to printing process. By selecting appropriate cells and
clarify their potential application in regenerative hydrogels, loading the bio-inks into the bio-printing
medicine. However, despite the important basic sci- system is needed. The next step is sending the
ence achievements in the field, clinical applications of designed paths to the controlling system and build-
decellularized tissues are not available yet [17]. Also, ing structures by depositing bio-inks. The bio-printed
the use of dECM scaffolds for bioengineering of constructs are examined through a microscope. As
human scale patient-specific organs using human more researches are performed implementing the
pluripotent stem cells (hPSCs) has been considered as techniques of 3DBP, this will ensure an enhancement
a major platform for therapeutic applications [18]. of the printing resolution and quality and also the
Interestingly, the concept of organ printing has lately actualization of the current challenges, furnishing the
taken the center stage due to recent 3D bio-printing capacity to print more complex 3D constructs.
(3DBP) advancements [19–22]. Due to the advances in the additive manufacturing,
3DBP is considered to have significant impact in some of the most well-known for bone tissue engi-
tissue engineering area. Bio-printing is a growing neering, including stereo-lithography, selective laser
field that is leading to a global revolution in medical sintering, fused deposition modeling and three-di-
sciences and has gained significant attention world- mensional printing are reviewed [32, 33]. Moreover,
wide because of tremendous transformation it will some basic physical principles along with primary
have in the treatment of diseases [23]. applications of aforementioned techniques have been
Bio-printing can be defined as the simultaneous reviewed beside a list of bone tissue engineering-re-
printing of living cells and biomaterials like hydro- lated biomaterials [33]. Various types of bio-inks used
gels, bioglasses, bio-ceramics and collagen by a pre- for bone bio-printing are reviewed as well as their
scribed layer-by-layer stacking organization, using a major challenges and future strategies [34]. The
computer-aided transfer process for fabrication of recent advancements, limitations, challenges and
bioengineered constructs. Initially the study of cells future strategies in bone bio-printing are summarized
was performed applying 2D structures; nonetheless, in recently published reports [32, 34, 35].
with the initiation of 3D printing to the engineering of There are several studies about 3DBP which cover
tissues, it became feasible for researchers to use 3D diverse tissues [36–43]; meanwhile, to the best of our
scaffolds. Even though vascularization, controlled there isn’t any review study about bone and skin
cell dispersion, innervation and cell deposition with tissues bio-printing. In fact, this paper focuses on
high resolution, in complicated 3D tissues are still tissues that successfully passed the commercializing
current technical challenges faced in 3DBP, however, procedures not only by printing techniques, but also
compared with other techniques it presents many by the prior scaffold fabricating methods. The
advantages such as scalability, simplicity and cost authors tried to include the skin and bone-related
efficiency. Due to these advantages, 3DBP develop- literature that published till preparing the paper with
ment and application has been increasing constantly the ‘‘bio-printing, 3DBP, 3D printing’’ key words,
over the past few years [24]. Also, 3DBP techniques whereas the unrelated papers in tissue engineering
have extensive applications in tissue engineering fields were excluded.
[25–28], transplantation clinics [23–29], drug screen- Our aim was to focus on studies with commonly
ing, high throughput assays [30] and cancer research employed cells, materials and printing methods in
[31]. skin and bone bio-printing (Fig. 1).
Since the human organs are different in terms of
the size, 3DBP is an appropriate and versatile method
J Mater Sci (2020) 55:3729–3749 3731

Figure 1 Various cells,

materials and printing methods
used in bone and skin tissue
bio-printing.

Bio-inks and their performances novel idea that can result in more effectiveness and
lowering of costs.
Among all the bio-inks used for 3DBP, there are some
that are distinguished based on their merits. In some
studies that refer to this issue, specific applications, Skin tissue 3D bio-printing
new methods and spectacular properties have been
reported [44–50]. In certain biomedical appliances, Many tissue fabrication techniques have been
conductivity is of extreme importance while in scaf- employed in the creation of skin tissues which cul-
folds, cell support is vital [24]. In a recent study, a minate in the introduction of tissue substitutes, such
formidable bio-ink was designed for reconstructing as autologous split-thickness skin graft [58], allografts
cardiac tissue [51], it was produced to supply the [59], acellular dermal substitutes and cellularized
correct conductivity and evade postponed electrical graft-like commercial products [59] such as Derma-
confluence in cardiac cells. This new gold nano-rod- graft and Apligraf [60]. New, bio-printing technology
integrated gelatin methacryloyl-based bio-ink was was utilized for skin tissue fabrication, Pourchet et al.
shown to be precisely printable, cytocompatible and [61] produced a two-layered (dermis and epidermis)
boosted the performance of cardiac cells. 3D cell-printed full-thickness skin. The bio-ink was a
Nerve [52], kidney [53] and cartilage [54] rejuve- mixture of gelatin and fibrinogen; moreover, they
nation and restoration including bionic ear [55] are embedded human dermal fibroblasts in the men-
other precise utilizations investigated for bio-ink tioned mixture and printed to produce a dermis
improvement. Impressionable electronics for bio- construct. Next stage was to generate the skin sub-
electronic boundaries are also presently under com- stitutes with 5 mm thickness which seeded the
prehensive investigation [56, 57]. Also, 3DBP of human epidermal keratinocytes over the dermis
commercialized tissue engineering products is a construct. Within 26 days of culture, natural human
3732 J Mater Sci (2020) 55:3729–3749

skin histological-like characteristics of the 3D cell- fibrinogen, calcium chloride (CaCl2) and human
printed skin exhibited. Interestingly, immunofluo- keratinocytes. Based on in vitro and in vivo evalua-
rescence results using different differentiation mark- tions, it was shown that the 3D cell-printed human
ers exhibited high expression of Loricrin, showing skin substitute was very similar to natural human
the barrier function of the skin, which is related to the skin tissue, and extremely differentiated dermis and
stratum corneum formation. Table 1 lists the studies epidermis layers clearly were noticed. They con-
on skin tissue 3DBP. structed an intelligible and efficacious 3DBP tech-
Cubo et al. [62] produced a full-thickness human nique and bio-inks that permitted the creation of a
skin similar to one-step manufacturing one, which double-layer human skin, adopting human plasma
used 3DBP technique. A combined structure was and primary human fibroblasts (hFBs) and human
comprised of four different elements—human keratinocytes (hKCs). The printed tissue was very
fibroblasts, human plasma supplemented with similar to natural human skin and indistinguishable

Table 1 Summary of skin tissue 3DBP studies representing the cell types and source, growth factors, animals and the print technique used
in some skin tissue engineering studies

Biomaterials/bio-ink Cell type Cell source Growth factor/ In vivo Bio-printing References
biomolecules animal model technique

Gelatin–fibrinogen– Fibroblast, Human skin – – Extrusion [61]

alginate keratinocyte
Human plasma/fibrin Fibroblast, Human skin – Nude mice Extrusion [62]
keratinocyte
Gelatin Keratinocyte – – – [63]
methacrylamide
(GelMA)
Collagen hydrogel Fibroblast, Human skin Human keratinocyte – Solid [64]
keratinocyte growth supplement freeform
fabrication
Gelatin–silk fibroin Child foreskin Human FGF-2 Rat Pneumatic [65]
(SF) fibroblasts (CFFs) bio-printing
Chitosan– Fibroblast skin cells Human – – – [67]
polyelectrolyte
gelatin hydrogel
Collagen hydrogel Melanocytes, – – – Extrusion [68]
precursor fibroblast,
keratinocyte
Gelatin–alginate Bone marrow Rat – Mouse Extrusion [69]
mesenchymal stem
cells
Gelatin and sodium Epidermal Mice Mouse plantar dermis and – Extrusion [70]
alginate progenitor cells epidermal growth factor
Collagen and AFS or MSC Amniotic fluid Thrombin Mice Inkjet [73]
fibrinogen
Collagen hydrogel/ Fibroblast and Human skin – – Extrusion and [74]
gelatin/PCL keratinocyte inkjet
Matriderm Fibroblast and Mouse Hydrocortison Male BALB/ Laser- [75]
keratinocyte embryo c-nude assisted
human skin mice
Hydrogel fibrinogen Fibroblast and Human skin Thrombin Porcine Inkjet [76]
and collagen type 1 keratinocyte wound (in situ)
model
J Mater Sci (2020) 55:3729–3749 3733

from dermo-epidermal analogs, precedent hand- from 3D-printed gelatin–silk fibroin base scaffolds.
crafted in their laboratory and employed successfully Also, incorporation of fibroblast growth factor
in the clinic. This method set up a novel technology 2 (FGF-2) could further enhance the treatment effi-
that permitted the production of skin substitutes in cacy. Skin scaffolds shown to contain sulfonated
reasonable amounts at shorter times. It showed moieties in order to raise scaffold hydrophilicity. The
decreased production cost and an improved pro- immobilized growth factor FGF-2 facilitated a sus-
duction line by utilizing an automotive and stan- tained release kinetics, as well as being able to initiate
dardized system of producing skin counterparts, cell proliferation and migration in vitro. Printed
thereby overcoming some of the challenges encoun- scaffolds exhibited favorable results in vivo. Prolif-
tered by the present physical production process. eration rate in this study showed a significant raise
Zhao et al. [63] synthesized varying concentrations from 40 to 75% by using FGF-2. Tissue morphology,
of gelatin methacryloyl (GelMA) hydrogels for a collagen fibril assembly, blood vessel formation and
monolayer skin modernization process. GelMA the expression of various corresponding markers
hydrogels have good physical properties, and sub- grew impressively. These data demonstrated that
sequently they systematically change the concentra- recombinant FGF-2 delivered by the scaffold could be
tion of GelMA in order to control the adhesion, a viable and innovative therapeutic strategy for sev-
proliferation and differentiation of keratinocytes. A ere skin wound.
hydrogel scaffold was used to reach a keratinocyte Rutz et al. [66] introduced a versatile and cell-
suspension, which was used to develop the recon- compatible bio-ink from a variety of amine-contain-
struction of the classified and functional epitromes. ing polymers and their mixtures, synthetically and
The result of this study indicated that the physical naturally. It was shown that 35 formulations of bio-
and biological properties of the resultant hydrogels inks can be customized with regard to composition
could be adequately controlled to meet the require- (additives and composites), the degree of crosslink-
ments for epidermis formation, by altering the con- ing and polymer concentration in order to optimize
centration of GelMA pre-polymer solution. GelMA structural and biological performance, while main-
hydrogels supported the formation of a stratified taining printability.
epidermis with a certain barrier function, e.g., elec- Also, Ng et al. [67] optimized a scaffold based on
trical resistance and prevention of water loss. Higher polyelectrolyte–gelatin–chitosan (PGC) hydrogel by
concentrations of hydrogels indicate the hardness of 3DBP. In order to form polyelectrolytic compounds,
materials for cell adhesion and the formation of sin- the chitosan was modified with the different gelatin
gle-layer keratinocytes and cell adhesion, coupled functional groups at pH of 6.5. In this work, to
with prolonged resistance to collagenase achieve an excellent biocompatibility with fibroblast
degradation. skin cells, the PGC hydrogels were modified at room
Lee et al. [64] constructed a two-layer skin via a temperature for the 3DBP procedure. In fact, PGC
3DBP procedure using skin dermal matrix formed by hydrogels have a high viscosity that is suitable for the
collagen. In fact, replacing the natural human skin by printing procedure at room temperature. The PGC
3D-printed one is not impossible morphologically hydrogels were optimized for the bio-printing of skin
and biologically, while the 3D-printed one was rein- designs. Their scaffolds in 400 lm for three layers
forced via histological and immunofluorescence were representative of the outer epidermal layer and
properties. In order to cover the full thickness of the part of the dermal area. Their findings suggested the
transdermal and localized wound forms, this tech- potential use of polyelectrolyte–gelatin–chitosan
nique has a wide range of applications in skin design hydrogels for skin bio-printing applications.
of toxicity investigations and wound healing. Their Min et al. [68] developed a 3DBP procedure that
study illustrated that cell viability and function were there was capability of producing a thick skin with
affected little by proteins and printing cells in nano- pigmentations. Multiple layers of collagen hydrogel
droplets form. Both keratinocytes and fibroblasts precursors with fibroblasts were printed using
enjoyed sufficiently high rate of viability in this study sodium bicarbonate as the cross-linker. For the skin
[66]. pigmentation induction on the subsequent air–liquid
Xiong et al. [65] study revealed that the rate of full- interface, melanocytes and keratinocytes were
thickness wound healing accelerated by utilizing sequentially printed on top of the dermal layer. To
3734 J Mater Sci (2020) 55:3729–3749

clarify the formation of distinct skin layers also to equipment and tailored bio-inks to support the cor-
recognize the pigmentation presence, histological rect functioning of cells. Reliable in vitro skin models
analysis was done. The printed skin product illus- are urgently required, especially in the cosmetic
trated that final differentiation of the keratinocytes business, for testing cosmetic ingredients as required
caused the formation of the stratum corneum in the by legislation in Europe.
dermal and epidermal layers. Moreover, the epider- In a novel study, Skardal et al. [73] applied bio-
mal layer included melanocytes that showed the printing technique for the full-thickness skin wounds
dermal–epidermal junction with freckle-like pig- treatment in nu/nu mice. They used fibrin-collagen
mentations, without the use of chemical stimuli or gel filled with amniotic fluid-based stem cell (AFSs)
external ultraviolet light. For therapeutic and and mesenchymal stem cells (MSCs) to print them on
research purposes, explanation of 3DBP technique as the wound site.
a productive on-demand option is available and Although Kim et al. [74] invented a novel 3D cell-
presented the ability of engineering ephelides pro- printing single-step process approach for human skin
duction in the biomimetic skin. Additionally, Li et al. engineering with a functional transwell system. They
[69] designed and manufactured the gelatin/alginate established a hybrid 3D cell-printing system that
scaffolds via 3D bio-printer and investigated its bio- facilitated the employment of extrusion and inkjet
compatibility and the histocompatibility over the skin modules simultaneously. The collagen-based con-
wound healing duration using bone marrow-derived struct facilitated this procedure with polycaprolac-
mesenchymal stem cells (BMSCs). tone (PCL) mesh that interrupted collagen
Huang et al. [70] produced a 3D ECM mimic con- contraction during tissue maturation. Moreover, the
struct for the sweat glands regeneration. Sweat inkjet-based disbursing unit was used to evenly dis-
glands perform a vital thermoregulatory function in tribute keratinocytes. This skin model disclosed
mammals; like other skin attachments, they are made promising biological properties that comprised of a
up of epidermal progenitors. Also, adult epidermal steady fibroblast-stretched dermis and stratified epi-
progenitors could be specified to differentiate to a dermis layers 14 days thereon. This method also
sweat gland cell lineage but this remains largely possessed 50 times lower cost and 10 times less
unexplored and whether they have low regenerative consumption of medium than in a stereotyped cul-
potential in response to injury is still questionable. ture. All in all, due to one-step procedure possibili-
Additionally, in order to create a functional in vitro ties, authors advised their cell-printing approach for
cell-laden 3D extracellular matrix mimics (3D-ECM), different human skin replicas engineering.
3D printing technology was used with composite Michael et al. [75] produced a completely cellular-
hydrogels based on gelatin and sodium alginate, due ized auxiliary skin by employing a laser-assisted bio-
to their chemical and structural similarities to ECM printing (LaBP) method. The unique aspect of LaBP is
components. Facilitating cell spreading and matrix the opportunity to situate diverse types of cell in a
formation because of the biological 3D structure precise three-dimensional pattern in space. They
could maintain cell viability. In this study, Ng et al. fixed fibroblasts and keratinocytes atop a sustaining
[71] demonstrated a bio-printing technique that can matrix Matriderm in order to construct skin surro-
be utilized for the production of films for skin wound gates. These skin surrogates were later verified
healing. Application of these bio-printed films could in vivo, using the dorsal skinfold chamber in nude
be in skin tissue engineering area. mice. Full-thickness skin wounds were then implan-
For the first time, Rimann et al. [72] provided an ted with the grafts, and these were completely bound
all-in-one solution for the production of a skin-like to the neighboring tissue when explanted after
soft tissue model with human primary fibroblasts 11 days. The printed keratinocytes established a
and keratinocytes. The defined printing method and multiple-folded epidermis with a commencement of
the advanced bio-ink were cell compatible and differentiation and stratum corneum. The prolifera-
allowed long-term culture models to be generated. tion of the keratinocytes was principally identified in
Further optimization of their model can possibly the percussive fundamental layers. Test tube controls,
promote the full standardization of the production of which were cultured at the air–liquid interface, also
3D tissue model. Also, future advances in this tech- revealed proliferative cells, but these were somewhat
nology will depend on standardizing bio-printing situated in the entire epidermis. The presence of
J Mater Sci (2020) 55:3729–3749 3735

E-cadherin as an indication for adherens junctions eight different types of cells. This study promoted the
and consequently the formation of tissue could be idea of utilizing bio-printing in the clinics.
seen in the epidermis both in vivo and in vitro. In In fact, skin bio-printing is a novel technique which
both conditions, the printed fibroblasts partially must be brought to the clinic (Fig. 3).
remained above the substratal Matriderm where they After a bio-ink pre-cellularization using a novel
formed collagen, while a part of them wandered into passive blending unit method, Thayer et al. [77]
the Matriderm. The blood vessels were seen to produced skin analogs. This method was designed to
develop from the base of the wound and its edges simplify the blending steps of a cell suspension into
toward the printed cells. In summary, Michael et al. an extremely viscous bio-ink. In this study, a bio-ink
[75] successfully exhibited the 3D printing of a cell based on nano-cellulose/alginate was used. The
composite through LaBP and the successive forma- analogs of skin tissue could be grown for up to
tion of tissue in vivo. These discoveries denote the 4 weeks. Histological results showed both tissue-
precondition for the generation of a composite tissue- specific extracellular matrix (ECM) markers deposi-
like skin, comprising of various types of cells in a tion and cell viability.
sophisticated 3D pattern. Albanna et al. [78] described a novel model of a
In vivo skin 3DBP procedure is shown in Fig. 2. mobile skin bio-printing process that quickly man-
In a different study, Binder [76] aimed at devel- ages comprehensive injuries on site. The biomaterials
oping a prototype skin bio-printer that could act as a used consisted of fibrinogen from bovine plasma and
test bed for the core components of an in situ printing thrombin from bovine plasma lyophilized powder.
system. A movement system was constructed capable Immunohistochemistry for human cells showed that
of 1.57 lm precision. The printer enjoyed a cartridge- 3–6 weeks after printing, as well as endogenous cells,
based delivery system capable of delivering up to human fibroblasts and keratinocytes were present in

Figure 2 In vivo study procedure of printed skin scaffolds. The first step is skin biopsy. Then cell isolation and expansion is the second
one. After cell delivery, cell and scaffold transplantation are necessary. Finally, data collection and analysis of procedure is expected.
3736 J Mater Sci (2020) 55:3729–3749

Figure 3 3DBP skin procedure. Reproduced with permission selectable. Whether printing on wound or printing on a feeder layer
from Universidad Carlos III de Madrid (UC3M) and Lawrence and assembling it on wound area is available.
[139]. Based on skin wound area, the wound repair strategy is

the dermis and epidermis of the wound, respectively. dimensionally stable compared to the serious con-
This research discussed the proof-of-concept valida- traction associated with the collagen-based skin
tion of mobile in situ skin bio-printing process with structure. At the other side, extensive keratinocyte
embedded imaging technology to quickly manage migration is observed by day 21 with the observed
full-thickness wounds on site. It was observed that self-assembly of keratinocytes by full coverage of the
the treatment with autologous fibroblasts and ker- 3D bio-printed construct’s pore. Proteomics data
atinocytes, supplied immediately to targeted wound analysis demonstrating striking resemblance of the
places depending on wound size and topology, con- developed 3D human skin model with a number of
sisting of improved wound healing and standard skin-specific pathways and required expression of
in situ skin formation. proliferation and cornification markers depicting full
Admane et al. [79] demonstrated that the special stratification of the advanced 3D human skin model
undulated pattern of the dermal–epidermal junction and extensive transcriptomics.
in the 3D human skin is physiologically relevant to All the mentioned artificial materials are usually
human skin and the bio-printed skin structure was non-biodegradable, and subsequent removal of such
J Mater Sci (2020) 55:3729–3749 3737

temporary wound dressings from the wound site is anisotropic behavior and partly failed at the interface
mandatory. Progression of biodegradable films is of their interlayer bonds. This study used 3D print-
necessary in the skin tissue engineering field [80]. ing, in other words, a print-based additive layer
Albeit solvent casting is a simple construction tech- manufacturing (ALM) technique for fabricating an
nique to produce such films, requirement qualities applicable porous scaffold adequate for the applica-
such as mechanical strength and water transmission tions of the engineering of bone tissue. In brief, the
rate cannot easily guarded through a solvent casting characterization of precursor flow ability, using two
technique. Hence, the bio-printing technique could be common funnel tests, qualitatively comparable with
used to manipulate the ultimate tensile strength, observed printability can be assumed as an exclusive
moisture permeability and the water uptake ability of vital prerequisite since it required recoating of the
the film. build bed which finally distinguished several critical
physical criteria such as mechanical strength,
microstructure and porosity.
3D bio-printing of bone tissue A glance at Table 2 provided reveals that there are
several studies in bone tissue engineering field. Bru-
Bone tissue engineering has been widely studied nello et al. [83] experimented with 3D printing of
using 3DBP. Leukers et al. [81] studied scaffolds powder for the purpose of the bone tissue engineer-
based on hydroxyapatite by using 3D printing for ing. Powder-based 3D printing is propounded as a
engineering of bone tissue. They brought forth a special encouraging bone remodeling technique, as
special test-part in which mouse calvaria 3T3-E1 the exterior frame, interior structure, permeability
(MC3T3-E1) cells were cultured on the scaffolds and and 3D-printed physical properties of bone replace-
maintained under static and dynamic setups, fol- ments can be modified and hence used for particular
lowed by a histological examination which was per- purposes. 3DBP of stem cells and polymer/bioactive
formed to determine the growth of these cells. In glass compound scaffolds for the engineering of bone
brief, the dynamic culture process resulted in a tissue was accomplished by Murphy et al. [84]. They
potent population versus the static culture process. used 3DBP techniques for manufacturing of PCL/
The cells were developed into the structure forming a bioactive borate glass composite, as well as human
nearly indirect communication with the hydroxyap- adipose stem cells (ASCs) in their work, by applying
atite (HA) granules, by creating a scaffold layout with a two-syringe system for fabrication of a scaffold with
inclined layers of 45°. This design facilitated the a bio-polymer/bio-glass composite. As a scaffold,
seeding procedure and increased cell attachment material of this composite dissolved in an organic
because the cells made the structure more integrated solvent, whereas concurrently printed cells remained
and prevented them from sliding down the structure. suspended in the MatrigelÒ. They noted that the
In a static culture, cells are deposited on the interface borate glass content could have an impact on the
of the HA granules in layers, whereas in a dynamic printability of composite paste, the scaffold temporal
seeding process, the cells grow within the cavities of bioactivity, degradability and cell survival in the
the HA granules. scaffold. The extrusion bio-printing technique has
The dynamic cultured cells are significantly dif- important features, which can produce a scaffolding
ferent from the static cultured cells. The powder- structure that supports cells and provides shape and
based 3D printing process developed micro-porosity mechanical integrity. Extrusion bio-printers normally
of the scaffold and as a result, it enhanced the scaf- have more than one syringe, one of them dedicated to
fold surface available for the medium flow and dis- print scaffolding structures. Molten deposition of
solution. Cox et al. [82] also presented the property of polymer and fused deposition modeling (FDM) with
bone tissue scaffolds manufactured by 3D printing a polymer wire feed were the options applied for this
from a composite of HA and polyvinyl alcohol matter, also the pore size factor is considerable
(PVOH). However, mechanical stability, microstruc- because it has a potential to affect the bone growth
ture and porosity of scaffolds produced by 3D after implantation.
printing were affected by the presence of HA: PVOH Byambaa et al. [85] produced bone-like
ratio precursor materials. By testing their compre- microstructure tissue constructs which contained
hensive strengths, these constructs showed perfusable vascular lumen by 3DBP. The bio-printed
3738 J Mater Sci (2020) 55:3729–3749

Table 2 Summary of bone tissue 3DBP studies representing the cell types and source, growth factors, animals and the print technique
used in some bone tissue engineering studies

Biomaterials/bio-ink Cell type Cell Growth In vivo Bio-printing References

source factor/ animal technique
biomolecules model

HA MC3T3-E1, post- Mouse – – Fused deposition [81]

printing modeling 3D
printer
Composite of hydroxyapatite (HA) and – – – – Additive Layer [82]
poly(vinyl) alcohol Manufacturing
(ALM)
Polycaprolactone (PCL)/bioactive borate glass Human adipose Human – – Extrusion-based [84]
composite stem cells (ASCs) 3D bio-printing
Gelatin methacryloyl (GelMA) hydrogel Human umbilical Human VEGF bFGF – Extrusion-based [85]
vein endothelial direct-writing
cells and bio-printing
hMCSCs
PLA-based scaffold conjugated with nHA hMSCs, post- Human VEGF – Fused deposition [87]
printing modeling 3D
printer
Poly(ethylene glycol) dimethacrylate MSCs Human – – Thermal inkjet [88]
(PEGDMA) peptides with nanoparticles of bio-printing
bioactive glass (BG) and hydroxyapatite
(HA)
PCL polymer and a sacrificial Pluronic F127 Cells encapsulated Human – Rat Integrated tissue- [89]
hydrogel composite hydrogel human AFSCs, organ printer
gelatin/fibrinogen/HA/Glycerol (ITOP)
Biphasic calcium phosphate (BCP) (with a MSCs Human VEGF Rat Direct-write [90]
composition of hydroxyapatite (HA) and encapsulated assembly
b-tricalcium phosphate (b-TCP) (robocasting)
technique
PCL/alginate MC3T3-E1 cells, Mouse – – Fused deposition [92]
post-printing modeling 3D
printer
Alginate–PVA–HA hydrogel Cell encapsulation Mouse – – Selective laser [93]
MC3T3 sintering
process
Tricalcium phosphate (TCP), hydroxyapatite Adipose-derived Human bFGF – Fused deposition [94]
(HA), bio-oss (BO), or decellularized bone stromal/stem modeling
matrix (DCB). cells (FDM) process
Poly(e-caprolactone) scaffolds modified with Rabbit bone Rabbit – Rabbit – [95]
hydroxyapatite and poly(propylene marrow stem femur
fumarate) cells BMSC defects

constructs were used as biomimetic in vitro matrices hydrogel formulations with a chemically conjugated
to co-culture human umbilical vein endothelial cells vascular endothelial growth factor (VEGF) to pro-
(hUVECs) together with human mesenchymal stem mote vascular spreading, cell-laden GelMA cylindri-
cells (hMSCs) in a naturally derived hydrogel. A cal parts loaded with silicate nano-platelets were
central cylinder with %5 GelMA hydrogel and low applied, the engineered construct could support cell
methacryloyl substitution was printed. For the pur- survival and proliferation during in vitro maturation.
pose of osteogenesis induction and synthesizing
J Mater Sci (2020) 55:3729–3749 3739

Kim and Kim [86] examined a combination of 3D Wang et al. [90] constructed a 3D-printed bio-ce-
printing, electrospinning and physical punching ramic scaffold with phage nano-fibers to dominate
process techniques to provide a composite of PCL/ obstacle of bone tissue formation. The 3D-printed
alginate construct along nano-fibrous content also scaffold contained biphasic calcium phosphate (BCP)
modified mechanical strength. This was achieved by with a composition of HA and b-tri-calcium phos-
sandwiching layers of micro-sized PCL structures phate (b-TCP) at a mass ratio of 60/40. Uniform
between electrospun layers of PCL/alginate and structure along interconnected macroscale and
punching the final scaffold to produce micro-sized microscale pores on the columns of the scaffold are
pores moving through the stack of 3D-printed and features of the mentioned construct. To achieve
electrospun materials. Interestingly, PCL/alginate modification of scaffold osteogenesis and also its
composite scaffolds against pure PCL scaffolds vascularization, nano-fiber phages loaded with Arg-
showed a considerable 7 days increased cell viability, Gly-Asp (RGD) were combined with chitosan and
calcium deposition and alkaline phosphatase (ALP) adhered to the construct pores through electrostatic
activity at 14 days and a high increase in water interactions. After implantation of this construct in an
absorption capacity due to the improved animal model, it was observed that the host cells
hydrophilicity contributed by the content of alginate interfered with the scaffold and established a vascu-
scaffold. lature, with MSCs undergoing osteogenesis. Even
PLA-based scaffold employing an integrated pre- though the host cells had their survival within the
cipitation modeling 3D printer has been developed cell-laden construct impaired; however, they slowly
by Holmes et al. [87]. To support reconstruction of formed the vasculature. Costantini et al. [91] con-
the ossified bone, like vascular cell growth, scaffolds structed a 3D-printed biomimetic hydrogel scaffold
were designed with highly interconnected 3D containing different combinations of GelMA, chon-
microvascular-mimicking channels. The constructed droitin sulfate amino ethyl methacrylate (CS-AEMA)
scaffolds were also chemically conjugated with nano- and hyaluronic acid methacrylate (HAMA). By
hydroxyapatite (nHA) to induce osteodifferentiation applying of two coaxial-needle bio-printing system,
of seeded hMSCs. SEM imaging illustrated printing they reached cell high density, increased cell viabil-
of vertical micro-channels having both a 500 and ity, high printing resolution and post-printing.
250 mm radius, surrounded by a porous bone matrix. Kim et al. [92] manufactured a SF/HA composite
Gao et al. [88] applied inkjet 3DBP to co-print an of hydrogel, made by hyaluronic acid-dopamine and
acrylated polyethylene glycol (PEG) hydrogel with also with surface modification of HA nanoparticles,
acetylated peptides. Composite hydrogel filled with managed to facilitate distribution of the HA content.
hMSCs applied to initiate simultaneous photo-poly- The hydrogel composite demonstrated excellent cell
merization of the hydrogel during printing following proliferation, an in vivo analysis required to entirely
exposure to ultraviolet light. The constructed scaffold investigate its bone tissue engineering potential.
demonstrated high biocompatibility with a cell via- Bendtsen et al. [93] created a modern formulation
bility of 87.9 ± 5.3% 24 h after printing. Mentioned of a scaffold made of alginate/PVA/HA hydrogel.
constructs containing hMSCs were cultured for This scaffold had the appropriate rheological prop-
21 days in both osteogenic and chondrogenic media. erty for 3D printing of MC3T3 cells. Nyberg et al. [94]
Osteogenic and chondrogenic gene expressions were printed a 3D porous PCL scaffold applying a FDM
noticed to be highly enhanced from day 7–21, as well process. To functionalize them, mineral additives
as a major collagen and extracellular matrix deposi- were mixed in that had been widely utilized com-
tion was observed. mercially and clinically: TCP, HA, Bio-oss (BO) and
Kang et al. [89] developed an interwoven scaffold decellularized bone matrix (DCB). In order to inves-
which was containing cell-laden hydrogels, PCL tigate properties of osteoconductivity, each scaffold
polymer and a sacrificial pluronic F127 hydrogel composites were seeded with an adipose-derived
used as a multi-head bio-printer. The Pluronic F127 stromal/stem cells in vitro and their differentiation
has been included in several other composite con- into osteoblasts was evaluated. The content of cal-
structs to facilitate the development of provisional cium—normalized to DNA—was especially elevated
structural support or vascular channels. in PCL-TCP, PCL-BO and PCL-DCB groups relative
to PCL only.
3740 J Mater Sci (2020) 55:3729–3749

Buyuksungur et al. [95] printed 3D PCL scaffolds reinforced with carbohydrate particles (cHA) using
adapted with HA and poly-propylene fumarate the additive manufacturing (AM) technology. A
(PPF). In order to produce a mechanically strong software application was used for digital surfacing in
implant with uniform pore size and porosity, gov- the mass proportions of 100/0, 95/5, 90/10 and
ernable surface hydrophilicity and osteoconductivity, 80/20 for application in bone tissue engineering,
cylindrical disks of PCL were printed by FDM and seeking higher proposition strength of PLA. As it is
modified with nHA and PPF. The cytotoxicity, irri- evident, biomimetic application can produce high-
tation and inflammation analyses showed that the strength biomechanical implants with appropriate
scaffolds were biocompatible. Also, PCL/nHA and mechanical features. The combination of polymers
PCL/nHA/PPF scaffolds were implanted in the leads to a rise in diversity of components and appli-
rabbit’s femurs for in vivo testing, with and without cations of biomaterials increased.
seeding of rabbit BMSCs and evaluated after 4 and In another additive manufacturing technique for
8 weeks by histological test, micro-CT and mechani- scaffold fabricating, Zhao et al. [115] constructed
cal test. As determined by bone mineral density and bionic porous titanium scaffolds by the selective laser
micro-CT, scaffolds that were seeded by BMSC melting (SLM) procedure. In those studies, different
demonstrated progress in bone tissue regeneration. bionic bone scaffolds were manufactured by com-
After eight weeks of implantation, the values puter-aided design (CAD). This structure with novel
obtained from mechanical analysis were remarkably porosity was plotted by using parameterization
better than those of the healthy rabbit femur and modeling. At all stages of construction from the
demonstrated a high capacity for patient-specific design phase of the evaluation of the scaffold porous
bone defect. structures, the parametric modeling of porous tita-
In addition to these researches, at present time nium bone scaffold with competent mechanical and
many researchers are attracted to the study of bone biological virtues was obtained. Meanwhile, in
3DBP [96–113]. The 3DBP bone transplantation pro- another study, Lai et al. [116] created a novel porous
cedure is shown in Fig. 4. Based on this figure, a poly(lactide-co-glycolide) (PLGA)/TCP/Mg (PTM)
computer-aided design of bone graft is an initial scaffolds using low temperature, rapid prototyping
phase for fabricating of bone grafts procedure. (LT-RP) technology with the formulation of Mg
Instances of successful 3D printing for bone powder, PLGA and b-TCP. The release of Mg ions
application are that of the 3D printing of bone scaf- was studied, and physical characterization of PTM
folds with hybrid biomaterials. Oladapo et al. [114] scaffold in vitro assay was performed. The PTM
designed a new hybrid material bone implant, by scaffolds were implanted in a rabbit model for eval-
combination of polylactic acid (PLA) matrix- uation of the osteogenic and angiogenic properties of

Figure 4 3DBP bone transplantation procedure. Patients suffering growth factors in order to produce a well printable bio-ink. After
from bone disorder, supposed to data gathering by computer-aided completion of printing procedure, transplantation surgery is the
instruments such as MRI. Provided bone graft design will be final step.
transmitted to a bone scaffold by selecting appropriate cells and
J Mater Sci (2020) 55:3729–3749 3741

the implant. Their results proved that the PTM scaf- how much the bio-printed cells can perform their
fold had designed in the bio-mimic the structure and in vivo functions under optimized microenviron-
modified mechanical properties. Ultimately it was ments [22]. Artificial tissues are seeded by either
confirmed that the PTM scaffolds are desirable com- printing functional primary cells with supporting
posite biomaterials for solving challenging bone cells [75, 121–126] or printing progenitors or stem
defect that would have great excellent property for its cells for further differentiation [127–132]. Direct
clinical usage. printing of primary cells can rapidly increase the
Roopavath et al. [117] prepared a 3D-printed complexness of bio-printing.
hydrogels by using an extrusion-based 3D printing
technology. The material of this hydrogel is based on
HA that had been approved clinically. SEM and Prospective market
Micro-CT results provided revealed that the scaffold
enjoys from a better rate of porosity. Mechanical tests 3DBP is presently increasing greatly toward a large
were employed to evaluate the porosity effects on the industry as a result of its variation and potential
compressive properties of 3D-printed structures. implementations. 3DBP market size is hoped to
Eventually printed HA hydrogel made of a patient- obtain a $10.8 billion worth in 2021 from a $2.2 billion
specific bone graft was tested in a series of studies in stance in 2012 [133]. Presently, many companies
patients. The results confirmed the promising engage in 3DBP production, for the purpose of tissue
potential of this 3D-printed material in manufactur- engineering applications [134].
ing a bio-mimic porous structure-based anatomical Furthermore, in 2014, a bio-printed human liver
bone models and preoperative 3D planning. tissue, named exVive3DTM Liver, which was manu-
factured for drug toxicity evaluation, was introduced
[135]. However, the product provided for in vitro
3D bio-printing challenges drug screening, the successful development of a
commercially available liver tissue, is still pending
The 3DBP has created a huge impact in the tissue [24]. Microfluidic systems [136] and layer-by-layer
analysis field and is turning into a practical strong assembly [137] will have an effect on the bio-fabri-
tool to produce tissues of human body. cation of microstructures in the future. It is
The main challenge of 3DBP is the need for in vivo inevitable that advances in bio-fabrication will also
vascularization in order to provide the cells with profit related fields such as imaging and diagnostic
adequate nutrition, growth factors, oxygen and applications too [24]. It is predicted that the pro-
remove carbon dioxide and wastes. Of course, the gressive trend of 3DBP methods’ prevalence will lead
future developments of bio-printing can also poten- to in situ bio-printing developing, which could be
tially overcome these vascularization challenges considered as an upward procedure from benchside
[118]. In vivo, capillaries are mostly located at a to the bedside [78, 138].
100 mm distance from a majority of cells in order to
enable enough diffusion for the cells to survive [119].
For greater distances, like in thick tissues in printed Conclusion
organs, supplementary modes for diffusion may be
required. To surpass this huddle, Hutmacher et al. In this paper, the different procedures of 3DBP of
[120] suggested an artificial vascular system. skin and bone tissues, results, advantages and dis-
Also, the bio-printing process is not currently advantages have been reviewed with details. This
automated and plenty of manual operations sepa- review emphasizes on the 3D bio-printed skin as a
rated in various steps may result in slow processing novel technology that provides the scaffold using
speed, thereby increasing the prospect for mistakes biomaterials such as gelatin, fibrinogen, GelMA,
and errors [21]. In order to form a highly mimetic chitosan and collagen. In this technology, applying
tissue or organ on a macroscale, bio-printed cells cells, such as fibroblast, keratinocyte, melanocyte and
should proliferate. When selecting cells, two main HUVEC by growth factors like FGF-2, thrombin and
factors are considered: how the bio-printed cells can hydrocortisone into a 3D environment, provides a
imitate the physiological state of cells in vivo and similar setting close to the natural skin tissue. Skin
3742 J Mater Sci (2020) 55:3729–3749

constructs can be beneficial for patients, whom Eng 16:247–276. https://doi.org/10.1146/annurev-bioeng-0

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Acknowledgements ttps://doi.org/10.1097/tp.0000000000000811
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A word of appreciation must be given to Aron Zhao LN, Wang ZB, Zhang JS, Tang ML, Mei J (2014)
Munggela Foma for his invaluable assistance and Decellularized kidney scaffold-mediated renal regeneration.
comments on the first phase of paper editing. Also, Biomaterials 35(25):6822–6828. https://doi.org/10.1016/j.b
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Compliance with ethical standards
lularization of human and porcine lungs: bringing the
Conflict of interest The authors declare that they matrix to clinical scale. J Heart Lung Transpl
have no conflict of interest. 33(3):298–308. https://doi.org/10.1016/j.healun.2013.10.
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