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Moldovan NI, et al.
combined with fibrillar meshes, would be “scaffold-free,” where main (yet small) blood vessels can be printed into
should not be entertained. tissue constructs, to further allow sprouting from and to
capillary systems. Cleary, the fourth dimension comes
1.1.3 Scaffold-in-scaffold: Integrated or Composite 3D into play, namely the time-component of maturation.
Bioprinting
When the hydrogels alone do not attain enough strength 2. Tissue Assembly
by polymerization, another option is to print harder or In this section, we describe 3D tissue engineering methods
stiffer scaffolds as cage-like system first and then to fill that do not make overt use of the ink and paper analogy.
them with cell-laden bioinks on hydrogel-basis [28] . For In most cases, the methods of this type could be called
this, the terms of “composite” , “integrated” or even “scaffold-free,” indicating a version of biofabrication where
[29]
[30]
“hybrid” bioprinting have been coined. only cells are used, and the needed matrix is produced by
[31]
the cells themselves. With regard to the configuration of
1.1.4 Advantages and Limitations of Hydrogels-based the cells in the initial assembly step, this approach comes
Bioprinting in two variants: A planar and a spherical mode.
Although only a few decades old, the concept of
bioprinting and the instruments operated on this principle 2.1 Planar Biofabrication
inspired from additive manufacturing have progressed This mode works with cell sheets that have a preferred two-
impressively and already generated convincing proofs of dimensional distribution. In essence, this biofabrication
concept. Responding to a large societal interest, a market method is a lamination approach and hinges on the
emerged for commercialization of a variety of bioprinting adhesion of cells to a substrate and the ECM produced
materials and instruments. However, with bioink-based by them. Thanks to a thermosensitive polymer (NiPAAM
bioprinting, it is not trivial to satisfy simultaneously the and pNIPAAM) as a support structure, a reduction of
requirement of both printability and biocompatibility . temperature below standard cultivation at 37°C will lead
[22]
Essentially, bioinks are soft biomaterials, which need to to a repulsion of the cell layer from the polymer, leading
permit extrusion or jetting, yet are required to maintain a to the detachment of the cell culture as a contiguous cell
printed shape. This is achieved by employing thixotropic sheet . Several cell sheets can be stacked, thus resulting
[32]
gels that show shear-thinning during an extrusion process in a multilayered tissue [33] . The approach, originally
and regain larger stiffness immediately after deposition proposed by Michael Sefton for drug delivery purposes,
or a by the post-printing chemical crosslinking process. later developed as a tissue engineering mode and was
[34]
These bioink modifications have consequences for driven forward by Owaki et al. .
[35]
cellular viability and proliferative and migratory behavior.
Cell damage and post-printing dysfunctionality may 2.2 The Spheroidal Mode of Cell Assembling
occur for a variety of reasons, such as high shear stress,
lack of growth factors or suitable ECM, or limitation of 2.2.1 Make and Cast
intercellular communication . Despite promising results
[9]
in this direction, an ideal bioink is yet to be found. Taking The 3D nature of tissues’ architecture is well captured
into consideration that there are so many different cell by employing cell spheroids. Frequently referred to also
types in the body, all with their refined needs regarding as “microtissues” [36] they are increasingly preferred to
the microenvironment, it appears that a variety of suitable isolated cells to improve the efficacy of cell therapy ,
[37]
bioinks still have to be developed to address the needs of or particularly as tumor models (oncospheres) [38] .
different cell types. In addition, the constructs obtained When these spheroids are grown from stem cells, they
by the current bioprinting techniques display a simplistic are called “organoids” (or “embryoid bodies” in the
cellular architecture. For example, although pre-vascular case of embryonic or induced pluripotent stem cells),
tubes could be embedded in the structural bioink as representing a promising new development in tissue
“sacrificial” hydrogels [28] , their patterns lack the fractal engineering . Alternatively, cell aggregates can be also
[39]
organization of natural microvascular networks. prepared as cell cords, for example, for further processing
In summary, this approach relies on hydrogels as into spheroids .
[40]
primary shape generators and cell carriers. While it A completely different bioprinting approach is based
remains promising for large, cell-homogenous, matrix- on the notion that the cell clusters forming spheroids
rich tissues, representing mostly the skeletal-muscular merge when brought into contact with each other .The
[41]
system, it is still struggling to solve its hard to conciliate fusion of spheroids is based on the same principles that
requirements, related to the hydrogels printability on govern their formation in the first place: Minimization of
one side, and cellular needs on the other. More work a system’s potential energy, generated by the adhesive
will need to be invested into self-organizing systems, interactions between cells, through intercellular adhesion

International Journal of Bioprinting (2019)–Volume 5, Issue 1 3