Special Issue: Tissue Engineering

Opinion

The Synergy of Scaffold-Based and

Scaffold-Free Tissue Engineering Strategies
Aleksandr Ovsianikov,1,7,* Ali Khademhosseini,2,3,4 and Vladimir Mironov5,6

Tissue engineering (TE) is a highly interdisciplinary research field driven by the Highlights
goal to restore, replace, or regenerate defective tissues. Throughout more than Integrating different technologies and
materials into one operational proce-
two decades of intense research, different technological approaches, which
dure can produce 3D tissue engineer-
can be principally categorized into scaffold-based and scaffold-free strategies, ing (TE) scaffolds with enhanced
have been developed. In this opinion article, we discuss the emergence of a properties.

third strategy in TE. This synergetic strategy integrates the advantages of both Scaffold-free TE, based on the assem-
of these traditional approaches, while being clearly distinct from them. Its bly of building blocks such as cell
characteristic attributes, numerous practical benefits, and recent literature sheets and spheroids, was established
as an alternative strategy, providing
reports supporting our opinion, are discussed in detail. several advantages over conventional
scaffold-based methods.
To Scaffold or Not to Scaffold
Bottom–up assembly of multiple func-
Current approaches in tissue engineering (TE) are mainly represented by two distinct and
tional units was introduced as a pro-
somewhat opposing strategies, each having their characteristic advantages and posing some mising approach toward engineering
intrinsic limitations (Box 1). complex tissue constructs and creat-
ing biomimetic hierarchical
architectures.
The scaffold-based strategy relies on the use of biomaterials to create a temporary structure
supporting cells throughout the tissue formation. The scaffold (see Glossary) can be a classical A synergetic TE strategy, resulting
3D construct with interconnected pores, a hydrogel with cells embedded in it, or a combination from the integration of the scaffold-
of the two. The use of a decellularized extracellular matrix (ECM) of a tissue is another based and scaffold-free approaches,
has emerged.
promising trend [1]. The scaffold-based approach is very versatile with respect to the
3D bioprinting and robotic tissue
assembly may solve multiple current
Box 1. Conventional Tissue Engineering Strategies bottlenecks in TE via automated pro-
Tissue engineering is a biomedical approach concerned with the development of functional cell-containing constructs duction of tissue constructs in a reli-
that are able to restore, maintain, or improve damaged tissues or whole organs. Current approaches in TE are mainly able and reproducible fashion.
represented by two somewhat opposing strategies:

The scaffold-based approach relies on the use of appropriate ‘templates’ supporting living cell attachment, proliferation,
and subsequent formation of 3D tissue. The possibility to use specially designed materials is the basis of the most
important functions of the scaffold: (i) matching the degradation profile of the scaffold to the formation of de novo ECM 1
Additive Manufacturing Technologies
by the cells, that is, maintaining the compliance of the construct at all times, which is especially important for tissues
(AMT) Group, Institute of Materials
exposed to load; and (ii) providing a biomimetic environment for cells and/or the delivery and controlled release of growth
Science and Technology, Vienna
and differentiation factors to facilitate tissue formation. In addition, robust 3D scaffolds can protect cells from possible University of Technology (TU Wien),
damage via external factors. Getreidemarkt 9, 1060 Vienna, Austria
2
Center for Minimally Invasive
‘Scaffold free’ is a bottom–up strategy employing cell sheets, spheroids, or tissue strands as building blocks. It relies on Therapeutics (C-MIT), California
the inherent ability of these building blocks to fuse and form larger constructs. In contrast to the scaffold-based NanoSystems Institute (CNSI),
approach, due to the high initial cell density, the proliferation and migration of cells are not decisive factors, so the time Bioengineering Department, Chemical
necessary for tissue formation can be reduced dramatically. Two important shortcomings of this approach are (i) the and Biomolecular Engineering
inferior mechanical properties of individual building blocks, leading to possible cell damage during their manipulation, Department, Radiology Department,
University of California, Los Angeles,
and (ii) the immobilization time necessary for the initial fusion of these building blocks and deposition of ECM to obtain a
Los Angeles 90095, CA, USA
cohesive construct. A remarkable advantage of this bottom–up approach is its capability to address the architecture of 3
Nanotechnology Center, King
the complex tissues and organs by controlled assembly of heterogeneous building blocks consisting of different cell
Abdulaziz University, 21589 Jeddah,
types.
Saudi Arabia

348 Trends in Biotechnology, April 2018, Vol. 36, No. 4 https://doi.org/10.1016/j.tibtech.2018.01.005

© 2018 Elsevier Ltd. All rights reserved.
 4
construct’s mechanical properties and degradation profile, as well as the possibility to deliver Department of Bioindustrial
Technologies, College of Animal
biomolecules either by controlled release from the materials or by immobilizing them on the Bioscience and Technology, Konkuk
surface. In addition, rapidly progressing 3D printing technologies offer a wide range of University, Hwayang-dong, Gwangjin-
possibilities from using bio-inspired composites to realization of multimaterial constructs gu, Seoul 143-701, Republic of Korea
5
Private Institution Laboratory for
and shape-morphing systems [2]. Biotechnological Research, 3D
Bioprinting Solutions, Moscow,
However, the use of scaffolds does present some challenges. For example, porous 3D Russian Federation
6
Institute for Regenerative Medicine,
scaffolds are notoriously cumbersome to seed with cells because, especially in case of large Sechenov First Moscow State Medical
constructs, the cell distribution tends to be inhomogeneous and the initial density is low. By University, Moscow, Russian
contrast, hydrogels can be formed in the presence of cells, facilitating their homogeneous Federation
7
ORCID: 0000-0001-5846-0198
distribution and somewhat higher loading densities. However, stiffer hydrogels with mechanical
properties adequate for realization of stable constructs are often suboptimal when it comes to
cell migration and spreading [3]. Combining hydrogels with robust macroporous scaffolds can *Correspondence:
produce mechanically stable 3D constructs, while their pores, filled with soft cell-containing aleksandr.ovsianikov@tuwien.ac.at
(A. Ovsianikov).
hydrogels, provide a highly biomimetic environment similar to that of the natural ECM [4–6]. This
approach is especially favorable in case of 3D bioprinting because it can deposit different cells
and materials at the defined locations of the TE construct [7]. Sequential multimaterial bio-
printing of hydrogels has also been used to create relatively thick vascularized TE constructs [8].
Recently, a novel method enabling continuous multimaterial bioprinting, as well as switching to
core–shell or hollow filament deposition, was introduced [9]. Coaxial extrusion of cell-containing
hydrogels was also used to create small-diameter blood vessel TE constructs [10].

Scaffold-free TE has its origins in self-assembly [11]. It emerged as a powerful strategy using
prefabricated multicellular building blocks, such as cell sheets, spheroids, and, more recently,
tissue strands. The capacity of these building blocks to fuse into larger cohesive constructs and
produce ECM is the essential attribute of the scaffold-free approach. Among these building
blocks, tissue strands represent the newest development; they are prepared by culturing cells
within a tubular alginate capsule, which is later removed by a sodium citrate solution [12]. These
strands can be prepared from heterocellular cell suspensions, and were shown to completely
fuse after 7 days [13]. Different methods for producing cell sheets and spheroids have been
used historically, but cell sheets are now conventionally prepared by culturing a monolayer of
cells in petri dishes coated with a thermoresponsive polymer [14]. It has also been reported that
by stacking sheets prepared from endothelial cells and fibroblasts, 3D tissue constructs with
prevascular networks can be produced in vitro [15]. Furthermore, patterned substrates have
been used to grow sheets of cells aligning along a certain direction to produce anisotropic
tissue constructs [16]. Largely due to the convenience of handling, multicellular spheroids are
arguably the most popular building block in scaffold-free TE [17,18]. Cell spheroid production
methods are highly elaborate and range from various antiadhesive cell-culture plates to
microfluidic and fully automated hanging-drop cultures [19,20]. Most importantly, spheroids
have been shown to exhibit improved biological properties with regard to regenerative capacity,
since they facilitate intense cell–cell interactions and resemble other physiological conditions of
complex tissues with 3D architecture [21]. Because of their high vasculogenic and angiogenic
potential, spheroids are especially advantageous when it comes to vascularization [22].
Facilitated by their shape, cell spheroids are suitable for bottom–up tissue self-assembly,
as was recently demonstrated by culturing spheroids produced from adipose tissue-derived
mesenchymal stem cells (MSCs) in a cylindrical mold in a study aimed at osteochondral defect
regeneration [23]. Spheroids are also used for additive manufacturing of tissue in the context of
bioprinting and automated tissue assembly. Computer-aided design (CAD)-based technolo-
gies potentially provide control over each individual building block within a complex 3D tissue

Trends in Biotechnology, April 2018, Vol. 36, No. 4 349

construct [18,24]. However, some concerns regarding the possibility of achieving adequate Glossary
mechanical properties with such scaffold-free tissue constructs have recently been voiced [25]. Extracellular matrix (ECM): the
noncellular component of natural
The advantages and drawbacks of scaffold-based and scaffold-free TE approaches appear to tissues and organs consisting mainly
of collagen, elastin,
be mutually exclusive. Nevertheless, in this opinion article, we suggest that a combined glycosaminoglycans, and other
synergetic strategy might represent an optimal solution capable of addressing a wide range molecules secreted by cells into the
of current TE challenges. By integrating multicellular spheroids with robust scaffolds, it is surrounding medium. The ECM
provides structural, biochemical, and
possible to constructively promote the advantages of current TE approaches, while mitigating
instructive support and signaling to
their drawbacks (Figure 1). To recapitulate, this synergetic TE strategy is using robust porous the neighboring cells.
3D scaffolds that contain multicellular building blocks and, due to high cell loading and porosity, Scaffold: in tissue engineering, a
facilitate their contact and fusion. Despite the use of scaffolds, cell migration and proliferation temporary construct supporting the
initial attachment and/or proliferation
are not decisive factors for the maturation of such TE constructs. By the same virtue, a more
of cells, and facilitating formation of
advanced stage of building block and tissue differentiation can be employed. In turn, by using functional 3D de novo tissue.
differentiated cells or directed differentiation of spheroids into microtissues containing ECM, the Spheroids: ball-shaped compacted
time necessary for tissue formation could be reduced dramatically. At the same time, the robust cell aggregates consisting of
monotypic or heterotypic living cells
3D scaffold serves to retain spheroids and protect them from the mechanical damage. At the
with maximal possible initial cell
initial stage, the mechanical properties of such construct are dominated by the scaffolds’ density. They have self-assembling
design and the stiffness of the utilized material, which can be adjusted independently of tissue capacities for tissue fusion and
spheroids. Furthermore, the use of scaffolds provides means for additional functionalization of forming larger tissue constructs and
are used as building blocks in
the constructs and delivery of relevant biomolecules, such as growth factors. This synergetic scaffold-free tissue engineering.
combination of scaffold-based and scaffold-free TE, usually seen as rival approaches, offers Two-photon polymerization (2PP):
numerous opportunities for overcoming their individual bottlenecks and developing new a 3D printing method relying on
solutions based on their added value. localized polymerization of a material
induced by nonlinear absorption of
laser light. An important distinction of
The Synergy of Scaffold-Based and Scaffold-Free TE Strategies 2PP compared with other methods is
This synergetic strategy is best illustrated with the example of the recently reported ‘lockyballs’ that it does not require layer-by-layer
deposition of material.
– microscaffolds containing individual spheroids. The spheroids are produced directly within
each microscaffold by incubating it in an antiadhesive microwell together with the cells for 12–
48 h (Figure 2A). The size of the spheroid is controlled by the number of cells seeded into each
microwell. Such constructs can be efficiently formed from mouse preosteoblasts as well as
human adipose tissue-derived MSCs (hMSCs) [26,27]. The spheroid fusion kinetics are similar
with and without the microscaffold. Importantly, in the case of hMSCs, the process did not

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Figure 1. Summary of Different Technological Tissue Engineering Approaches. Expanded cells (left) can be
used to (bottom) seed scaffolds to produce tissue-engineered constructs in the scaffold-based strategy; (top) produce
spheroids to assemble scaffold-free constructs; and (middle) produce spheroids directly within cage-like microscaffolds,
which are later assembled into a 3D construct and create cohesive construct via spheroid fusion in a synergetic tissue
engineering strategy.

350 Trends in Biotechnology, April 2018, Vol. 36, No. 4

(A) (B)

(C) (D) (E)

CD31 F-Acn
Nuclei Nuclei F-Acn

hν

hν

(F) (G) (H)

15kV X200 100µm UoC

Figure 2. Self-Assembly in the Synergetic Tissue Engineering Strategy. (A) Seeding procedure, from left to right:
microscaffold inserted into an antiadhesive microwell, cell seeding, and spheroid formation directly within the microscaffold
[27]; (B) fusion of microscaffolds carrying spheroids produced from adipose tissue-derived mesenchymal stem cells [27];
(C–E) formation of a CD31-positive interconnected network within a modular microgel construct [31]; (F) laser 3D printing of
a microscaffold by means of two-photon polymerization (2PP) technique; (G) scanning electron microscopy (SEM) image
of a microscaffold equipped with hooks – the lockyball [35]; (H) SEM image of an arrow-headed microscaffold [26] [scale
bar is 25 mm for images (C–E) and 100 mm throughout the rest of the figure]. Reproduced, with permission, from the
indicated references.

compromise the cell viability and the expression of genes important for osteogenic and
chondrogenic lineages [27].

The shape and the open porosity of the microscaffold allow spheroid fusion, whereas at the
same time such microscaffolds are capable of shielding the individual spheroids from mechani-
cal damage (Figure 2B). The scaffolds’ design and material can be varied independently of their
cellular content, enabling adjustment of the degradation profile and the mechanical properties
of the final construct beyond of what the cells alone would allow. Depending on the target
application and cell type, the scaffold material can be designed to deliver and release special-
ized biomolecules such as growth and/or differentiation factors. For example, despite the
capability of organoid building blocks to form complex tissue structures through self-assembly,
biomaterials add value to this approach by providing means to guide cell behavior and function
[28]. Spheroid fusion provides high initial cell density and, therefore, can dramatically minimize

Trends in Biotechnology, April 2018, Vol. 36, No. 4 351

the time necessary for tissue formation. The modular nature allows bottom–up assembly of
tissue constructs. This approach should not be confused with the conventional assembly of
microgels and microcarriers seeded with cells, since the resulting constructs neither possess
comparable cell density nor rely on spheroid fusion [29,30].

The success of TE construct goes hand-in-hand with adequate vascularization. Apart from the
possibility to functionalize the microscaffolds with proangiogenic molecules, the modular nature
of the method might in itself be advantageous for vascularization. Using the example of
microgels, modular constructs facilitate their rapid permeation by prevascular networks
[31]. In a co-culture of MSCs and endothelial cells, the endothelial cells formed a CD31-
positive network, engulfing and bridging the microgels (Figure 2C–E). Although the study was
focused on relatively small single-cell-containing modules, we speculate that a similar scenario
could be realized with multicellular microscaffolds. For example, subcutaneous grafting of
scaffold-free endothelial cell spheroids can give rise to capillary networks and anastomoses
with mouse vasculature [32]. Self-assembly provides considerable flexibility in using different
cell types and controlling their ratio. They can either be provided in a form of spheroid co-culture
or introduced by combining microscaffolds containing different cells.

The Enabling Technology behind the Microscaffolds

The size of the microscaffold is dictated by the diameter of the spheroid, which is usually less
than 300 mm. The prerequisite of large open pores implies that the scaffold-constituting
filaments are only several microns wide, putting stringent constraints on the resolution of
the technology used to produce the microscaffolds. The demonstrated lockyballs were
produced using the two-photon polymerization (2PP) technique, which can achieve spatial
resolution below 100 nm [33,34]. Another outstanding distinction of 2PP compared with other
3D printing methods is that it does not require layer-by-layer deposition of material or the use of
temporary supports, since the structures are produced in the volume of the photosensitive
material (Figure 2F). These technological features also help to create functional elements such
as the hooks of the lockyball (Figure 2G). The hooks facilitate interlocking between the individual
building blocks during the bottom–up assembly, providing the possibility of rapid tissue
assembly and fixing the shape of the construct ahead of spheroid fusion [35]. This characteristic
might be advantageous for engineering load-bearing tissues, such as cartilage and bone. 2PP
is capable of producing other functional elements, such as burrs, as well as introducing
additional inner microscaffold structure to further adjust its mechanical properties
(Figure 2H) [26]. More advanced maturation of tissue spheroids by induced tissue-specific
differentiation might reduce their capacity for rapid tissue fusion [36]. Thus, encaging tissue
spheroids into microscaffolds does not compromise their fusion and also enables additional
functionalities.

The Synergetic Tissue Engineering Strategy beyond Microscaffolds

Combining multicellular spheroids with porous 3D microscaffolds, facilitating the fusion of
neighboring spheroids, nicely illustrates the emergence of the synergetic strategy in TE.
However, bottom–up assembly of such tissue modules is not an essential prerequisite of this
strategy. Several attempts at combining robust 3D scaffolds with spheroids can be found in the
literature throughout the recent years. In an early example, Kim and colleagues [37] described
vascularized adipose tissue using 3D poly(caprolactone) scaffolds containing spheroids pro-
duced from bone marrow hMSCs. These hybrid constructs were assembled from separate
units, each carrying nine spheroids, by stacking them in a Lego-like fashion (Figure 3A). Cellular
adhesion to the scaffolds and fusion of spheroids improved the integrity of assembled hybrid

352 Trends in Biotechnology, April 2018, Vol. 36, No. 4

constructs. In this way, stable 3D constructs with high initial cell density resulting from spheroid
fusion were produced. This work also demonstrated that the possibility of poly(caprolactone)
functionalization with basic fibroblast growth factor enabled adequate vascularization of the
resulting adipose tissue in vivo (Figure 3B,C). This finding underlines the synergetic advantage
of using scaffolds as a means of delivering growth factors in a sustained manner.

4mm
(B) m
2m
(3) Woven scaffold
Arcular face

1mm
Compacted collagen
sheet

MSCs collect
(4) Macropore channel
day 3

(C) (D)

(A)
100 µm

Combined construct Zonal construct 100%

90%
SZ
Incorporated GAG/total GAG

SZ + DZ 80%
DZ 70%
60%
50%
Pellets
40% Constructs
30%
20%

(E) 10%
0%

Figure 3. Synergetic Tissue Engineering Strategy Beyond Microsaffolds. (A) Robust 3D scaffold combined with mesenchymal stem cell (MSC) spheroids,
providing high cell loading and attachment between separate elements, (B) optical, and (C) confocal microscopy images of a construct after 4 weeks of in vivo culture,
showing adipose tissue formation and vascularization [37]; (D) woven collagen scaffolds designed to carry spheroids demonstrate strong immunostaining for aggrecan
after 28 days in culture [41]; (E) scaffolds containing spheroids derived from deep cartilage zone (DZ), superficial zone (SZ), and a combination thereof did not preserve
the initial cell distribution pattern, but showed improved incorporation of glycosaminoglycans compared with scaffold-free culture after 24-day in vitro culture [40].

Trends in Biotechnology, April 2018, Vol. 36, No. 4 353

Literature reports suggest that mesenchymal cell condensation, that is, formation of spheroids,
often referred to as micromass culture or pellets in cartilage TE, is a decisive factor for their
chondrogenic differentiation [38]. At the same time, in the case of cartilage repair, it was
indicated that during the initial implantation stages, spheroids will likely require surgical
unloading or additional support to protect the immature scaffold-free tissue construct from
mechanical damage [25]. The synergetic TE strategy provides an elegant solution to this
problem.

A report by Schon and colleagues investigated the possibility to place such predifferentiated
cartilage spheroids into the vertical pores of a 3D-plotted scaffold [39]. The spheroids fused to
produce a coherently cellularized hybrid 3D construct after 21 days in dynamic culture. The
manual placement and assembly of spheroids was indicated as one of the main drawbacks and
rate-limiting steps of the process. Later, the same approach was used to combine scaffolds
with spheroids produced from cells derived from superficial and deep zones of the cartilage in a
spatially defined manner (Figure 3E). Although the zonal cell distribution pattern was not
preserved after an extended in vitro culture, the incorporation of glycosaminoglycans was
substantially improved in scaffolds compared to spheroid/micromass pellet culture alone [40].
A very recent study took advantage of mesenchymal condensation-driven chondrogenesis by
placing micromass pellets within scaffolds woven from collagen fibers (Figure 3D) [41]. The
scaffold geometry allowed only limited spheroid fusion, so this work does not fully comply with
the synergetic strategy definition. However, the authors acknowledged that lateral spheroid
fusion would be advantageous and proposed improved scaffold geometry for future
experiments.

Most methods for spheroid formation lose no cells or very few cells, which is especially
appealing for cells with limited availability, as in the case of primary cells. Therefore, combining
spheroids with 3D scaffolds results in close to 100% seeding efficiency [39,40]. The important
difference with the microscaffold-based synergetic approach is that spheroids are not formed
directly within the scaffolds, but are instead produced separately and then combined with it.
The essential common attribute is that the fusion of spheroids is not being compromised
despite the use of (micro)scaffolds, placing both cases in the domain of the synergetic strategy
in TE (Box 2).

Box 2. Synergy Is in the Air

The search for alternative approaches capable of overcoming the limitations of the prevailing TE strategies is well
documented. Multiple comments have called for combining different techniques to achieve synergetic approaches
showing added value. Discussing various scaffold fabrication techniques, Billiet and colleagues [58] noted ‘The
combined effect of different techniques will most likely exhibit positive cooperative effects. Thus, instead of focusing
on the exploitation of one single technique, it would be most fruitful to combine the positive effects of different
techniques into one operation procedure.’

More recently, Derby [59] predicted that ‘a hybrid approach intermediate to the scaffold-directed approach (seeded
cells) and scaffold-free approach (organized cellular subcomponents) is likely to be developed’.

Nichol and Khademhosseini [60] discussed modular, bottom–up TE as a promising approach for creating complex
tissues. One of the major challenges they identified was the possibility of creating tissue constructs robust enough
mechanically to withstand clinical implantation and interact properly with the native tissues.

Kachouie and colleagues [61] voiced an opinion in support of directed tissue assembly, anticipating that ‘there will be
increased integration of directed assembly-based approach with conventional scaffold-based approach to create more
complex functional tissues with physiological architecture for clinical applications’.

354 Trends in Biotechnology, April 2018, Vol. 36, No. 4

Translational Prospects of the Synergetic Strategy Outstanding Questions
Despite the enormous amount of new knowledge and undisputable progress achieved during What methods will be used to most
the past few decades, TE and regenerative medicine still face plenty of challenges. Clinical efficiently combine spheroids and
robust scaffolds, often produced by
translation encounters considerable regulatory and reimbursement hurdles, strongly correlat- processes using elevated tempera-
ing with the size of the potential patient market. To achieve disruptive breakthroughs in TE, tures, such as fused deposition
novel unconventional strategies have to be pursued. The emerging synergetic strategy, modeling?
conceptualized in this opinion article, allows circumventing the major drawbacks of current
What is the optimal spheroid precul-
TE methods, while simultaneously fostering their advantages (Figure 1). Therefore, we expect
ture time before they are combined
that it can strongly boost the progress in TE and help to develop relevant clinical solutions. with the 3D scaffold and allowed to
fuse? How does it vary for different cell
In particular, the bottom–up approach based on the use of microscaffolds enables rapid or scaffold types?
bottom–up assembly of tissue constructs. Such a modular process is highly advantageous
Currently, 2PP technology, used for
with respect to scalability [25]. Furthermore, we speculate that by varying the microscaffold
producing microscaffolds, aligns the
properties, the system can be optimized for different cell and tissue types, without substantial third strategy of TE with the field of
alteration in the TE construct assembly procedure. The dimensions of microscaffold permit their bottom–up modular tissue self-assem-
use with minimally invasive arthroscopic procedures, already well established in orthopedic bly. What other technologies could be
used to create such microscaffolds?
surgery. The synergetic strategy in general can also be used for bioprinting and robotic
assembly, where individual elements are positioned in accordance to a predefined blueprint.
This specific case would fall under the definition of bioassembly, as discussed in recent
publications on biofabrication terminology [42,43].

However, implementing this emerging synergetic TE strategy is not without challenges. The
2PP technology, currently used for 3D printing of microscaffolds, is inherently slow due its high
spatial resolution. We estimate that even with an advanced 2PP printer developed at TU Wien
(Vienna, Austria), which is around 100 times faster than any commercially available device, the
time required to produce enough microscaffolds to fill a 5-mm deep cylinder with a diameter of
3 mm would exceed 37 h. This is a significant, but not a decisive bottleneck, because the
microscaffolds can be produced in advance and stored until use. A more important issue is the
availability of biodegradable materials compatible with 2PP technique and supporting the
required level of spatial resolution. All microscaffolds reported so far were produced from
nonbiodegradable materials. However, recently reported biomaterials developed for 2PP could
be suitable for this purpose [44–46].

Currently, there is no evidence that higher-throughput technologies, such as microfluidics,

emulsion templating, or self-folding, can produce microscaffolds suitable for the synergetic TE
strategy [31,47–49]. However, over the long term, these methods might be developed to mass-
produce robust scaffolds capable of hosting spheroids and not compromising their fusion.

As we describe here, bottom–up assembly of microscaffolds is not a mandatory attribute of the

synergetic TE strategy. Spheroids can also be combined with 3D biodegradable scaffolds
produced by conventional methods. Manual placement of spheroids into the scaffold pores
limits the throughput of such procedure. However, this issue is already solvable with currently
available technologies, since several recent reports utilized various robotic systems capable of
automated manipulation and placement of spheroids [24,50,51]. Combining such systems with
a 3D printer producing robust macroporous scaffolds is straightforward.

Concluding Remarks and Future Perspectives

The field of TE continues to evolve and diversify (see Outstanding Questions). The classic
scaffold-based approach is becoming more sophisticated, and scaffolds have gradually trans-
formed from temporal supporting structures into so-called instructive and interactive ‘living

Trends in Biotechnology, April 2018, Vol. 36, No. 4 355

biomaterials’ [52]. Scaffold-free TE strategies are also rapidly progressing, and modern micro-
fluidics technologies enable the biofabrication of more complex types of tissue spheroids [53].
Recent technological advances can substantially accelerate spheroid formation by using
acoustic tweezers [54]. Another potential direction is functionalizing scaffolds and tissue
spheroids with ECM molecules, enzymes, and growth factors [55]. Coating 3D scaffolds with
a component of the bacterial cell wall can induce and control a macrophage response and help
to vascularize subcutaneously implanted TE constructs [56].

Incorporating magnetic nanoparticles opens new opportunities to manipulate spheroids and

microscaffolds in bottom–up tissue assembly [47,57]. We envision that the synergetic TE
strategy will enable rapid in situ tissue assembly, benefiting from the development of new
materials and designing new specific types of microscaffolds encaging tissue spheroids. In the
short term, one of the most realistic implementations of this synergetic strategy is cartilage and
bone TE using minimally invasive procedures.

Integrating 3D printing of scaffolds with robotic deposition of spheroids will create more
complex mechanically anisotropic TE constructs and precise placement of different cell types
in 3D. In conclusion, the rapidly emerging synergetic third strategy, integrating scaffold-based
and scaffold-free approaches, represents an exciting, truly convergent research direction with
strong potential for enabling disruptive solutions and advancing the fields of TE and regenera-
tive medicine.

Acknowledgments
A.O. would like to acknowledge the financial support of the European Research Council (Starting Grant 307701) and
Austrian Science Fund (FWF, Proj. Number I2444-N28).

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