Recently, specialized applications of 3D printing have extended into the biological
realm. As the field is so new, related activities are being approached from groups
with disparate specialties and backgrounds. Therefore, few firm, unambiguous and universally
understood definitions have been established. Many have local and connotative meanings,
and most have both unique and overlapping aspects to their evolving applications.
Also, questions arise regarding the distinction between some of the almost homonymous
terms – and even such confusion as to the meaning of the prefix ‘bio’ in terms. For
example, some may think the difference between 3D biomaterial printing and 3D printing
is a biological nature and/or biocompatibility of materials in the printed object.
While for others, the difference reflects a biomedical application of the object post-printing.
Our intent is to introduce common usages of 3D bioprinting-related terms in the context.
3D printing
3D printing is now a well-established technology and rapidly gaining utility in industry [1,2].
As an implementation of ‘additive manufacturing’ and ‘direct digital manufacturing’
it refers to a particular means of product fabrication; specifically, processes where
successive layers or rows of material are deposited under computer control to directly
create a 3D object. There are a variety of mechanisms for this controlled deposition
fabricating the object directly or via a printed mold from which the final product
is cast.
4D printing
In 4D printing, the fourth, or extra, dimension is typically time-dependent. Products
here are a special case of ‘self-actuating materials’ [3–5] providing a smart, environmentally
responsive change in structure or functionality that is engineered and self-actuated.
Thus, an intrinsic feature of a 3D-printed material leads to subsequent, progressive
changes in, for example, the shape or topology of the printed object. Careful definition
of the post-printing change is required, as for example, dropping a printed object
on the floor may cause a change in its shape, but it is neither designed nor self-originated.
These changes in shape or size or functionality are usually influenced by such inputs
as heat, light, humidity or air pressure.
Medical 3D printing
One exciting example of this biomedical application of 3D printing is 3D printed pills
– a new dosage form in late stages of development [6]. 3D printed pills provide many
advantages, from improved chemical stability, pill dissolution and medication adsorption
to multiple unit-dosing of disparate medications. They also promise to support pills
as easier to take and allow here-to-for impossible pill design. They may even be customizable
– supporting personalized medicines and therefore more inexpensive, accurate and effective
dosing. An example of ambiguity in terms is that many refer to the action of 3D printing
parts for prosthetic implants as 3D biomaterial printing or biofabrication and as
an implementation of medical 3D printing. Regulatory designations beyond the scope
of this paper include 3D printing toward active implantable medical devices, nonimplantable
medical devices or in vitro diagnostic medical devices. Medical 3D printing has also
been used to refer to patient-specific anatomical models, often derived from patient-specific
image data sets, used to guide surgical strategies. Technically, no living or biological
components are used in printing of a model; typically resins or thermoplastics are
used. However, the medical-related application lends itself to the terms used.
3D biomaterial printing & bioadditive manufacturing
The generation of complex 3D biomedical devices and enhanced scaffolds has driven
the need for 3D printing with biocompatible (or ‘biofunctionalizable’) materials such
as natural and synthetic polymers, polymerizable fluids, ceramics and metals. In this
(most common) usage, it is the product’s compatibility with cells, tissues and humoral
systems that drives the ‘bio’ component of the term. ‘Biocompatible implants’, ‘biopapers’
and other scaffolds require appropriate macro-, micro- and nano-level properties.
While cells are not typically a component of such printed devices, biocompatibility
is required to support subsequent cell-interaction. Examples of these properties include
micro-architecture supporting cell adhesion and matrix integration, as well as heterogeneous
multifold scaffolds providing appropriate release kinetics of loaded biomolecules
enabling synchronization of regenerating tissue [7–9]. In biomaterial applications
some refer to any method of solid free form fabrication toward a biological application
as 3D printing, while others reserve it for liquid binder-based inkjet technology [10,11].
Related expressions in this arena include ‘additive biomanufacturing’ and ‘bio-medical
additive manufacturing’. The 3D biomaterial printing and bioadditive manufacturing
terms are employed by some as 3D bioprinting [12–14].
Biofabrication & biomanufacturing
The general objective is the production of complex biological products from such raw
materials such as (in)organic molecules, extracellular matrices, biomaterials and
living elements (i.e., cells) [15,16]. Historically, manufacturing refers to the formation
of a complete, usable product while fabrication efforts lead to the formation of a
part or sub-assembly used in a more comprehensive manufacturing program. For example,
a company purchases many fabricated parts that are then assembled into a manufactured
car. With these traditional definitions in mind, 3D printing of a biomaterial scaffold
would be referred to as biofabrication while integration of this scaffold with cells
and/or other components would constitute biomanufacturing of the final product. With
living systems, these classical definitions may be blurred as many biological ‘parts’
are contained, functional products. For example, a biological ‘subassembly’, such
as a prevascularized matrix, intended to be used in manufacturing a larger, more complex
and complete tissue product can itself be used as is. However, biomanufacturing may
also refer to processes in which biological systems manufacture a product, such as
operation of a cultured cell system to secrete biomolecular products, an activity
traditionally termed biosynthesis [17]. Regardless, practically speaking, technologies
employed to originate a biological structure, such as molding, casting, stereolithography,
cell seeding and 3D bioprinting reflect fabrication approaches. Meanwhile, approaches
enabling the integration of these fabricated structures into or production of a product
(e.g., pick-and-place, systems engineering) reflect more manufacturing approaches.
It is likely these terms will become more defined as the field evolves [17]. Reflecting
this broader evolution, new 3D bioprinter designs have emerged using a manufacturing
robot as the core fabricating technology [18].
Bioprinting, 2D bioprinting & 3D bioprinting
All three terms refer to biofabrication through the deposition of micro-channels or
-droplets of living cells with or without additional structural materials. The most
common term, 3D bioprinting (3DBP), describes the fabrication of 3D, engineered living
(often cell-based) models, tissues and organs. These are being used for drug discovery,
pharmaceutical and environmental toxicology assays, in vitro models of organism development
and disease, and production of engineered human tissues and organs [5,19]. In 3DBP,
the cell-laden fluids or bioinks are built upon each other and become any number of
very small or rather large biological structures. 2D bioprinting employs related equipment
to organize cells in monolayers for such applications as cell-based assays and models [20].
The actual mechanics of this deposition of cells and matrix vary greatly between bioprinters
and bioprinting applications. But regardless of the printing specifics, these printed
objects composed of cells, biopolymer hydrogels or synthetic matrix materials present
revolutionary promise in research, diagnostics and therapeutics [9]. Many are working
on standardized 3D human tissues for predictive toxicology and preclinical testing
because 3DBP tissues recapitulate many aspects of in vivo tissue architecture and
function. It is believed they will provide many distinct advantages over nonhuman
animal models. The most common bioprinting, ‘scaffold-based bioprinting’, involves
fluids composed of cells, nutrients and (biomedical) matrix materials. ‘Scaffold-free
bioprinting’ [21] involves the manipulation of concentrated cells and their own extracellular
matrix in a ways that exogenous natural or synthetic matrix materials are not required
for immediate structural integrity.
4D bioprinting
As 3DBP is so new, we might expect that a further development, 4D bioprinting (4DBP)
would be in a stage of definition and development. As in 4D printing, we are here
concerned with programmed, anticipated and self-actuated changes occurring well after
the printing operation (Box 1) [3,22].
One example of 4DBP is producing an assembly of micro-droplets of cells in the general
shape of final structure, designed such that they will eventually coalesce and shape-morph
into the final intended structure. Some limit the change, as in 4D printing, to the
construct’s size, shape or organization. Others suggest that the self-actuated changes
in 4D bioprinting might allow developments other than shape-morphing. These other
developments include cellular differentiation, polarization and tissue patterning
as well as matrix evolution or functionality development – all of which reflect the
robust adaptability intrinsic to most biological systems [23]. Importantly, from a
fabrication perspective, 4D bioprinting will undoubtedly entail strategies for constraining
constructed environments in such a way to direct the 4D/emergent behavior/activity/phenotype
to a desired outcome (Box 2).
Biomimetic 4D printing
Related to both 4D printing and 4DBP, biomimetic 4D printing involves predicted shape-morphing
of composite-material printed objects following the dynamic architectures of living
organisms. One example of this is 3D morphology development through post-printing
changes in the printed objects hydration. In this case, the engineers have programmed
lamellar hydrogel-printed structures with restricted, asymmetrical swelling. This
controlled directional swelling of specifically aligned parts of the printed object
determines a final shape after the object absorbs water [24]. The difference between
biomimetic 4D printing and simple 4D printing appears to be the mechanism of 4D change.
In this particular case, the anisotropic hydration initiated warping of the printed
product, mimicking that in a plant.
Bioinks
The fluids that 3D bioprinters deposit have been referred to as bioprinting inks or
bioinks [25] and are corollary to biopaper. They are basically a fluid containing
nutrients and/or matrix components and/or cells. In fact, a critical step in the bioprinting
process involves selection or design of this bioink, as its composition is based upon
the type of printing employed, and the sequence of product construction (Box 3). Several
(biomedical) matrix materials have been specified for scaffold-based bioprinting [26,27].
They range from hydrogels built from such materials as alginate, collagen, fibrin,
gelatin methacrylate, hyaluronic acid and block co-polymers to microcarriers or decellularized
matrices. Some printing techniques present a strong reliance upon the nutrient and
factor components of the bioink.
One ambiguous aspect of the term is that some use it (or variations) to refer to a
cell-free material. Some condition the term for applications which may or may not
contain living cells as ‘cell-encapsulating inks’ or ‘acellular inks’ [8]. Others
do not. A matrix-containing fluid employed in a bioprinting process to simply generate
an empty space or separate other active elements of the object can be referred to
as a ‘sacrificial bioink’ [26].
Conclusion
Many activities related to the 3D printing of a variety of materials for biotechnical
and biomedical applications are now in development or actual use [28]. Many of the
terms employed to identify them and their materials are in a stage of refinement.
From the manufacturing of devices to synthetic polymers destined for biomedical implantation
(biofunctionalizable 3D biomaterials) to complex fluids supporting living cells during
synthetic tissue construction (a bioink), we find specific terms identifying a number
of distinct materials designed for very different 3D bioprinting processes.
Box 1.
4D bioprinting characteristics.
Smart, environmentally responsive biological structure
Composed of various cells and biocompatible matrices
Undergoing designed and self-originated development
Responding post-printing to an environmental change
Box 2.
Proposed types of 4D bioprinting.
Shape change: a smart biopolymer and cells which changes its 3D configuration (or
shape) upon stimulation
Size change: an in vivo printed cell device (e.g., hydrogel) is implanted in the body,
then biological activities leave tissue as its absorbed
Pattern change: cell micro-droplets (± exogenous matrix) printed in a particular pattern,
then stimulated to a pre-envisioned new pattern
Phenotype change: pre-engineered changes in nonstructural but biologically-relevant
characteristics, such as the cellular assembly’s CD marker type or cell polarization
Box 3.
Bioink design properties.
Cell-specific formulation needs
Heightened pH buffering demand
Addressing component 3D gradients
Specifically control or inhibit apoptosis
Support or inhibit further differentiation
Printer-determined hydrodynamic stress
Co-culturing and tissue environment effects
Serum- and xeno-free and protein-free ideal
Address altered cell metabolism rates and flux
High plastic mass-to-medium volume ratio effects
Unique matrix and matrix-active component effects
Active and passive rheology requirements and effects