Perspective

https://doi.org/10.1038/s41551-019-0471-7

Opportunities and challenges of translational

3D bioprinting
Sean V. Murphy 1
*, Paolo De Coppi2 and Anthony Atala1

3D-printed orthopaedic devices and surgical tools, printed maxillofacial implants and other printed acellular devices have been
used in patients. By contrast, bioprinted living cellular constructs face considerable translational challenges. In this Perspective,
we first summarize the most recent developments in 3D bioprinting for clinical applications, with a focus on how 3D-printed
cartilage, bone and skin can be designed for individual patients and fabricated using the patient’s own cells. We then discuss
key translational considerations, such as the need to ensure close integration of the living device with the patient’s vascular
network, the development of biocompatible bioinks and the challenges in deriving a physiologically relevant number of cells.
Lastly, we outline untested regulatory pathways, as well as logistical challenges in material sourcing, manufacturing, standard-
ization and transportation.

T
hree-dimensional printing—a type of additive manufactur-
ing—creates 3D objects through the digitally controlled
deposition of successive layers of material. The United
States Food and Drug Administration (FDA) has cleared several
3D-printed devices for clinical use, including orthopaedic devices,
patient-matched implants, surgical guides and restorative devices
such as dental bridges1,2. However, to date most medical applica-
tions of 3D printing have involved static non-living constructs
designed to function as structural or space-filling prostheses3.
Personalized 3D-printed titanium plates, specifically modelled via
image reconstruction following magnetic resonance imaging (MRI)
and computed tomography (CT), have significantly improved out-
comes in patients undergoing tissue reconstruction after the sur-
gical removal of bone tumours4. Similarly, 3D-printed genioplasty
template systems can provide greater accuracy in the repositioning
of the chin than traditional intraoperative measurements5. In 2013,
a 3D-printed and personalized bioresorbable external airway splint
was implanted in an infant with tracheobronchomalacia6. In 2015, a
further three infants received patient-matched 3D-printed splints7.
These constructs, although non-living, were designed to prevent
external airway compression over a predetermined period before
they are resorbed by the body to accommodate airway growth. 3D
printing has also been applied in the layer-by-layer manufacturing
of oral pills (such as Spritam, the commercial name for the drug
levetiracetam) that deliver a high drug dosage while being porous
enough to dissolve quickly8.
These recent developments in the design and fabrication of non-
living 3D-printed implantable constructs illustrate the potential of
3D-printing technology for the production of advanced patient-
specific devices. By contrast, the bioprinting of living cellular con-
structs has faced significant hurdles, in particular: the transition
from synthetic materials—including metals, ceramics and plas-
tics—to biologically functional materials while maintaining control
over the mechanical properties at the microscale and macroscale;
achieving tissue designs with physiological heterogeneity; develop-
ing methods to derive and expand functional cells from stem cells;
and interfacing bioprinted tissues with a physiological vasculature
network. 3D-printed tissues at the preclinical stage of development
still lack essential functional elements, such as vasculature, innerva-
tion, lymphatics, and the number and diversity of functional and
supporting cell types required for tissues and organs that are large
and complex. Owing to these challenges, early successes in the clini-
cal translation of 3D-printed living tissues are likely to involve rela-
tively simple tissues (rather than tissues with complex geometries
at the scales that are clinically relevant). In this Perspective, we pro-
vide an overview of the translational opportunities and challenges
of 3D-printed tissues and organs. We first discuss recent advances
in the 3D bioprinting of cartilage, bone and skin, and why these
bioprinted tissues have progressed to the preclinical stage, and then
outline challenges in the fabrication of more complex 3D-printed
tissues and organs.
Clinically relevant 3D-bioprinted tissues
The capabilities of 3D bioprinting are at a stage where multiple
biomaterials and cell types can be patterned into constructs
approaching clinically relevant sizes and geometries. 3D bioprinted
tissues, such as cartilage, bone and skin, exemplify the potential of
the technology.
Cartilage. Cartilaginous tissues are avascular and aneural struc-
tures with a relatively low density of chondrocytes. Their simplic-
ity limits potential hurdles in the fabrication of human cartilage at
scale. However, because of the heterogeneity (zonal structure) of the
tissue9, it is challenging to reproduce functional articular cartilage.
The zonal structure of native articular cartilage includes areas with
different cell morphologies and cell arrangements, as well as with
different extracellular matrix (ECM) arrangements, constituents
and distribution. Such heterogeneity in structure is associated with
the tissue’s tensile properties, which enable it to resist the sheer, ten-
sile and compressive forces imposed by articulation10,11.
Chondrocytes can be harvested from different zones of the car-
tilage12 and, by using 3D printers, deposited in hydrogels (such as
gelatin and alginate, cartilage-derived ECM, and nanofibrillated
cellulose and alginate13–16), with the cells maintaining high viability
and zone-specific gene-expression profiles17,18. Deposition of human
chondrocytes within a shear-thinning nanofibrillated cellulose can

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA. 2Stem Cells & Regenerative Medicine
1

Section, University College London, Great Ormond Street Institute of Child Health, London, UK. *e-mail: semurphy@wakehealth.edu

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NaTure BIomeDIcaL EngIneerIng Perspective
a b
Day 0
2 mm

10 mm 5 mm
Red: cells
Green: PCL/TCP 5 months

5 mm

10 mm 10 mm

c d
i ii

iii iv

Fig. 1 | Bioprinted cartilage, bone and skin tissues. a, Anatomically shaped 3D-printed cartilage structures (a human ear, left; and a sheep meniscus,
right). b, A bioprinted human-scale calvarial structure amenable to facial reconstruction. Visualized motion program for 3D-printing of the calvarial bone
construct (green and red indicate the dispensing paths of, respectively, the polymer mixture and cell-laden hydrogel) (top left). Photograph of the printed
construct (left). Implantation in a calvarial bone-defect region in Sprague Dawley rats (top right). Construct 5 months post-implantation (centre right).
Histology image of the construct five months after implantation (bottom). c, In situ skin-bioprinting concept. Wounds are first scanned (i) to obtain
precise information on wound topography (ii), which then guides the print heads to deposit specified materials (iii) and/or cell types (iv) in appropriate
locations. d, Skin bioprinter. Wound scanning with a hand-held ZScanner Z700 scanner (top left). The geometric information obtained by scanning is then
input into software for orienting the scanned images to the standard coordinate system (top right). The scanned data with its coordinate system is used to
generate the fill volume and the path points for the nozzle head (bottom left). Printing of the fill volume (bottom right). Panels reproduced from ref. 16,
ACS (a); ref. 26, Springer Nature Ltd (b); and ref. 49, Springer Nature Ltd (c,d).

also be combined with crosslinkable alginate to print anatomically
shaped cartilage structures, such as a human ear and a sheep menis-
cus, with high fidelity and stability16 (Fig. 1a). Inkjet 3D-printing
has also been used to repair cartilage in preclinical models19, achiev-
ing a tissue construct with a compressive modulus of the same order
of magnitude as hyaline cartilage20. Another approach involves the
fabrication of tissue constructs using micromass chondrocyte pel-
lets to form cartilage strands, with tubular permeable alginate cap-
sules working as a reservoir for cell aggregation and tissue-strand
maturation. This approach resulted in strands with a diameter of
~500 μm and with significantly increased cell density, as well as
improved post-transplantation maturation and function of the
engineered tissue21.
Combining multiple cell types may also increase the efficiency
of the bioprinted cartilage. The co-printing of multipotent articular
cartilage-resident chondroprogenitor cells, bone marrow mesen-
chymal stromal cells (MSCs) and chondrocytes by using a gelatin
methacryloyl-based hydrogel, led to generation of constructs with
a layered distribution of collagens and glycosaminoglycans, defin-
ing a cartilage with superficial and deep regions, each with distinct
cellular and ECM composition22. The integration of MSCs into a
layered construct of natural and synthetic biomaterials can direct
those cells to differentiate into zone-specific chondrocytes, and to
create a native-like articular cartilage with mechanical and bio-
chemical properties varying with depth23,24. Similarly, hyaline-like
cartilaginous tissue expressing collagen type II has been made via
the bioprinting of induced pluripotent stem cells (iPSCs) within a
nanocellulose alginate bioink25.
Further advances in 3D bioprinting will enable the creation of
patterns of growth factors, mechanical gradients and stem cells
in each cartilage zonal region, improving the function of biofab-
ricated cartilage tissue. In fact, it has been shown that 3D-printed
cartilage can have the histological and mechanical characteristics
of human auricles after implantation in vivo26. And a tissue-engi-
neered trachea from a 3D-printed biodegradable reticular polycap-
rolactone (PCL) scaffold suspended in culture with chondrocytes
was used in the replacement surgery of the native trachea of rab-
bits27. Owing to potential limitations in the yield, proliferation and
metabolic activity of chondrocytes derived from older patients28,
bone-marrow-derived MSCs can be used as a cell source to
derive functional chondrocytes. The induction of chondrogenesis
in MSCs can also be readily achieved by the addition of specific

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Perspective
growth factors29,30, and can be modulated by controlling the biome-
chanical environment31.
Bone. Although bone is the most commonly transplanted solid tis-
sue (only blood transfusions are more common32), traditional tech-
niques for bone-tissue biofabrication are associated with significant
limitations and complications, such as the paucity of available autol-
ogous materials and donor-site morbidity (especially for segmental
defects33). Significant efforts have thus focused on the study of the
unique structure and mechanical properties of bone.
Multiple types of biomaterial scaffold afford control over the
mechanical properties and degradability of the constructs follow-
ing transplantation34. Two commercially available void-filling bio-
material products—Infuse (Medtronic), an absorbable collagen
sponge that releases bone morphogenetic protein-2 (rhBMP-2);
and Actifuse (Baxter International), a silicated calcium phosphate–
hydroxyapatite graft—can accelerate bone-tissue healing in non-
weight-bearing defects, but they are not printable.
Attempts are underway to combine the natural regenerative
potential of bone with the benefits of bioprinting, such as the con-
trol over the shape and precise placement of cells and biomateri-
als in bioprinted scaffolds or bioengineered grafts. Several scaffolds
have been developed using synthetic biomimetic nanoceramics,
particularly calcium phosphate ceramics (such as hydroxyapatite,
biphasic calcium phosphate or tri-calcium phosphate; TCP), and
fabricated into bone-like architectures through 3D bioprinting35.
For example, a calcium-phosphate–collagen composite bone scaf-
fold has been fabricated using a modified inkjet-based 3D printer36,
and the implants were confirmed to be osteoconductive and biode-
gradable in a segmental murine femoral defect. Also, a case report
described the computer-aided design and fabrication of a medical-
grade PCL–TCP biodegradable scaffold implanted to reconstruct
a complex cranial defect37. The scaffold appeared to be well-inte-
grated at the site after six months, with the onset of bone consolida-
tion detected via CT.
To reproduce the appropriate mechanical properties of cortical
bone, many 3D-bioprinting approaches rely mainly on hard scaf-
folds; hence, they fail to fully recapitulate the properties of the cel-
lular component. This is a significant limitation. Although many
fabrication techniques can produce constructs with suitable inter-
connected porosity and mechanical properties, they require the
application of temperatures, solvents or other conditions that can
adversely affect living cells. The post-production seeding of cells
then results in problems associated with non-uniform cell distribu-
tion and poor cell attachment.
By taking advantage soft materials amenable to cell growth and
tissue formation, bioprinting techniques may meet the mechanical
needs of hard bone tissue while promoting tissue regeneration. One
approach is the development of hybrid or composite structures with
a strong, mechanically robust structure, filled with precisely placed
cells deposited within osteoconductive hydrogel bioinks. These
composite scaffolds could enable the fabrication of large and strong
scaffolds with specific patterns of cells and chemical factors that
elicit specific tissue development. In one approach, nanohydroxy-
apatite (nHA) and human osteoprogenitor cells were patterned and
assembled via laser-assisted bioprinting38, with the osteoprogenitors
maintaining their osteoblastic phenotype and functionality after
printing (the resulting tissue was, however, not evaluated in vivo).
Other methods can produce customized 3D porous structures by
building osteochondral tissue, fabricated via the sequential dispens-
ing of PCL and two alginate solutions containing osteoblasts and
chondrocytes39,40, or by using biofabrication to make a mechanically
reinforced template that supports the development of vascularized
bone41. In the latter, soft bioinks were supported by a network of rein-
forcing PCL microfibers to enable the fabrication of mechanically
reinforced constructs with decoupled biological and mechanical
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NaTure BIomeDIcaL EngIneerIng
functionality. These 3D-printed constructs can mimic the geometry
and bulk mechanical properties of trabecular-like endochondral
bone with a supporting marrow structure, and undergo endochon-
dral ossification following implantation. Scalable mandible and
calvarial structures have been 3D-printed using a similar approach,
with sizes and shapes similar to what would be needed for facial
reconstruction (Fig. 1b). Also, 3D-printed mandibular bone con-
structs, comprised of a mixture of PCL and tricalcium phosphate
(TCP) with26 or without42 cells and implanted in animal defect mod-
els, can form mature vascularized bone tissue (as shown with con-
structs retrieved 3–5 months after implantation).
The combination of multiple biofabrication tools and materi-
als seems to hold promise for the recapitulation of the mechanical
and biological properties that are essential for bone; but engineer-
ing bone tissue with histomorphometry and function approaching
those of native bone tissue remains elusive. The approaches with
best results remain those that successfully direct bone healing to
fill the injury. Yet the use of bioprinting to guide the innate heal-
ing process to fill large bone injuries with an anatomically accurate
shape (rather than approaches seeking to replicate the developmen-
tal stages of bone formation) has clinical potential.
Skin. The skin’s relatively thin (~2.5 mm), layered and structured
nature, combined with easy access to cell sources, has facilitated the
early adoption of 3D bioprinting technology for the manufacturing
of skin tissue. There are two main strategies for the 3D bioprint-
ing of skin constructs: in vitro bioprinting and transplantation of
the printed tissue into the defect site; and direct in situ bioprint-
ing, where the cells and materials are printed directly into the defect
site. The feasibility of using bioprinting to fabricate skin constructs
in vitro was first shown with multilayered engineered tissue com-
posites of human skin fibroblasts and keratinocytes deposited layer-
by-layer within a collagen hydrogel, resulting in an inner layer of
fibroblasts and an outer layer of keratinocytes42. Since then, owing
to laser-based bioprinters43,44 and inkjet-based printers45,46, in vitro
skin bioprinting has increased in complexity and accuracy. Areas
up to 100 cm2 of bilayered skin using primary human fibroblasts
and keratinocytes, obtained from skin biopsies, can now be printed
in less than 35 minutes47. Human skin can also be fabricated in
amounts and timeframes that should be appropriate for clinical and
commercial applications.
In situ bioprinting of the skin construct directly on the wound
site relies on the patients’ body serving as the ‘bioreactor’ for the
functional maturation of the bioprinted tissue. In wound healing,
the main advantage of this approach is the rapid coverage of large
wounds with permanent skin tissue, and their accelerated healing.
In comparison to the transplantation of in vitro fabricated con-
structs, in situ bioprinting avoids the risk of damaging the thin and
fragile construct during transport and handling, and avoids poten-
tial issues related to the correct placement and orientation of a con-
struct with complex 3D topology. In one of the first descriptions
of in situ bioprinting, human keratinocytes and fibroblasts were
printed directly into a full-thickness mouse skin-wound model48.
Wounds were first scanned to obtain precise information on wound
topography, which then guided the print heads to deposit speci-
fied materials and cell types in appropriate locations (Fig. 1c). The
first layer of a fibrinogen–collagen hydrogel precursor containing
fibroblasts was bioprinted, followed by the simultaneous deposi-
tion of thrombin from nozzles to form a fibrin–collagen hydrogel;
an additional layer of keratinocytes was then bioprinted on top of
the fibroblast layer via a similar deposition approach. The same
approach was also applied in a porcine model with large full-thick-
ness wounds (Fig. 1d), where in situ bioprinting led to the complete
re-epithelialization of the large wound after 8 weeks49.
Our group has previously used an in-situ skin bioprinter to
deposit amniotic-fluid-derived stem cells on full-thickness skin
NaTure BIomeDIcaL EngIneerIng
Table 1 | Specific needs in the 3D-printing of tissues, and potential solutions
Specific needs
Materials Accurate reproduction of the functional and
biomechanical properties of tissue
Compatible properties for bioprinter deposition
Robust and controllable post-printing mechanical
properties
Physiological, biochemical and mechanical interactions
with the cellular component
Appropriate scaffold-degradation times
Cells Minimally invasive or non-invasive cell sourcing
Autologous or non-immunogenic cell sources
Ex vivo expansion of the cells without loss of phenotype
or function
Vascularization Oxygen and nutrient availability across thick tissues
Fabrication of multiscale hierarchical networks with
complex structures
Direct vascular fabrication or patterning of cells and/or
factors that mature either in vivo or in bioreactors
wounds in mice, using either a fibrin–collagen bioink50 or a hyal-
uronic acid-based gel with tuneable properties tailored for extended
cytokine release51. Although the stem cells did not permanently
integrate into the regenerated skin, the secretion of trophic fac-
tors accelerated wound-closure rates and promoted angiogenesis.
Similar strategies would avoid the sourcing of cells from patients
with significant loss of healthy skin tissue.
No bioprinting approach can yet fully replicate the morphologi-
cal, biochemical and physiological properties of native skin. The
incorporation of additional cell types and the patterning of more
representative ECM components is necessary. For example, achiev-
ing stratified tri-layered structures containing epidermis, dermis
and hypodermis would be beneficial, as well as the incorporation of
components of the vasculature, nerves, sweat and sebaceous glands,
hair follicles and pigmentation. Moreover, future skin constructs
should facilitate the proper development and regulation of hair fol-
licles, pigmentation and epidermis formation and maturation. This
will need the understanding of the roles of multipotent progeni-
tor cells present in the upper permanent region of the hair follicle,
which contribute to the formation of hair follicles and sebaceous
glands52, and of the interaction of these cells with melanocyte stem
cells during skin homeostasis and repair53.
Current challenges and potential solutions
Most bioprinted tissues and organs are small in scale, contain only
one or two cell types, consist of relatively simple structures, and
provide limited functionality. Bioprinted tissues generally lack
vascular networks, and thus rely on diffusion for nutrient supply.
The bioprinting of more complex and larger tissues with robust,
tailorable mechanical properties and cell-compatible materials
requires methods for the derivation and expansion of multiple
types of functional, progenitor and supporting cell types, as well as
strategies for the integration of a vascular network for oxygen and
nutrient supply (Table 1).
Nature Biomedical Engineering | VOL 4 | April 2020 | 370–380 | www.nature.com/natbiomedeng
Perspective
Potential solutions
Hybrid constructs, consisting of patterned synthetic and natural
materials23,24,40
ECM-based hydrogels54,91–93
Chemical or non-chemical modification of ECM and hydrogel
materials63,64
Synthetic peptides that mimic ECM functional motifs65,66
‘Smart’ materials whose properties change over time or after the
application of external stimuli122,123
Small-molecule control over cell function, such as conditional cell
reprogramming70,71
Mesenchymal cells72,73
Induced pluripotent stem cells80,81
Perinatal/adipose-derived cells74,75
ECM-based 3D hydrogel culture systems91–93
Microchannel embedding26,102,103
Patterning of angiogenic growth factors or cells104
Sacrificial templates for the fabrication of perfusable microchannel
networks42
Direct bioprinting of vasculature by using cell patterning or tissue
spheroids109
Bioinks. Making tissue constructs with the desired functional and
biomechanical properties from available biomaterials remains a
challenge. One approach involves hybrid constructs of patterned
synthetic and natural materials (such as ECM-derived hydrogels):
the synthetic materials provide physical integrity and control
over the scaffold’s mechanical, structural and geometrical prop-
erties (such as the elastic modulus, tensile strength, porosity and
alignment) at the macroscopic level, and the natural materials
provide an appropriate structural and biochemical environment for
cell encapsulation and placement. Hence, the use of hydrogels based
on ECM components in native tissue is desirable for cell encapsu-
lation because they facilitate the provision of tissue-specific nutri-
ents and cellular products after printing. For example, a hydrogel
composed of skin-derived decellularized ECM was included in
a 3D cell-printing process that enabled the precise generation of
cell-laden constructs by inducing the simultaneous gelation of the
printed bioinks54.
However, a major limitation is the need for these materials to be
deposited by bioprinting techniques that often rely on melt-extru-
sion, on materials with sheer-thinning properties or on selective
crosslinking approaches55. A further limitation is that these materi-
als must also establish physiological, biochemical and mechanical
interactions with the cellular component. The approach of combin-
ing a synthetic material for mechanical strength, together with a
softer hydrogel for cell encapsulation and deposition, may lead to
a construct in which the cells are not exposed to the appropriate
mechanical stimulation necessary for the maturation of tissues such
as articular cartilage and muscle. Furthermore, many tissues have
biological and biomechanical heterogeneity, and interface with dif-
ferent tissue types. In this context, 3D bioprinting enables the for-
mation of concentration gradients of materials, cells and biological
factors, and hence the reproduction of these heterogeneous tissue
properties. Examples of solutions to these problems are micro-
fluidic switching nozzles that swap between two different inks on
373
Perspective NaTure BIomeDIcaL EngIneerIng
a b

Rotating
impelier
Inlet 2

2 cm 2 cm
Inlet 1
Mixing
volume

Nozzle
outlet

4 cm

Printed
structure

Fig. 2 | 3D bioprinting of biomaterials with graded properties. a, A mixing nozzle that can be used to print materials at the microscale, with tuneable
gradients of differing material properties. b, Images of the cross-section of a 3D rectangular lattice with a continuously varying compositional gradient,
showing continuous change in fluorescent pigment concentration under bright light (top left) and UV radiation (top right). 2D lattice structure shows
discretely varying fluorescence gradient at eight different mixing ratios under bright light (middle) and UV radiation (bottom). Dashed white lines mark
the regions of different mixing ratios. Figure reproduced from ref. 57, NAS.

demand56, and mixing nozzles that can be used to print materials at
the microscale with tuneable gradients of differing material proper-
ties57 (Fig. 2). Some specific applications may require more complex
printing patterns, such as in the reproduction of the zonal mechani-
cal structure of articular cartilage23,24, the fabrication of vascularized
bone constructs with hierarchical organization58 and the fabrication
of muscle–tendon interfaces59.
Although ECM-derived hydrogels are biocompatible, their weak
mechanical properties limit their contribution to the physical prop-
erties of the tissue. Still, the hydrogels can be chemically modified to
enable the material to crosslink and alter, for instance, its mechani-
cal strength or degradation time60. For example, synthetic hydrogels
based on poly(ethylene glycol) (PEG) have been modified to cova-
lently tether ECM-derived biomolecules to the hydrogel network
via monovalent, divalent or multivalent reactive groups, such as
acrylate, amine, thiol, azide, maleimide and biotin–streptavidin. In
a similar approach, a photocrosslinkable hyaluronan (HA)–gelatin
hydrogel was developed for use in the bioprinting of decellularized
ECM materials61,62. This protocol implemented a methacrylate-
based photopolymerization system for a two-step photocrosslink-
ing process, allowing the extrusion of the material through a syringe
or printing head and the subsequent increase in elastic modulus63.
The incorporation of additives such as graphene oxide nanosheets
can also significantly increase the tensile strength of soft hydro-
gels64. Although modifications such as these have expanded the util-
ity of hydrogels for 3D printing, the ability to significantly improve
the mechanical strength of ECM-derived hydrogel scaffolds
remains limited.
Another approach involves the use of synthetic peptides that
mimic functional motifs of the ECM. The most common peptide
is Arg-Gly-Asp (RGD), which is found on multiple ECM proteins
and is involved in cell adhesion. There are many other self-assem-
bling peptides with a range of properties and functions65,66 that can
influence cell adhesion, shape, migration and differentiation, and
that are known to be involved in the binding and release of growth
factors and in the stimulation of tissue repair and regeneration67.
Also, stimuli-responsive systems can be made responsive to factors
such as temperature, pH, and biochemical or biological stimuli,
including specific enzymes, antibodies or cells68,69. The ability to
tightly control the density, patterning, structure and orientation of
synthetic peptides within a bioprinted 3D matrix provides opportu-
nities for inducing cell responses that would not normally be evoked
by native matrix molecules. Still, this approach has, to date, been
limited to the coating and functionalization of artificial surfaces and
has yet to be incorporated into a 3D bioprinted construct.
Another important factor is the requirement for the degrada-
tion time of the scaffold to be commensurate with the timing of
host remodelling of the construct. The implanted construct should
facilitate host ECM production and remodelling, to allow for the
replacement of any synthetic or temporary support structures in a
time scale that neither delays nor inhibits the formation of native
tissue structures, nor poses any substantial risk of premature scaf-
fold failure. There is thus a need for ECM modifications or ECM-
mimicking materials that provide stronger mechanical strength and
that maintain a cell-friendly environment, as well as for synthetic
materials that support cell delivery and post-printing bioactivity
and function. Controlling the spatial hierarchies of materials, cells
and biological factors, and the dynamics of tissue fabrication offers
many possibilities that remain to be exploited.
Cell sourcing. The production of an adequate number of regen-
eration-competent cells that do not elicit an immune response
following transplantation is an ongoing challenge. 3D bioprinted
tissues have typically contained only a small number of accessible
cell types. One of the simplest methods of obtaining cells involves
the harvesting of autologous primary cells (often mature, terminally
differentiated cell types) from the patient, followed by their lim-
ited expansion in vitro prior to tissue fabrication. For example, for
cartilage tissue engineering, primary chondrocytes are the choice
because they comprise the native tissue population and are isolated
easily as homogeneous cell preparations that can be expanded ex
vivo. However, articular chondrocytes cannot be easily harvested in
significant numbers and, in addition to donor-site morbidity, de-
differentiate following in vitro expansion.
Some of the limitations of expanding primary cell types can
be overcome. One approach is to use conditional reprogramming

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NaTure BIomeDIcaL EngIneerIng
to temporarily immortalize primary cells and thus increase their
expansion potential. Conditional reprograming often includes the
use of small molecules or other factors to promote cell proliferation
or other cellular functions. For example, culturing primary human
epithelial cells with fibroblast feeder cells combined with the Rho-
associated kinase (ROCK) inhibitor Y-27632 induces continued
cell proliferation while retaining the normal karyotype and remain-
ing non-tumorigenic70,71. Other solutions to limited cell availability
include the use of progenitor cells or stem cells. The most com-
monly used progenitor cells are MSCs, which can be isolated from
many tissue types, expanded and differentiated either in vitro or
in vivo into osteogenic, chondrogenic, tenogenic, myogenic, adipo-
genic and marrow-stromal lineages72–74. Perinatal or amniotic-fluid-
derived stem cells75, which can also be expanded and differentiated
into multiple lineages, are alternative multipotent stem cell sources.
Multipotent stem-cell populations will continue to have impor-
tant applications in the biofabrication of mesodermal-derived tis-
sues such as cartilage, bone and muscle. However, these cell sources
are associated with complications, in particular their propensity to
undergo senescence following multiple expansion rounds in vitro76.
Donor age can also negatively affect cell expansion and differentia-
tion77–79. Since the discovery of iPSC in 2007 (refs. 80,81), their easy
accessibility, expandability and ability to give rise to almost any cell
types has motivated the investigation of autologous iPSC-derived cell
types for regenerative-medicine applications. The first clinical study
involving the evaluation human iPSC-derived cells, specifically the
use of human iPSC-derived retinal pigment epithelial cells for the
treatment of macular degeneration82,83, initiated in 2014, highlighted
several challenges and risks that will need to be addressed before
the clinical application of iPSCs can take off. Most importantly, the
potential for tumorigenicity remains a significant risk84; in fact, an
iPSC trial was put on hold following the identification of two genetic
variants in a patient’s iPSCs85,86. Also, although they have the ability
to expand indefinitely, the in vitro culture conditions and passage
number have a significant impact on iPSC phenotype, including the
incidence of mutations87,88. To avoid this problem and to reduce the
cost of preparing autologous iPSCs, there are attempts to establish
iPSC banks, so that cell donors and recipients can be matched on
the basis of the major histocompatibility complex class-I and class-II
human leukocyte antigens (HLAs) involved in the immune recogni-
tion of foreign antigens. This may be easier to achieve in countries
with small genetic variability (such as Japan, where it is estimated
that 10, 75 and 140 cell lines would match about 50%, 80% and 90%,
respectively, of the Japanese population89,90).
Protocols for the isolation and expansion of new primary cell
sources are highly sought after. Advances over the past few decades
have seen the isolation, characterization and expansion of primary
cell types that had been thought to be impossible to culture out-
side of the body. However, many cell types cannot yet be expanded
with retention of their phenotype and function. A greater under-
standing of growth factors, culture media and enzymatic subculture
techniques significantly contributed to the current ability to expand
primary cells. Also, the more recent applications of 3D cell-culture
techniques (especially those with mixed cell populations in which
stromal and vascular cell types are combined with other functional
cell types in 3D culture conditions) provide contact-specific and
biochemical supports for the maintenance of cell viability and func-
tion in vitro. Additionally, physiologically accurate biomaterial-
growth environments are replacing the common tissue-culture
plastic. For instance, recent advances in ECM and 3D hydrogel cul-
ture systems91–93 have enabled the culture and expansion of clonal
primary adult liver stem cells without changes to cell phenotype94.
This includes 3D-culture environments that match the physical and
biochemical composition of the cells’ native environment, includ-
ing tissue-specific motifs and tissue-derived ECM that provide
near-physiological interactions with the embedded cell types. 3D
Nature Biomedical Engineering | VOL 4 | April 2020 | 370–380 | www.nature.com/natbiomedeng
Perspective
bioprinting and 3D multicellular building blocks comprising cells
and their ECM, and in the form of cell aggregates, strands, fibres
or more complex organoid or microtissue structures, can be com-
bined, as was shown with the mixing of dispensed cell spheroids and
cell cylinders into a hydrogel bed via the use of extrusion bioprint-
ers95–97 (this approach exploited the self-organizing capacity of mul-
ticellular spheroids). Moreover, the development of small molecules
of low molecular weight may regulate biological processes such as
survival, proliferation and function, and may allow for significantly
greater expansion of cell numbers without loss of phenotype and
function. A combination of approaches is likely to be applied to spe-
cific cell types in the future, with culture conditions designed for
each end-stage application.
Vascularization. Tissue-engineered constructs in vitro, for instance
in perfusion bioreactors98, can be supplied with oxygen and nutri-
ents. But after implantation in vivo the supply of oxygen and nutri-
ents is often limited by diffusion kinetics. Because in engineered
tissues oxygen diffusion is often slower than its consumption,
oxygen is the limiting factor in cell survival99; hence, as the thick-
ness of the tissue exceeds the limits for nutrient diffusion, the need
for vascularization becomes a critical factor. This aspect becomes
increasingly important for tissues with a high volumetric oxygen-
consumption rate, such as cardiac, pancreas and liver tissues. For
instance, few cells tolerate distances greater than 200 μm from
a blood vessel100, with sensitive cells such as islets undergoing
necrosis when the diffusion distance exceeds ∼100 μm. Conversely,
cartilage cells are generally more resistant, maintaining viability in
grafts thicker than 1 mm (ref. 101). Therefore, the fabrication of a
functional 3D-printed tissue will require the incorporation of mul-
tiscale vascular, lymphatic and/or neural networks. Currently, the
majority of tissues fabricated for transplantation using 3D printing
lack these components.
A major roadblock in the fabrication of engineered tissues
concerns the replication of the complex hierarchical structure of
a native integrated vascular network spanning arteries and veins
down to the smallest capillaries. Two main approaches have been
designed to overcome current limitations: the embedding of micro-
channels to improve the diffusion of nutrients and oxygen in the
absence of a vasculature26,39,40,102,103 (Fig. 3a), and the patterning
of angiogenic growth factors or cells within a tissue construct to
facilitate vascular development, either in vitro or following trans-
plantation104. However, although both of these approaches allow for
the fabrication of larger constructs, they ultimately rely on endog-
enous vascularization.
Although current technologies excel at fabricating a specific type
of materials within a relatively small range of scales, the complex
hierarchical 3D architecture of a multiscale vascular network can-
not yet be recapitulated. One promising route involves the com-
binatorial use of bioprinters with different working principles for
the deposition of multiple materials and cell types, and at differ-
ent scales. In this regard, direct ink writing has led to remarkable
achievements in the high-resolution patterning of matrix materi-
als105. One example is omnidirectional printing (ODP)105, whereby
fugitive bioinks are printed within a photocurable gel reservoir that
physically supports the patterned features, thereby allowing truly
omnidirectional free-form fabrication. The fugitive bioink can
then be removed to yield a desired microchannel network within
the matrix (Fig. 3b). Because the nozzle can simultaneously move
in three dimensions, the major advantage of ODP, compared with
multidimensional bioprinting, is that ODP is not limited to stan-
dard layer-by-layer fabrication. This allows ODP to broaden the
network-design space and to deposit materials in highly anisotropic
structures. Also, in ODP the microchannel diameter can be con-
trolled via dynamic-pressure variation; hence, a single nozzle can
pattern microchannels of varying size.
375
Perspective NaTure BIomeDIcaL EngIneerIng
a b

Fluid filler

PCL Matrix

Ink
3D-printed Nozzle direction
construct
el)
nn
ha
oc
icr
(m
re
Po

Cell A

Cell B
c
t = 0 days t = 2 days

Cell ink 1 Lumen

Vasculature

Cell ink 2 HNDFs

ECM
HUVECs

Fig. 3 | Overcoming diffusion limitations and the need for vascularization. a, A patterning approach for generating a 3D architecture that includes
multiple cell-laden hydrogels, a supporting PCL polymer and microchannel pores for improving the diffusion of oxygen and nutrients. b, Fabrication of
vascular structures via omnidirectional printing. Deposition of a fugitive ink into a physical gel reservoir (top left) allows hierarchical and branching
networks to be patterned. Voids induced by nozzle translation are filled with liquid that migrates from the fluid-capping layer (top right). Subsequent
photopolymerization of the reservoir yields a chemically crosslinked hydrogel matrix (bottom left). The ink is liquefied and removed under modest vacuum
to expose the microvascular channels (bottom right). c, A 3D-bioprinting approach in which vasculature, cells and ECM are co-printed to yield engineered,
vascularized and heterogeneous cell-laden tissue constructs (left, schematic; right, micrographs). Scale bars, 300 μm. Panels reproduced from ref. 26,
Springer Nature Ltd (a); ref. 105, Wiley-VCH (b); and ref. 107, Wiley-VCH (c).

With current technology, it is technically challenging to print
functional capillaries at the micrometre scale. One alternative is to
let capillaries develop by first fabricating a vascular network that
then matures in vivo or in bioreactors106. For example, by drawing
on techniques based on sacrificial template materials, one can fab-
ricate a perfusable microchannel network within the tissue to, for
instance, develop a construct consisting of two endothelialized flu-
idic channels with a fibrin–endothelial-cell mixture located between
them. This design results in the formation of adjacent capillary
networks and, consequently, in the generation of a multiscale vas-
cular network that connects millimetre-scale vessels with adjacent
microvasculature. Another alternative involves the bioprinting of a
vasculature network by using patterned cells or tissue spheroids. For
example, multiple biomaterials encapsulating MSCs and fibroblasts
within a customized ECM can be co-printed alongside embedded
vasculature subsequently lined with endothelial cells107,108 (Fig. 3c).
This approach enabled the creation of thick tissues (larger than 1
cm) replete with an engineered ECM, embedded vasculature and
multiple cell types. Another example of such a bioprinting strategy
involved multiple vascular cell types aggregated as multicellular
vascular tissue spheroids and printed layer-by-layer concomitantly
with agarose rods as a template109. The closely placed vascular tis-
sue spheroids underwent tissue fusion and self-assembly into small
segments resembling a branched vascular tree. Another strategy
involves microscale continuous optical bioprinting, which offers
speeds, resolutions and flexibility that are superior to those of con-
ventional bioprinters110. In this approach, multiple vascular cell types
are encapsulated directly into hydrogels with precise distribution
and without the need of sacrificial materials. Notably, anastomosis
can occur between the bioprinted endothelial network and the host
circulation.
Despite advances in the direct fabrication of larger conduits, in
the incorporation of perfusable microchannel networks and in the
stimulation of vasculogenesis, current fabrication approaches can-
not incorporate a complete multiscale vascular network suitable for
the provision of oxygen and nutrients into clinically relevant sized
3D-printed tissues. The patterning of tissues at scales ranging from
micrometres to centimetres will require significant technologi-
cal innovations in material deposition and cell deposition. Recent
advances in microextrusion technology have enabled the pattern-
ing of multicomponent constructs containing both synthetic and
natural materials with resolutions down to 2 μm for biomaterials
alone and down to 50 μm for encapsulated cells26. Further progress
will require the ability to deposit an even wider range of material
types concurrently with increased printing resolution and printing
speed. One promising approach involves parallelization schemes
that use multiple nozzles to deposit materials at the same time: with
multinozzle arrays designed with hierarchically branching chan-
nels, each print-head distributes bioink from a single microchannel
into repeatedly bifurcated branches, to deposit bioink simultane-
ously from multiple nozzles during printing111. This approach can
print, at high-throughput, planar and multilayered architectures
composed of single and multicomponent materials. However, it has
yet to be demonstrated with materials containing cells. Also, bio-
logical advances could lead to improvements in the bioprinting of
vasculature: for example, relationships between endothelial cells

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NaTure BIomeDIcaL EngIneerIng
and parenchymal constituents, and between interstitial and per-
fusate flows and hydrostatic pressure, are relevant for the optimal
self-assembly of capillary-sized vessels112–115. Although biological
responses to multivariate stimuli are difficult to model and predict,
such knowledge should enable the engineering of vascular struc-
tures that favour rapid angiogenesis.
Another promising general approach is ‘modular tissue design’,
where ‘tissue modules’ are scaled to incorporate relevant vascular-
ization strategies that consider the limitations of each approach. For
example, small tissue modules may incorporate a series of perfus-
able microchannels combined with approaches for the stimulation
of angiogenic sprouting and growth. These small tissue modules
could then be assembled with a larger network of directly fabricated
vessels, and patterned with a branching network of endothelialized
microvasculature connected to millimetre-scale vessels capable of
anastomosis. This will require the development of approaches for
the high-throughput, high-resolution bioprinting of multiscale vas-
cular networks within instructive bioinks that promote angiogenic
sprouting and neovascularization.
Outlook
To envisage how future advances will influence the next generation
of 3D-printed tissues, we can look at current technologies for the
3D-printing of cartilage, bone and skin constructs. They can serve
as benchmarks for the evaluation of the status of bioprinting tech-
nology. By understanding the factors that have contributed to their
success, and by anticipating advances in the bioprinting of materi-
als and cells, we can postulate which of the current limitations may
be overcome in the short term, and what the impact will be in the
development of the next generation of 3D-printed tissues. Common
properties of successfully 3D-printed tissues include: (i) the repli-
cation of the 3D architecture, size and form of native tissues that
(ii) contain appropriate mechanical properties at the microscale and
macroscale, (iii) including the cell types necessary for tissue func-
tion and for the maintenance of tissue homeostasis, and (iv) the
reproduction of tissue or organ function at a level sufficient for the
replacement, restoration or supplementation of the in vivo tissue.
In our opinion, the continued development of materials suitable
for the deposition, with a 3D printer, of cells that can also support
cell function, tissue structure and biomechanical properties would
have significant impact. Also, the further development of bioprinters
could accelerate the fabrication timeline, and provide the resolution
needed for the fabrication of functional tissues at a clinically relevant
scale. Approaches that accelerate manufacturing processes, such as
advances in bioprinter or material technologies, could, in future,
overcome the slow speed of current layer-by-layer printing. Although
not yet tested for the fabrication of medical devices, the continuous-
liquid-interface-production approach can in principle print complex
solid parts at rates of hundreds of millimetres per hour116.
The limits of biomimicry. Whether the current approach of mim-
icking biological structures will continue to lead to optimal function
and design efficiencies in larger and more complex tissues is unclear.
Manufacturing close reproductions of the cellular and extracellular
components of a tissue or organ has often resulted in approximations
of tissue function. However, biomimicry may reach a point where
increased complexity no longer improves functional outcomes.
Also, technological or economic limitations may require a departure
from biomimicry in the process of enhancing tissue function, tis-
sue scale or printing throughput. Therefore, new design approaches
that prioritize construct function and biofabrication efficiency may
be needed, possibly with 3D-bioprinted constructs that are efficient
in replacing or supplementing tissue function but that do not pos-
sess the size, shape or form of the functional tissue type (for exam-
ple, one could imagine 3D-printed constructs shaped as a sheet or
patch and designed to assist liver function or pancreas function).
Nature Biomedical Engineering | VOL 4 | April 2020 | 370–380 | www.nature.com/natbiomedeng
Perspective
This approach could significantly improve fabrication efficiency and
reduce fabrication costs, although it may need the determination of
appropriate sites for implantation, the promotion of anastomosis,
and the ensuring of long-term integration and function.
Automation and personalization. Implementing automated
designs in personalized manufacturing processes can be used to
understand which control mechanisms can be regulated to ensure
predictable outcomes. Currently, many personalized designs are
produced with more artistry than engineering, simply because of the
complexity of anatomical designs. Computational processes with
clear design parameters are made possible by modern 3D imaging
and modelling software, which can be adapted and automated to
generate personalized structures that match the patient’s anatomy
and injury needs117. Such an approach would map predetermined
hatching architecture, materials and cell types onto appropriate
regions of the bioprinted construct spatially defined by a 3D model
generated via medical imaging, typically via CT or MRI. A detailed
understanding of biomaterial self-assembly, of tissue healing and
of the integration of the bioprinted construct, will be needed to
develop the required design criteria for future bioprinting concepts
to reliably meet patient needs. This understanding is also necessary
for the clinical translation and regulation of personalized bioprinted
constructs. In many ways, bioprinting is a type of 4D printing, as the
cells reorganize and produce tissue, and degradable scaffold materi-
als break down over time after implantation. Building the repertoire
of knowledge needed to properly predict the outcomes of such com-
plex 4D prints118 will require research on the relevant in vitro and
in vivo interactions of cells over time.
Cost-effectiveness. There are substantial challenges associated
with the scaling and commercialization of bioprinted tissues. For
example, the tissue design, cell sourcing and fabrication logistics
developed for each organ in many current approaches are highly
personalized to the individual needs of each patient. Therefore, the
cost-effectiveness of personalized 3D-printed tissue replacements
will be a significant challenge. In the short term, companies consid-
ering investing in this area may consider the development of generic
or universal scaffolds, or of components that would reduce the costs
associated with the need for individualized materials and designs.
The use of acellular scaffolds that are then seeded with the patient’s
own cells may reduce costs while minimizing the risks of alloge-
neic rejection. When considering a personalized tissue-engineering
approach such as 3D bioprinting, the cost-effectiveness of a treat-
ment may require high upfront costs, but these must be balanced
against the alternative of costly life-long treatments (such as dialysis
or the management of diabetic complications). For many conditions,
a cost-effectiveness analysis may indicate that a single intervention is
economically preferable than life-long non-curative treatments.
Regulation. The implantation of 3D-bioprinted tissues and organs
will face regulatory challenges. Despite the variety of manufacturing
methods, the FDA currently assesses 3D-printed medical devices
and conventionally made products under the same guidelines. The
21st Century Cures Act119 describes regenerative-medicine therapies
that may be eligible for the designation of ‘regenerative medicine
advanced therapy’ (RMAT). These include cell therapies, therapeu-
tic tissue-engineering products, human cell and tissue products,
combination products that use any such therapies or products, and
gene therapies that lead to durable modifications of cells or tissues.
To help frame the future of 3D bioprinting for healthcare, the FDA
has released detailed guidance for 3D-printer manufacturers120.
There are a number of regulatory challenges that apply to func-
tional 3D-bioprinted constructs containing living cells and bio-
active materials. First, they are intrinsically different from other
clinical products, owing to the complexity of mechanisms and to
377
Perspective
as-yet-unknown long-term effects in human hosts. Second, the
mechanisms of action in tissue constructs depend on their multiple
active components, which makes it difficult to meet a regulatory
definition of potency. Third, the manufacturing and product char-
acterization are likely to be significantly more complex; seemingly
minor manufacturing changes may have large and unpredicted
effects on the characteristics of the product. Hence, a centralized
and defined regulatory pathway will be necessary to avoid overbur-
dened and redundant regulatory pathways. Regulatory frameworks
will need to describe clearly how regulators will apply existing
laws and regulations that govern device manufacturing to non-
traditional manufacturers such as medical facilities and aca-
demic institutions that create 3D-printed personalized devices.
Encouragingly, the current guidelines and the regulatory pro-
cesses proposed by the FDA and by other national regulatory bod-
ies suggest that these considerations are known. Also, some of the
regulatory challenges could be addressed via the technological
convergence of production methods, and via the increased stan-
dardization and identification of best practices for the design and
production of bioprinted constructs.
Standardization. There are currently no standards for 3D bioprint-
ing technology, for bioprinting materials (including bioinks and cell
sources) or for the overall bioprinting process. Despite the existence
of standards for additive-manufacturing terminology (ISO/DIS
17296-1) and the recent first guidance documents for 3D-printer
manufacturers120, there is an urgent need for increased levels of
standardization and for production guidelines for bioprinting.
A degree of standardization of the bioprinting materials (analogous
to standardized cell-expansion basal media or to modular bioink
chemistry) would have a beneficial impact on product-development
time. The standardization of materials and manufacturing processes
would also accelerate the clinical translation of bioprinted con-
structs, and enable manufacturers to optimize their processes for a
specific construct or product and to minimize the resources required
for the development of base materials and technical processes.
Standardized cell-culture media and bioinks, and standardized
quality-control systems for in-line sensing (required to collect qual-
ity-control data throughout the manufacturing process and to ensure
product safety, efficacy and reproducibility), are also necessary for the
reduction of the high costs and prolonged development times asso-
ciated with the manufacturing of regenerative-medicine products121.
Logistics. Because products that contain living cells and tissues are
environmentally and time-sensitive, logistical demands for these
products are especially challenging. The logistical chain can be par-
ticularly complex when considering the manufacturing of patient-
specific tissues or organs, as it is unlikely that patient-care facilities
will possess 3D-printing capabilities or be capable of the clinical-
grade production of the necessary cells and materials. Current cold-
storage shipping procedures, originally developed decades ago, do
not meet the precision or performance required for living biological
products. A new shipping system that ensures the required stabil-
ity, communication, data collection and documented compliance
will thus need to be designed. This includes the transfer of medical
data for the design of biofabricated tissues or organs. Furthermore,
because few facilities possess the expertise, technology and resources
to manufacture tissues and organs for transplantation, a centralized
logistical model may need to be used, at least initially; several major
facilities could then be strategically located. Hence, patient-derived
cells and materials would need to be transported to a centralized
biofabrication facility, and the biofabricated organs would then be
shipped back to the patient. The shipping logistics could take a page
from traditional organ-procurement and transplantation programs,
which focus on best systems and practices for organ procurement,
multi-site coordination, preservation and transportation.
378 Nature Biomedical Engineering | VOL 4 | April 2020 | 370–380 | www.nature.com/natbiomedeng
NaTure BIomeDIcaL EngIneerIng
In summary, a clinically relevant bioprinted construct will most
probably need to reach thresholds for functional and supporting
cell types (including stem cells), have biomechanical properties that
closely mimic those of the native organ at both the microscale and
macroscale, and feature intact vascular networks spanning arteries,
veins and capillaries. These criteria have been partly met for bio-
printed cartilage, bone and skin constructs. Although the scale and
functionality of 3D-printed tissue constructs continues to improve,
significant challenges remain for the bioprinting of more complex
tissues with greater physiological demands. Progress towards the
multiscale and multicomponent fabrication of clinically relevant
bioprinted tissues and organs will require the incorporation of
advances in the isolation and expansion of populations of primary
cells and stem cells, the development of ‘smart’ biomaterials, and the
integration of complementary bioprinting technologies.
Received: 13 July 2018; Accepted: 30 September 2019;
Published online: 6 November 2019
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Acknowledgements
P.D.C. is supported by the National Institute for Health Research
(NIHR-RP-2014-04-046) and by the NIHR Great Ormond Street Hospital Biomedical
Research Centre.
Author contributions
S.V.M, P.D.C and A.A. discussed the content, researched the literature and wrote
the manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Correspondence should be addressed to S.V.M.
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