TIBTEC-1268; No. of Pages 6
Opinion
Bioprinting scale-up tissue and organ
constructs for transplantation
Ibrahim T. Ozbolat
Biomanufacturing Laboratory, The University of Iowa, Iowa City, IA, 52242, USA
Bioprinting is an emerging field that is having a revolutionary impact on the medical sciences. It offers great
precision for the spatial placement of cells, proteins,
genes, drugs, and biologically active particles to better
guide tissue generation and formation. This emerging
biotechnology appears to be promising for advancing
tissue engineering toward functional tissue and organ
fabrication for transplantation, drug testing, research
investigations, and cancer or disease modeling, and has
recently attracted growing interest worldwide among
researchers and the general public. In this Opinion, I
highlight possibilities for the bioprinting scale-up of functional tissue and organ constructs for transplantation and
provide the reader with alternative approaches, their
limitations, and promising directions for new research
prospects.
Bioprinting: a promising technology to revolutionize
medicine
Bioprinting can be defined as the spatial patterning of
living cells and other biologics by stacking and assembling
them using a computer-aided layer-by-layer deposition
approach to develop living tissue and organ analogs for
tissue engineering, regenerative medicine, pharmacokinetic, and other biological studies [1]. It uses four
approaches to deposit living cells: inkjet [2], extrusion
[3], acoustic [4] and laser [5] based. Given its great benefit
in spatially arranging multiple cell types to recapitulate
tissue biology, bioprinting is a game-changer in the rapid
development of tissue constructs and is receiving enormous attention. Although bioprinting of functional 3D
whole organs for transplantation remains in the realm
of science fiction, the field is moving forward, providing
hope that shortages in tissue grafts and organ transplantation will be mitigated to some extent in the future
[6]. While current tissue-engineering strategies cannot
enable fabrication of fully functional tissues or organs
[7], bioprinting enables precise placement of biologics to
recapitulate heterocellular tissue biology to some degree.
Current technology enables the development of organ or
tissue constructs that do not require substantial vascularization, as well as mini-tissue models mimicking the
Corresponding author: Ozbolat, I.T. ([email protected]).
Keywords: bioprinting; bioprinting for transplantation; vascularized-tissue printing;
in situ bioprinting.
0167-7799/
ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2015.04.005
biology of natural counterparts for pharmaceutical testing
or cancer studies [8].
Here I present recent approaches to the bioprinting
scale-up of functional tissue and organ constructs for
transplantation, including bioprinting vascularized tissue
and organ constructs in vitro and in situ bioprinting technology to build tissues directly in defect sites. I discuss
major roadblocks to this approach and provide potential
solutions and future directions.
Bioprinting of vascularized tissue and organ constructs
in vitro
Organ bioprinting holds great promise for the future, but
whole-organ bioprinting has remained elusive due to
several limitations associated with biology, bioprinting
technology, bioink material, and the post-bioprinting maturation process [9]. The bioprinting of functional tissues is
an intermediate stage toward achieving organ-level complexity. In vitro fabrication of functional tissues is a
sophisticated phenomenon comprising a hierarchical arrangement of multiple cell types, including a multiscale
network of vasculature in stroma and parenchyma, along
with lymphatic vessels and, occasionally, neural and muscle tissue, depending on the tissue type. In vitro engineered
tissue models that incorporate all of these components are
still far on the horizon. Bioprinting technology offers a
great benefit in the hierarchical arrangement of cells or
building tissue blocks in a 3D microenvironment, but the
bioink and the post-bioprinting maturation phase are as
important as the bioprinting process itself. The bioink
material is crucial because it should provide the spectrum
of biochemical (i.e., chemokines, growth factors, adhesion
factors, or signaling proteins) and physical (i.e., interstitial
flow, mechanical and structural properties of extracellular
matrix) cues to promote an environment for cell survival,
motility, and differentiation [10]. In addition, the bioink
should exhibit high mechanical integrity and structural
stability without dissolving after bioprinting, enable differentiation of autologous stem cells into tissue-specific cell
lineages, facilitate engraftment with the endogenous tissue without generating an immune response, demonstrate
bioprintability with ease of shear thinning, rapid solidification and formability, and be affordable, abundant, and
commercially available with appropriate regulations for
clinical use [11]. A variety of bioinks, including naturally
and synthetically derived materials, has been used for
tissue regeneration, as detailed in the literature
[12,13]. The post-bioprinting process is also crucial and
necessitates mechanical and chemical stimulation and
Trends in Biotechnology xx (2015) 1–6
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TIBTEC-1268; No. of Pages 6
Opinion
signaling to regulate tissue remodeling and growth, the
development of new bioreactor technologies enabling rapid
maturation of tissues, multiscale vascularization for survivability of tissues, and mechanical integrity and innervation for transplantation.
Although several researchers have studied bioprinting
of tissue constructs, the fabrication of scale-up tissues with
a high volumetric oxygen-consumption rate, such as cardiac, pancreas, or liver tissue, is still a challenge. One major
roadblock is associated with the integration of the vascular
hierarchical network spanning arteries and veins down to
capillaries. To bioprint vascularized thick tissues, highly
repeatable and straightforward technologies and protocols
should be developed in logical steps, from simple to complex. Since it is difficult to print capillaries at the submicron scale using current technology, an alternative could be
to bioprint the macrovasculature and then leave nature to
create the capillaries. To this end, two alternative
approaches have been considered: (i) indirect bioprinting
by utilizing a fugitive ink that is removed by thermally
induced decrosslinking, leaving a vascular network behind
[14,15]; and (ii) direct bioprinting of a vasculature network
in a tubular shape [16–19] (Figure 1).
Several recent attempts have been made to bioprint a
fugitive bioink to create vascular channels [15,20–22]. Cell
laden hydrogels were used to fabricate the tissue construct,
and integration of the vascular network demonstrated
increased cell viability inside the construct; regions near
channels exhibited significant differences compared with
regions away from channels. Although most researchers
have attempted to create a vascular network on a macroscale and generate an endothelium lining inside the lumen
via gluing endothelial cells through perfusion, Lee et al.
[21] took one step forward and successfully achieved angiogenesis by sprouting endothelial cells within a fibrin
network loaded with other pericyte-like supporting cells
(Figure 1A,B). Their study demonstrated that endothelial
spouting generated a considerable increase in the permeability of the tissue construct. More advanced angiogenesis
and vasculogenesis have already been developed in microfluidic devices; several supporting cells have been
attempted and used in cancer metastasis studies [23]. Despite the flexibility in bioprinting channels and the ability
to create angiogenesis, this technology still faces major
challenges. First, loading cells in hydrogels does not support cell–cell interactions, because those take place in vivo,
and limited phenotypic stability and activity of cells are
observed during prolonged in vitro incubation. Second,
while fibrin is a suitable environment for angiogenesis
because it has a crucial role in blood clotting [24], it is
not a convenient environment for tissue-specific cells, such
as islets in a pancreas or follicles in a lymph node; a
scaffold-free environment should be considered for these.
A recent article [25] demonstrated contiguous vascularization of cell aggregates in tumor spheroid models and robust
angiogenesis into the fibrin matrix where spheroids were
encapsulated, showing the possibility of generating anastomosis of vascular networks of stromal and parenchymal
tissues in vitro.
The other approach is direct bioprinting of a vascular
network via: (i) bioprinting of scaffold-free branched
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vascular tubes [19] using tissue spheroids as building
blocks [26] that are printed inside a mold pattern; and
(ii) bioprinting of vasculature using direct extrusion of a
tubular network [16–18]. A recent study [3] demonstrated
hybrid biofabrication of vasculature in tandem with tissue
strands, where fibroblast tissue strands quickly fuse to
each other, maturate, and form the tissue around the
vasculature (Figure 1C–E). Tissue strands were made
scaffold free and used as building blocks to construct the
scale-up tissue due to their quick fusion, folding, and
maturation capabilities. This approach demonstrated the
proof of concept toward larger-scale perfusable tissues;
further work is needed to demonstrate a complex vascular
network within larger tissues with vascularization on
multiple scales that can be envisioned using a MultiArm BioPrinter [27]. Although vascularization is important for larger-scale tissue constructs for transplantation,
anastomosis to the circulatory system and functionality
post-transplantation should also be considered. The vascular network should be designed and bioprinted so that it
can be sutured to a vascular network easily, and it should
have certain properties, such as enough mechanical properties to satisfy suture retention and burst pressure, sufficient intactness of endothelium to prevent thrombosis, and
a high patency rate to support occlusion-free circulation
[28]. Compared with indirect bioprinting of a vascular
network, the direct bioprinting of vasculature can be more
convenient, suturing to the host at the time of implantation.
From in vitro to in situ: regenerating tissues through
direct bioprinting into defect sites
Bioprinting living tissue constructs or cell laden scaffolds
in vitro has been well studied, and thin tissues or tissues
that do not need vascularization, including skin, cartilage,
and blood vessels, have been grown [12]. However, in situ
bioprinting can enable the growth of thick tissues in critical
defects with the help of vascularization driven by nature in
lesions. Therefore, it is a promising direction for the bioprinting of porous tissue analogs that can engraft with
endogenous tissue and regenerate new tissue along with
vasculogenesis through the migration of progenitor cells
into the tissue construct and sprouting of capillaries from
the endogenous tissue.
The idea of in situ bioprinting was first proposed by
Weiss using inkjet technology [29]; however, translating
bioprinters into operating rooms was considered to be
challenging due to the perception that surgeons can be
considered artists and prefer off-the-shelf solutions, such
as using prefabricated tissue constructs and cutting or
carving them into a form to be implanted into the defect
site. Limited research has been performed on in situ bioprinting since Weiss proposed this technology. Inkjetbased bioprinting of skin cells has been tested for burn
wounds [30], and laser-assisted bioprinting has been performed to test the feasibility of printing nanohydroxyapatite particles on a mouse model [31]. The idea of bioprinting
skin cells (fibroblasts and keratinocytes zonally) has been
considered feasible for transitioning the technology to
clinical settings, with the hope of repairing major wound
defects of soldiers on the battlefield.
TIBTEC-1268; No. of Pages 6
Opinion
Trends in Biotechnology xxx xxxx, Vol. xxx, No. x
(A)
(C)
Vascular channels
Stage 1
Fibrin
Collagen
Detachable
nozzle
Co-axial
nozzle
Embed endothelial
and supporng cells
Stage 2
Capillary network is formed
within fibrin scaffold
(D)
Stage 1
Tissue
strands
Stage 2
Stage 3
Stage 3
Key:
Endothelial cells
Supporng cells
Angiogenic sproung is formed
from vascular channels to the
capillary network
Vasculature
Assembly through
hybrid bioprinng
(B)
Paral fusion of ssue
strands around the
vasculature
Complete fusion of ssue
strands ghtly adhering
to the vasculature
(E)
Capillary
network
Sproung
Endothelium
100µm
TRENDS in Biotechnology
Figure 1. Vascularized tissue construct bioprinting in vitro. In the first approach, (A) a fugitive ink is bioprinted to create vascular channels inside a collagen support
structure along with deposition of endothelial and fibroblast cells in a fibrin scaffold, resulting in capillary network formation in fibrin scaffold followed by endothelial
sprouting from vascular channel to capillary network in 9 days, where (B) a immunohistochemistry image shows integration of large sprouts from the parental vascular
channel (red) to the capillary network (green). In the second approach, (C) a hybrid bioprinting approach can be applied using a Multi-Arm BioPrinter, where (D) the tissue
assembly is created by placing the vasculature in the middle, resulting in fusion and maturation of scaffold-free tissue strands around the vasculature, further resulting in
(E) the tight fusion of the fibroblast tissue to the vasculature in 1 week [3]. The co-axial nozzle used in that study can enable continuous printing of a single lumen
vasculature throughout the fabrication of larger-scale hybrid tissue constructs. Reproduced and adapted, with permission, from [21] (B).
In situ bioprinting is challenging, and further systematic research is required to take the technology into a
robust state. There are major limitations associated with
its biological, biomaterial, and engineering aspects, such as
printing difficulties on nonhorizontal surfaces, the need for
highly advanced robotics bioprinters coupled with computer-aided design technologies (i.e., scanning the defect site
and providing an interactive user interface for surgeons),
the requirement for a highly effective extrudable bioink
enabling instant solidification in a living body (without the
need for an external solidifier, such as a ultraviolet light or
a chemical crosslinker), the need for a biologically appealing ink for enhanced tissue formation, the requirement for
technologies that do not interfere with in vivo conditions
and regulatory issues related to animals or humans necessitating safe delivery of the tissue construct under anesthesia. In addition, in situ bioprinting can sometimes
increase the duration and cost of surgery.
Bioprinting ex vivo on explants can be considered a
transitional stage (Figure 2A) in which tissue constructs
can be built and engineered inside explants [32]. In situ
bioprinting is promising for developing tissue analogs
directly on the defect model in operating rooms
(Figure 2B), which paves the way to developing associated
enabling technologies for humans in the future. When the
technology is translated into clinics, it will have several
benefits: (i) direct bioprinting of tissue constructs into
defects can eliminate the need for preshaping or reshaping
3
TIBTEC-1268; No. of Pages 6
Opinion
Trends in Biotechnology xxx xxxx, Vol. xxx, No. x
(A)
Cells migrang
from defect
(B)
Printed
cells
In situ
bioprinng
Bioprinter
nozzle
Defect
Explant
Fixture
Bioprinter
Bioprinter
nozzle
Bioprinter
nozzle
Stem cells
Plasmid
DNA
Calvarial
defects
Rat
Bioink
Printed ssue inside a
carlage defect
Biodegradable
microparcle
(C)
In situ hybrid
bioprinng in
operang rooms
Large
defects
Dura substute
Printed frame (biological or
synethc)
made of
Printed
hydrogel
composite biodegradable
loaded with hard polymer
stem cells and
genes
TRENDS in Biotechnology
Figure 2. In situ bioprinting. (A) Bioprinting cells into explants in situ can be considered an intermediate step toward (B) bioprinting tissue constructs directly into animal
models, where DNA can also be printed for bioprinting mediated gene therapy to transfect and differentiate printed stem cells into multiple lineages. This will have a great
impact on (C) transitioning in situ bioprinting technology into operating rooms for humans in the near future; for example, large, deep calvarial defects can be regenerated
on a synthetic or biological Dura substitute, where a hard polymeric biodegradable frame can be printed along with stem cells and genes loaded in hydrogels to generate
the vascularized bone tissues. Image in (C) reproduced courtesy of Christopher Barnatt.
the scaffold based on the defect geometry. This can avoid
the laborious nature of scaffold preparation and the risks
associated with contamination and limited activity of cells
in vitro; (ii) for bioprinting of cell-laden tissue constructs
for critical- or large-size defects, in situ bioprinting can
eliminate the need for differentiation of stem or progenitor
cells in vitro, which might be expensive and time consuming.
4
When bioprinting stem or progenitor cells in situ, cells are
exposed to the natural environment with growth factors that
can induce their differentiation into the desired lineages;
(iii) in situ bioprinting can enable the precise deposition of
cells, genes, or cytokines inside the defect, unlike manual
interventions, such as prefabricated scaffolds, in which the
shape can alter due to swelling, contraction, or deformations.
TIBTEC-1268; No. of Pages 6
Opinion
Localized control via bioprinting, such as printing different
cytokines at different layers, is an asset for future bioprinting research; (iv) standard defects made by surgery tools are
easily addressed by manipulation and bioprinting of tissue
analogs. However, naturally occurring defects resulting
from trauma, surgical excision, or other issues are random
in morphology and geometry and need to be captured precisely; laser- or image-based scanning systems can overcome
this challenge. In situ bioprinting can eliminate the need for
multiple operations because the tissue constructs can be
bioprinted into the defect immediately; and (v) twisting
multi-axis robotic arms can enable angled deposition and
printing of the bioink into nonhorizontally oriented defects.
Defects on the live model can be at random locations, and it is
not convenient for the surgeon to change the position of the
anesthetized model during bioprinting.
As a result of these benefits, in situ bioprinting of tissue
analogs can be applied to various sites on the body, such as
deep dermal or extremity injuries and calvarial or craniofacial defects during maxillofacial or neurosurgeries. In the
future, in situ bioprinting technology could be considered
for humans (Figure 2C). For large calvarial defects that
need a great deal of microvascularization along with structural support, one can envision bioprinting a frame (using
hard osteoconductive polymers, such as a blend of polycaprolactone, copolymers, and hydroxyapatite) in tandem
with stem cells (loaded in blends of hydrogels, such as
collagen and fibrin) to be differentiated toward multiple
lineages, including osteoblasts and endothelial cells for
bone generation and angiogenesis, respectively [33]. In
situ bioprinting of porcine aortic endothelial cells in fibrin
gel has already demonstrated vascularization in the subcutaneous tissue of mice 1 week after implantation [34]. In
another long-term future application, in situ bioprinting
could assist placement of different cell types in different
compartments in large, deep defects for composite tissue
regeneration, where manual surgical interventions are
required for suturing supplement blood vessels and nerves.
In that case, bioprinting can be performed with a deposition head mounted on a six-axis robotic arm controlled by
the surgeon. The printed bioink should have a highly
porous microstructure or could be mixed with a sacrificial
(quickly bioresorbable) biomaterial, leaving porosity upon
transplantation, which would enable diffusion and interstitial flow of blood for survivability of cells until neovascularization is completed. A major challenge is the lack of
growth factors far from the defect periphery, which are
needed to induce differentiation of printed stem cells or
progenitor cells that migrate from the host tissue. This can
be overcome with gene therapy, where gene delivery can be
sustained and controlled using gene-activated matrices
(GAMs), such as microparticles loaded with genes, for
the long-term release of genes for the transduction and
differentiation of autologous cells into multiple lineages
[35]. Printing nonviral vectors in GAMs is safe and efficient
for transfecting target cells and surpasses the benefits of
direct delivery of growth factors during in situ bioprinting.
Concluding remarks
Here, I present the state of the art in bioprinting technology
for biofabrication of scale-up tissue and organ constructs.
Trends in Biotechnology xxx xxxx, Vol. xxx, No. x
Two approaches are discussed: (i) in vitro bioprinting of
tissue and organ constructs with a focus on alternative
technologies in bioprinting a multiscale vascular network
in tandem with the rest of the tissue construct, and (ii) in situ
bioprinting of tissue construct for regeneration of large
defects that will one day enable the repair of body parts
directly in patients in operating rooms. Although the technology shows a great deal of promise for bioprinting at
organ-level complexity, there is still a long way to go to
realize this ambitious vision. There is a need for advancement in several areas, including new cell sources and stem
cell technology, novel bioinks, and advanced fully automated bioprinter technologies and bioprinting processes. Overcoming current impediments in these technologies along
with advancements in in vivo integration is essential for
developing seamlessly automated technology from autologous stem cell isolation to tissue or organ construct transplantation.
Acknowledgments
This work has been supported by National Science Foundation Awards
CMMI 1462232, CAREER 1349716, Diabetes in Action Research and
Education Foundation, and the Grow Iowa Values Funds. The author
would like to express his gratitude to G. Dai, Christopher Barnatt, Yin
Yu, and Kerim Moncal for providing some of the high-quality images in
the figures.
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