TIBTEC 1600 No.

of Pages 16

Review

Tissue Engineering 3D Neurovascular Units:

A Biomaterials and Bioprinting Perspective
Geoffrey Potjewyd,1,2,3,@ Samuel Moxon,1,@ Tao Wang,2 Marco Domingos,3 and Nigel M. Hooper1,*,@

Neurovascular dysfunction is a central process in the pathogenesis of stroke Highlights

and most neurodegenerative diseases, including Alzheimer’s disease. The Advances in 3D biomaterials known as
hydrogels, with the structural proper-
multicellular neurovascular unit (NVU) combines the neural, vascular and extra-
ties of a solid and the capability to
cellular matrix (ECM) components in an important interface whose correct contain a high water content (99%),
functioning is critical to maintain brain health. Tissue engineering is now allow for 3D culture of NVU cells.

offering new tools and insights to advance our understanding of NVU function. Development of hydrogels with bio-
Here, we review how the use of novel biomaterials to mimic the mechanical and printable properties, referred to as
functional cues of the ECM, coupled with precisely layered deposition of the bioinks, allows for deposition of
layered 3D NVU models.
different cells of the NVU through 3D bioprinting, is revolutionising the study of
neurovascular function and dysfunction. Biofabrication strategies utilising bio-
printing and bioassembly can be used
to develop 3D NVU models.

Modelling the Neurovascular Unit in Health and Disease NVU models could incorporate chan-
nels seeded with vascular cells to
The neurovascular unit (NVU; see Glossary) is an organised multicellular and multicompo-
mimic the BBB.
nent network that is important to brain health [1]. This brain-localised unit is composed of neural
and vascular components, with the interface and interactions between these components Attachment of fluidic pumps to a vas-
being crucial to regulation of cerebral blood flow through neurovascular coupling, blood–brain cular channel can aid functionalisation
of endothelial cells and the BBB as a
barrier (BBB) function, neuroinflammation and neuronal function [1–3]. Damage to the NVU
whole.
can lead to restricted transport of oxygen and nutrients to the brain, impaired ability to clear
toxic compounds from the brain and/or allow for the infiltration of harmful molecules and
immune cells across the BBB [4], resulting in both onset and progression of neurovascular
dysfunction and neurodegeneration (Figure 1). The importance of vascular contributions to a
range of brain diseases, including Alzheimer’s disease, vascular dementia, Parkinson’s
disease and stroke, is becoming increasingly recognised [5,6]. 1
Division of Neuroscience and
Experimental Psychology, School of
Biological Sciences, Faculty of
The neural component of the NVU consists of neurons and glial cells (microglia, astrocytes, Biology, Medicine and Health, The
oligodendrocytes), while the vascular component consists of endothelial cells, pericytes and University of Manchester, Manchester
Academic Health Science Centre,
vascular smooth muscle cells (Figure 1A). The vascular component forms the BBB, a critical
Manchester M13 9PL, UK
interface enabling selective permeability of molecules between the brain and systemic blood 2
Division of Evolution and Genomic
circulation. Specialised junctional protein complexes between adjacent endothelial cells Sciences, School of Medical Sciences,
Faculty of Biology, Medicine and
help to maintain the physical and functional integrity of the BBB, tightly regulating permeability
Health, The University of Manchester,
through the BBB and transferring extracellular mechanical signals to the surrounding cells Manchester M13 9PL, UK
3
(Figure 1A). Another critical component of the NVU is the extracellular matrix (ECM), a Manchester Institute of
Biotechnology, The University of
complex, acellular structure that provides a physical support for the various cells, as well as
Manchester, Manchester M1 7DN, UK
biochemical and mechanical cues that influence their behaviour. These biochemical and @
Twitters: @Geoff3P, @DrSamMoxon,
physical interactions between neural and vascular NVU components provide the necessary @HooperLabManc
stimuli to aid neurogenesis, angiogenesis and maintenance of brain homeostasis, while the
*Correspondence:
ECM provides structural support and a platform for cellular migration and interaction. More
Nigel.Hooper@manchester.ac.uk
information regarding the NVU ECM is provided in Box 1. (N.M. Hooper).

Trends in Biotechnology, Month Year, Vol. xx, No. yy https://doi.org/10.1016/j.tibtech.2018.01.003 1

Crown Copyright © 2018 Published by Elsevier Ltd. All rights reserved.
 TIBTEC 1600 No. of Pages 16

Glossary
(A) (B) Alzheimer’s disease: a type of
dementia characterised by deposition
in the brain of tau protein and
amyloid-b peptide, which leads to
progressive neurodegeneration.
Angiogenesis: the formation of new
vasculature from pre-existing vessels.
Basement membrane: the
extracellular matrix component that
surrounds the vasculature and is
degraded during neurovascular unit
dysfunction.
Bioassembly: the self-assembly of
biological constructs following
automated distribution of cell-laden
units to form a construct.
Biofabrication: in the context of
tissue engineering and regenerative
medicine – automated manufacture
of tissues and organs.
Pericyte Oligodendrocyte
Endothelial cell Bioink: a biomaterial that can be
combined with cells or biological
JuncƟonal protein Astrocyte Neuron with matter and subsequently be 3D
complex with endfoot myelinated axon bioprinted into a defined structure.
Biopolymer: a polymeric unit used
Basal lamina/ AcƟvated
basement membrane Microglia for biological applications like the
microglia development of hydrogels and
bioinks.
Bioprinting: the additive
manufacture of tissue and organs
Figure 1. The Neurovascular Unit (NVU) in Health and Disease. The NVU is composed of neural (neurons,
through layered deposition of
microglia, astrocytes, oligodendrocytes) and vascular (endothelial cells, pericytes, smooth muscle cells) components,
biological material and cells ensuring
which interact to regulate cerebral blood flow. (A) In a healthy NVU, endothelial cells, pericytes, astrocytes and the
precise spatiotemporal distribution of
basement membrane form the blood–brain barrier (BBB), with myelinated neurons and glial cells surrounding the BBB in
cell types, thereby building a 3D
the brain extracellular matrix. Pericytes and astrocyte endfeet maintain contact with the basement membrane, with
construct.
endothelial junctional protein complexes intact and functional. (B) In a dysfunctional NVU, junctional protein complexes and
Blood–brain barrier (BBB): a
the basement membrane degrade, in addition to induction of a proinflammatory-activated microglial and astrocytic
cellular barrier of endothelial, smooth
response, leading to demyelination and death of neurons. This dysfunction contributes to neurovascular damage and
muscle and pericyte cells which form
neurodegeneration.
the brain capillaries and arterioles
that separate the brain tissue from
In the aforementioned diseases, the integrity of the NVU is compromised. Degradation of the blood.
junction protein complexes and the basement membrane [7] allows infiltration of systemic Extracellular matrix (ECM): tissue
component surrounding cells and
erythrocytes, leukocytes and antibodies into the brain and the subsequent induction of a
providing biomechanical and
proinflammatory-activated microglial and astrocyte response [2] (Figure 1B). The proinflam- biochemical cues.
matory response causes further BBB damage and neuron death as a result of demyelination of Hydrogel matrix: a highly water-
axons and direct damage to neurons (Figure 1B). This destruction of the NVU contributes to the saturated (99%) 3D matrix in which
cells can be encapsulated within.
neurodegeneration evident in Alzheimer’s disease and other forms of dementia [8,9]. Junctional protein complex:
intercellular protein junctions that
Thus, modelling NVU function and dysfunction is critical to understanding normal physiology, control selective permeability across
as well as revealing the molecular and cellular mechanisms underlying a range of neurovascular the BBB and include tight junctions,
adherens junctions and gap
and neurodegenerative diseases. Accurately replicating the interactions between the different junctions.
NVU cell types and ECM components within the brain is important to ensure the effectiveness Mechanotransduction: translation
of an NVU model. Despite the advances in brain imaging and the availability of animal models, of extracellular mechanical cues
through the cytoskeleton to induce
there is an urgent need to develop human cell-based in vitro models of the NVU to provide new
changes within the cell.
insight into neurovascular function, and its dysfunction in disease. Current cell-based models of Neural component: the neural cells
the NVU commonly integrate two cell types separated by a porous physical layer, such as a of the brain, including neurons and
transwell filter. Such models, however, fail to replicate the 3D complexity of the NVU.

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 TIBTEC 1600 No. of Pages 16

Box 1. The Neurovascular Unit Extracellular Matrix glial cells (microglia, astrocytes and
The ECM is a complex yet well-organised network of molecules and proteins, which provides a dynamic platform for oligodendrocytes).
cell-to-cell signalling and enables the formation and remodelling of tissue. The neurovascular-specific ECM is diverse, Neurodegeneration: the
with distinct differences in both the neural cell ECM and the vascular ECM, both of which can be altered structurally by degeneration of neurons caused by a
proteases that degrade certain ECM components [89]. Receptors on the surface of cells interact with the ECM, which variety of pathological influences (e.
leads to regulation of cytoskeletal coupling and allows cell migration [90]. For example, integrin receptors attach to the g., hypoxia, oxidative stress).
actin cytoskeleton, promoting changes in the ECM mechanical environment that affect gene expression and activate Neurogenesis: the growth of new
multiple signalling pathways through mechanotransduction [91]. neurons from neural stem cells.
Neurovascular unit (NVU): the
Brain ECM interface between the neural and
vascular components of the brain,
crucial for neurovascular coupling
A relatively large proportion (17–20%) of the brain consists of ECM [92]. The brain ECM has an architecture distinct from
and delivery of key nutrients and
that of systemic tissues, with fibronectin and type I collagen being practically absent, and an abundant expression of
oxygen from the circulatory system.
proteoglycans and type IV collagen, with a high expression of lecticans [93,94]. In addition, the brain ECM has a different
Rheology: the science of flow –
composition of growth factors, receptors and proteases [95]. The brain is an extremely soft organ with a low elastic
study of flow of a material in
modulus (0.1–1 kPa), reflected by the low proportion of type I collagen [30,91].
response to stress allowing for
determination of mechanical
Vascular Basement Membrane properties.
Stroke: ischemic or haemorrhagic
A thin fibrous segment of ECM surrounds the brain vasculature, referred to as the basement membrane. This ECM brain injury from an infarct that
component differs from typical neural tissue, with different protein expression and mechanical characteristics. The blocks cerebral blood flow; often
vascular ECM is primarily composed of collagen type IV and laminin, with fibronectin and proteoglycans integrated in the leads to vascular dementia due to
network [8]. These basement membrane components have been used in vitro to grow endothelial layers with increased the neurovascular damage caused.
functionality compared to those without these components [96,97]. Vascular component: the cells
(endothelial cells, pericytes, smooth
Physicochemical Hydrogel Properties Required for Building a Successful NVU muscle cells) that form the blood
vessels of the brain, the BBB and
the overall vascular microstructure.
The physical properties of the ECM are important when developing a model of the NVU. Studies have shown that
Vascular dementia: a form of
physical stiffness of the extracellular environment is optimal when it mimics that of the native tissue, with neural and
dementia where reduced cerebral
vascular tissues favouring 1 kPa environments [27,29–31]. The physical stiffness and rheological properties of the
blood flow and neurovascular
hydrogel used also affect the capability of a biomaterial to be bioprinted, adding additional variables to the implementa-
dysfunction lead to
tion of a bioink to develop a 3D NVU model.
neurodegeneration.
Vascularisation: the formation of a
capillary network within a model or
A promising area for development of NVU models is their biofabrication through 3D bio- tissue.
printing to produce a multicomponent NVU in which the contribution of the different cell types
to neurovascular function and dysfunction can be studied at the molecular and cellular level. In
this review we discuss (i) how this can be achieved by combining the different cell types with
appropriate biomaterials to mimic the ECM and (ii) the various advantages and disadvantages
of utilising the advances in 3D bioprinting to construct functional 3D NVU models (Figure 2, Key
Figure).

Cell Types Used for NVU Engineering In Vitro

An important parameter when designing an NVU model is the selection of cell components.
Endothelial cells and pericytes are the basic building blocks of the vascular component in the
NVU. The endothelial cells in the brain microvasculature are morphologically, biochemically and
functionally distinct from non-brain endothelial cells. Although they express conventional
adherens junction proteins such as VE-cadherin, the brain endothelial cells are also linked
to each other by tight junctions, which reduce paracellular transport between neighbouring
cells [10]. The tight junctions are formed by interactions between proteins such as occludins,
claudins and junctional adhesion molecules. Pericytes, along with the vascular smooth muscle
cells, are mural cells located directly on the capillary wall. In the brain, pericytes play a vital role
within the NVU by supporting angiogenesis, regulating capillary function and participating in the
formation and maintenance of the BBB [11]. The brain endothelium has significantly higher
pericyte coverage than peripheral tissues [12,13], suggesting a specific brain function for this

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Key Figure
Utilising Different Cells, Biomaterials and Bioprinting to Construct a Multicomponent 3D
Neurovascular Unit (NVU)

Biomaterials
∼1 kPa & shear thinning
Vascular bioinks Neural bioinks

NVU cells
Endothelial Pericytes Astrocytes Neurons
cells

BioprinƟng

MulƟcomponent
3D Neurovascular unit

Figure 2. Different NVU cell types can be combined with specialised biomaterials, known as bioinks, and bioprinted to manufacture multicomponent 3D NVU models.
Neural and vascular cells are encapsulated within bioinks suitable for their viability and growth. Bioprinting allows for precise positioning of the neural and vascular cells
to form appropriate interactive interfaces that mimic the in vivo situation.

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cell type. Recently, pericyte degeneration was shown to lead to neurovascular uncoupling and
neurodegenerative changes [14]. The microvasculature creates a perivascular niche, giving
neural stem cells the permissive cues for neurogenesis [15]. Factors secreted from endothelial
cells, such as vascular endothelial growth factor and chemokines, support neural stem cell
expansion and differentiation as well as neural recruitment and migration [15,16]. In the NVU,
astrocytes are the key cell type that mediate neurovascular coupling [17]. Astrocytes are the
most abundant glial cells in the brain. They possess projections called astrocytic endfeet
through which they interact with both endothelial cells and synapses on neurons, physically
linking neighbouring neurons with their surrounding capillaries, sensing changes in the neuro-
environment and adjusting microvascular function accordingly. Astrocytic endfeet cover about
99% of the abluminal surface of cerebral vessels critically involved in autoregulation of cerebral
blood flow by responding to local vasoactive factors [17]. Immune cells, such as microglia,
although not a structural component of the BBB, are often included in the NVU as they influence
barrier function in response to injury and disease [10] and are of particular relevance to
modelling the NVU in stroke and neurodegenerative diseases where inflammation plays a
major role in the initiation and/or progression of neurodegeneration [18].

Many different factors dictate the source of these cells for use in NVU models. These include
availability, cost, ease of use and the ability to replicate the (patho)physiology required. To date
the major sources of cells used for NVU research are immortalised cell lines (e.g., mouse brain
microvascular endothelial bEnd.3 cells or human neuroblastoma SH-SY5Y cells) and primary
cells obtained from rodent brain (e.g., neurons) or human umbilical vein (e.g., endothelial cells)
[10]. Immortalised cell lines offer advantages including fast growth, robustness, ability to be
passaged multiple times and a relatively low cost to acquire and maintain, and are extremely
useful for initial optimisation of a 3D NVU model [19]. However, immortalised cells often yield
lower outputs in functional tests and do not always express the same transporters and tight
junction proteins as in vivo and generally exhibit poor barrier function [19,20]. In BBB models,
primary cells have been shown to have higher functional output results than immortalised cell
lines [21] and may better reflect the in vivo situation. However, primary cells are difficult to purify,
may grow slowly and/or lose their phenotype in culture. Species differences also may impact on
an NVU model if, for example, human endothelial cells are combined with rodent neurons.

The availability of human induced-pluripotent stem cells (iPSCs) is now revolutionising our
approach to NVU models, allowing the opportunity to use previously unattainable human cells.
Advances in stem cell technology have made it possible to generate iPSCs from adults and
differentiate them into the various cell types (endothelial cells, pericytes, astrocytes, neurons
and microglia) that form the NVU [22–24]. The availability of disease-specific iPSCs carrying the
exact DNA mutation and other genetic information present in the patient donor without
overexpressing any mutant gene products allows the effect of the disease mutation on
NVU structure and function to be investigated in precise detail. Isogenic controls can be
generated in which the disease-specific mutation is changed to the wild-type sequence in a
particular gene (or vice versa where the wild-type sequence is changed to the disease
mutation), allowing for the effect of mutations within individual cell types to be assessed on
NVU function [20]. Indeed, the potential utility of iPSC cell models engineered to mimic the NVU
was recently identified as a key new research tool for studying neurovascular dysfunction [25].

Biofabrication of a tissue-engineered 3D NVU model, utilising hydrogel-based bioinks with

specific spatial distribution of the different cell types and with different combinations of the
normal and diseased NVU cells, will allow precise dissection of the contribution of each cell type
to the disease-specific dysfunction, such as neurovascular miscoupling, abnormal expression

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patterns and/or secretions of trophic factors from different cell types, as well as facilitating high-
throughput drug screening.

Biomaterials for NVU Scaffolds: Bioinks and Hydrogel Matrices

A bioink is a pregelled biomaterial, commonly with cells encapsulated, which can be bioprinted
and successfully gelled to form a solid construct. As the majority of bioinks suitable for NVU
tissue engineering are hydrogel based, unless otherwise stated this review will refer to hydrogel-
based bioinks, and the pregelation formulation of hydrogels, as a bioink. There are many
different biopolymers that can be developed into a bioink to form a hydrogel matrix for cells.
These range from natural proteins and polysaccharides, which are either constituents of the
ECM or mimic the native ECM properties, to synthetic biopolymers and peptides, which can be
tuned to mimic the native NVU ECM. Ideally the hydrogel matrix will allow cells to synthesise and
deposit their own native ECM, thus potentially replicating the physiological roles of the native
tissue ECM [26]. A list of natural and synthetic biopolymer-based bioinks that have suitable
properties for tissue engineering an NVU and have been utilised for bioprinting or 3D culture of
neural or vascular cells previously is provided in Table 1, along with a description of their
properties in relation to neurovascular cell culture and bioprinting.

For an NVU model the bioink should be able to facilitate cellular migration and adhesion,
vascularisation/angiogenesis and neurogenesis, as well as interactions between vascular
and neural interfaces. Physical properties (determined by rheology) are also extremely impor-
tant in dictating the effectiveness of the bioink for developing a hierarchal NVU structure, as well
as providing the appropriate physical and mechanical cues required for the NVU cells. When
developing a 3D-bioprinted NVU, rheological parameters need to be balanced with the
functional output required from the model. For example, bioinks with a higher yield stress
(and typically a higher stiffness) tend to print with better resolution than those with lower
viscosities, yet the NVU exists within a soft in vivo environment of 1 kPa (Box 1) [27–31].
Bioprinting and gelation in a layered manner provide the opportunity for tailoring the interfacial
interaction between different vascular and neural components in the NVU. Although a faster
gelation rate correlates positively to the definition and resolution of a printed bioink, it is inversely
correlated with angiogenesis and neurogenesis [32–35]. The key architectural properties for
NVU-appropriate bioinks can be divided into four subcategories: crosslinking, mechanical,
porosity and cell adhesion.

Crosslinking
The transition from solution to gel requires intermolecular associations through either physical
or chemical crosslinking. Physical crosslinking occurs through noncovalent interactions
between biopolymer chains [36,37], whereas chemical crosslinking requires the covalent
crosslinking between polymer chains to induce a solution to gel transition [26,28,38–41]. For
NVU modelling it is important to consider the potential issues caused by the crosslinking
strategy, with a risk from chemical crosslinking being cellular overexposure to the free radicals
produced in the redox reactions required to facilitate gelation [42,43].

Mechanical Properties
Although the brain and NVU tissue is exposed to a relatively small amount of external physical
stress – unlike bone, for example – there is a crucial role for mechanical stressors within the
NVU for translating extracellular signals into intracellular transcriptional responses, referred to
as mechanotransduction [44]. Blood flow and the surrounding ECM provide shear stress and
mechanical cues to the NVU, which regulate the expression of key junction proteins and
promote functional NVU cellular phenotypes [45]. These physical and mechanical stimuli can be

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Table 1. Bioinks Suitable for 3D Bioprinting Tissue-Engineered Neurovascular Units.
Bioink (Source) Gelation mechanism Reported elastic Cell-adhesion domains Printing conditions Advantages for neurovascu- Refs
moduli (kPa) lar tissue engineering

Collagen (natural; Thermal; pH 0.1–0.8 Inherent Commonly combined with Cell-mediated remodelling [34,41,58,98,99]
protein) other materials or through collagenases
crosslinkers to print

Gelatin (natural; protein) Thermal; photo-crosslinking 0.1–1 Inherent Prints close to gelation As with collagen; Caveat: [28,41,47]
temperature requires modification for
[269_TD$IF]37 C cell culture

Fibrin (natural; protein) Fibrinogen crosslinking with 0.3–4 Inherent Combines with other material Widely used for neural tissue [60,99–103]
thrombin to print engineering

Gellan gum (natural; Thermal; ionotropic; photo- 0.1–186 Requires chemical Prints with crosslinker; prints Tuneable; NVU-promoting [28,64,104,105]
polysaccharide) crosslinking modification or combining into support matrix cell adhesion domains
with other material chemically added

Hyaluronan (natural; Photo-crosslinking 0.01–3.5 Inherent Combines with other material Promotes cell migration, [24,60,106–108]
polysaccharide) to print or printed under UV proliferation, angiogenesis
exposure and neurogenesis

Self-assembling peptide Self-assembly 1 to >50 Requires chemical No modification required Tuneability; customisable [36,56,67–70,84,109]
(SAP) (synthetic) modification or combining bioprinting and cell adhesion
with other material domains

Elastinlike polypeptide Self-assembly; photo- 1 to >50 Requires chemical If not self-assembling, As with SAP; similar physical [38,85,109–111]
(synthetic) crosslinking; amine-reactive modification or combining requires combination or properties to vascular
crosslinking with other material printing with crosslinker basement membrane

Polyethylene glycol Photo-crosslinking; thermal; 0.9–132 Requires chemical No modification required As with SAP; well- [65,112–115]
(synthetic) ionotropic modification or combining documented usage
with other material
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replicated in vitro by selecting bioinks that have similar physical and architectural characteristics
to the native NVU ECM, and by introducing shear stress through flow-based bioreactor
platforms, exemplified in the following studies [46–50]. The combination of bioprinting to attain
specific NVU spatiotemporal distributions and the stimuli introduced through the material can
promote bioassembly of NVU cell types into interactive interfaces between neural and
vascular components.

Porosity
The porosity of a bioink can affect model permeability dramatically, with a low pore size
potentially resulting in poor delivery of nutrients and oxygen to cells within the middle of the
model leading to necrotic regions [51,52]. Pore size is also a crucial parameter for the growth of
neural and vascular cells within an NVU model, with larger pore sizes promoting vascularisation
and neuronal differentiation [40,51].

Cell-Adhesive Domains
Cell adhesion to the surrounding microenvironment – whether it be the ECM or a hydrogel
biopolymer – is important within the NVU, where this interaction allows for mechanotransduc-
tion. Functional adhesion domains are specific amino acid sequences present within the ECM
polymers that allow cells to bind and interact with their surrounding environment through cell
membrane receptors. Two of the most common amino acid sequences present in functional
adhesion domains that are relevant for the NVU are the tripeptide sequence Arg–Gly–Asp
(RGD) and the pentapeptide sequence Ile–Lys–Val–Ala–Val (IKVAV). RGD domains are the key
binding sites for integrins within multiple different ECM proteins [53] and IKVAV domains are
present within laminin glycoproteins and have been shown to promote neurite outgrowth,
synapse stabilisation and BBB performance [54–57]. Such peptide-based adhesion domains
can be synthetically produced and integrated into a polymer chain to provide the intended
physical attachment site. This has been manipulated to produce binding domains present on
native ECM polymers, but has also been used to create biomimetic alternative peptide
sequences to induce a similar effect; for example, the QK-peptide domain sequence is
biomimetic for the receptor-adhesive domain of vascular endothelial growth factor, providing
angiogenic stimuli to cells seeded with hydrogels containing this domain [38]. Bioinks with
relevant physical and biochemical properties for incorporation into NVU models are listed in
Table 1, along with their inherent suitability for cellular adherence or the possibility for adaption
to enable cellular adherence.

Hydrogel Bioinks for Development of NVU Matrices

Natural Polymers
A common strategy to functionalise hydrogels for NVU-bioprinting purposes is to physically
combine two biopolymers (rather than chemically modifying them) to produce a blended
hydrogel-based bioink, with the advantageous properties of each polymer contributing to
the favourable NVU properties required. This technique has been utilised commonly to create
collagen-based bioinks; although collagen has an inherent RGD adhesion domain, collagen
hydrogels typically lack the physical properties to be used as a bioink, with slow gelation times
[58]. Collagen has been used to create mixed hydrogel-based bioinks – with various other
matrices – to successfully bioprint neural and vascular cells, including agarose [34], gelatin
methacrylate [41], Matrigel [59] or alginate [57,59], altering its shear thinning properties to
create a bioink effective for NVU cell culture.

Another strategy is combining more than two polymers to produce a hydrogel with multiple
advantageous properties for tissue engineering an NVU model. An example of this is the

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combination of fibrin, hyaluronan (hyaluronic acid) and laminin to produce a 3D model for both
neural and vascular stem cells [60]. This hydrogel utilised the beneficial properties of fibrin for
neural stem cells, with interpenetrating networks of hyaluronan to aid crosslinking and enhance
the similarities of the gel with the native neural ECM. Hyaluronan biopolymers promote cell
migration and proliferation and – crucial to the development of an NVU model – also promote
angiogenesis and neurogenesis [24,61,62]. The addition of laminin with its own IKVAV
sequence provided the cells with functional adhesive domains. Although the hydrogel was
not optimised for bioprinting, the rheological properties of the hydrogel could be tailored to
produce a bioink and utilise the NVU-favourable components within the hydrogel.

Conversely, some hydrogels are bioprintable without modification or mixing but lack the
inherent cell-adhesive domain necessary for development of effective NVU models. Gellan
gum is an example of a polysaccharide used commonly for producing hydrogels, which when
unmodified does not have the functional domain for cellular adhesion, but can be 3D printed
with a crosslinker to form layered NVU structures [37,63]. The chemical modification of gellan
gum to contain the IKVAV cell-adhesive domain has allowed for the use of the polysaccharide
for bioprinting layered cortical neural models [64].

Synthetic Biopolymers
Synthetic hydrogels enable full control over production and functional parameters, by designing
the exact structural composition and functionalities of the hydrogel to the intended application.
This presents opportunities for developing NVU-suitable bioinks, as the exact bioprinting
properties and cellular-adhesive domains can be designed into the biopolymer structure. A
common class of synthetic biopolymer-based bioinks is self-assembling peptides, which form
ordered nanofibrous b-sheets that replicate the structure of the native ECM [36]. Self-assem-
bling peptides can be designed to assemble quickly and autonomously through physical
crosslinking interactions or, alternatively, designed to be crosslinked through inducing physical
or chemical interactions [38,65]. This biomolecular and cellular self-assembly is commonplace
in vivo and can be replicated in hydrogel constructs, where cell migration through the pores in
the hydrogel enables cell–cell interactions [66]. Because of their synthetic design, bioprintable
properties can be tailored into the synthetic hydrogel to develop a fully functional bioink [67],
and such materials have been utilised for NVU cell studies previously [56,68–70]. Synthetic
peptides can also be developed to replicate different aspects of tissues, with elastinlike
polypeptides being designed to incorporate angiogenic peptide domains for utilisation in
vascular cell 3D culture studies to promote vascular growth [38].

NVU Biofabrication
The NVU is complex, with multiple cells and cell–cell interactions (Figure 3) that present
particular challenges to in vitro modelling. To simplify an NVU model – or to focus on the
vasculature – several models compartmentalise the NVU by developing a BBB-only model.
Other approaches are to use a partial component of the BBB, that is, endothelial cells and
astrocytes/pericytes/vascular smooth muscle cells, with neural cells. These approaches do not
give the effect of modelling a whole NVU but can be very useful to investigate the BBB and
circumvent the difficulty of co-culturing up to five cell types in one model.

Within the context of tissue engineering and regenerative medicine applications, the definition
of biofabrication as a research field has been clarified as ‘the automated generation of
biologically functional products with structural [271_TD$IF]organization from living cells, bioactive mole-
cules, biomaterials, cell aggregates such as micro-tissues, or hybrid cell-material constructs,
through bioprinting or bioassembly and subsequent tissue maturation processes’ [71,72].

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essi ter
o l i fera expr diame ses
p r e i n
Mod
ify rot pillary llagena
on p a
j u nc nesis/c otein co
u c e o ge p r
Ind e angi nc on
iat f ju
Med ssion o
x p re
E

Figure 3. Cell–Cell Interactions in the Neurovascular Unit (NVU). The individual cell types of the NVU interact with
the other cellular components of the NVU through biochemical and physical cues, with both direct and indirect interactions
between neural and vascular components. Adapted, with permission, from [88].

Bioassembly utilises the self-assembling nature of cells to form interactive networks and
tissues, where 3D bioprinting can be utilised to place cells in an appropriate spatiotemporal
location for assembly. This can be used to develop a bioassembled vasculature through
patterned 3D bioprinting [73] or to develop hierarchal structures using a thermoplastic material
as a structural support in-between the layered deposition of bioink–cell formulations [74].
Bioassembly is an important design strategy for developing NVU models, where automated
deposition of cells containing units (in the form of hydrogels) can initiate the bioassembly of
neural–vascular interfaces and cell–cell interactions, which are crucial to the development of
hierarchal NVU models and the production of a BBB with functional phenotype.

There are many different bioprinting techniques, which have been clearly described in other
reviews [72,75]. Two main strategies have been employed for bioprinting NVU models: indirect
additive manufacturing (indirect bioprinting), where an additive-manufactured negative sacrifi-
cial mould is used to enclose and shape biomaterials and cells (Figure 4A,B) and bioplotting
(direct bioprinting), where bioink and cells are printed directly into a 3D structure (Figure 4C,D).
Both of these approaches to bioprinting have the potential to produce multicellular 3D NVU
models, with defined cellular compartments, critical cellular interactions and vasculature.

Bioprinting also offers the potential to introduce channels within the NVU model (Figure 4A,B,D)
– through use of sacrificial biomaterials in direct bioprinting – allowing for enhanced diffusion of
media, nutrients and oxygen throughout the model, mimicking capillary networks within the in

10 Trends in Biotechnology, Month Year, Vol. xx, No. yy

 TIBTEC 1600 No. of Pages 16

vivo NVU and avoiding the development of necrotic regions seen in organoid cultures and some
particularly fibrous hydrogel constructs [76,77]. This channelled network can then be seeded
with endothelial cells to produce a vascular network. By integrating a peristaltic or microfluidic
pump for perfusion, shear stress can be applied to the model, thus creating a bioreactor culture
platform [46–50]. This incorporates bioassembly to the model, with shear stress promoting the
formation of junction proteins and improving the functionality of the BBB and accompanying
NVU cells [45]. This approach also introduces potentially harmful or protective compounds and
drugs to combat NVU dysfunction through a physiologically relevant ‘systemic’ process,
mimicking the introduction of drugs through intravenous or oral administration.

Indirect Bioprinting
There are different approaches to creating NVU models from indirect bioprinting. The two most
common methods include additive manufacture of a ‘master’ mould, in which the subsequent
biocompatible negative mould is created and removed; the negative mould is then seeded with
cell-encapsulated hydrogel, creating a 3D model of the NVU, which can be crosslinked with
another cured hydrogel to create vascular channels within the model (Figure 4A) [47,78]. The
alternative method uses additive manufacture of a sacrificial biomaterial to create a channelled
network into which cell-laden hydrogel is then poured and cured around, enabling extraction of
the sacrificial material to produce channels and without damaging the surrounding NVU
construct (Figure 4B) [77,79].

Indirect bioprinting permits the construction of a 3D hydrogel construct without physically

printing cells, thus avoiding printing shear stress, which has been shown to affect cell viability
[80]. A disadvantage of manufacturing an NVU model in this way arises when there is a need to
deposit multiple cell types with different spatial distributions, as the mechanism requires crude
‘pouring’ of hydrogel rather than specific deposition. A further potential disadvantage to
creating two hydrogel constructs to piece together is that the crosslinking process could
damage encapsulated cells, as chemical or UV crosslinking may have to be employed to join
the precrosslinked individual layers [42,43]. Because of the crude aliquotting of cell-laden
bioinks employed in this method of bioprinting, the integration of neural and vascular compo-
nents is typically through either two biofabricated layers (one containing neural cells and one
containing vascular cells) or through the encapsulation of neural cells within the bioink (set in a
negative mould or around a sacrificial biomaterial), and subsequent seeding of vascular cells
within a fabricated vascular channel.

Direct Bioprinting
Direct bioprinting directly deposits a cell-laden bioink with the option of a sacrificial biomaterial
to create the vasculature (Figure 4C,D) [81]. A further possibility is to bioprint a cell-laden
hydrogel into a structure that incorporates pores into which vascular cells can be seeded,
avoiding the use of sacrificial biomaterials. The direct bioprinting of cell-laden hydrogels and
sacrificial channel network allows for the manufacture of very complex structures, with precise
spatiotemporal cell deposition to ensure the desired cell–cell interaction and neural–vascular
interface are achieved. The use of multiple bioprinting cartridges permits biofabrication of
complex and precise multicellular NVU models through cell-specific bioink encapsulation that
contain appropriate supplements for growth and optimal physical properties (i.e., rheology and
stiffness) to represent the different ECM structures of the NVU [75]. This approach is ideal for
the manufacture of NVU models, where an optimal model requires both neural and vascular
cell-laden ECM, with an interface for cell–cell interactions. Bioprinting also enables the devel-
opment of a reproducible model for comparative experiments between healthy and diseased
cell types.

Trends in Biotechnology, Month Year, Vol. xx, No. yy 11

 TIBTEC 1600 No. of Pages 16

Indirect 3D bioprinƟng
(A)

'Master' Stamp Crosslinking Channel cell

creaƟon creaƟon seeding

(B)

Channel Channel cell

flush seeding

Direct 3D bioprinƟng
(C)
Cells
Vascular Neural
cells cells

Biomaterials
(D) Vascular Neural
hydrogel hydrogel

Channel Sacrificial
flush biomaterial
Channel cell
Cross secƟon
seeding
(E) Self-healing
Crosslinking and fluid gell
removal of
self healing fluid gel

Figure 4. Bioprinting Strategies for Developing a 3D Neurovascular Unit (NVU). The combination of cells and
biomaterials enables the production of 3D NVU models with hydrogels and hydrogel-based bioinks encapsulating the cells
within a 3D environment. Hydrogels with physical properties suited to vascular and neural cells can be used to create
models with direct interface interaction. (A) Indirect bioprinting of a stamp from a ‘master’ mould allows for cell-laden
hydrogels to be poured into the mould to create a construct. The hydrogel can then be crosslinked through photoinitiation
or chemical reticulation (network formation) and two layers can be further crosslinked to create a channel between the two
manufactured hydrogels. Additional vascular cell seeding and a flow-based microfluidic pump can be incorporated. (B)
Bioprinting of a vascular network from a sacrificial biomaterial enables cell-laden hydrogel to be poured around the
sacrificial element, which is subsequently flushed away to leave the channel free for vascular cell seeding and introduction
of a microfluidic pump. (C) Vascular and neural cells can be encapsulated within a vascular and a neural bioink,

(See figure legend on the bottom of the next page.)

12 Trends in Biotechnology, Month Year, Vol. xx, No. yy

 TIBTEC 1600 No. of Pages 16

A common issue with direct bioprinting of NVU models is the difficulty achieving a defined Outstanding Questions
resolution from bioinks postprinting. This is why the development and optimisation of bioinks Can the complex interactions between
with high shear thinning properties to create clearly defined postprint resolution is an important multiple cell types in the NVU be faith-
fully reproduced in 3D models?
step in designing a 3D NVU model. A method to circumvent this issue and enable the
bioprintability of previously nonprintable hydrogels is through the incorporation of a suspended Will novel biomaterials replicate the
manufacture technique, which allows for the controlled gelation of hydrogels over time, rather mechanical and functional cues of
than relying upon bioinks with high shear thinning properties and fast crosslinking rates. This the natural ECM?
technique utilises the viscoelasticity and shear thinning properties of a self-healing fluid gel to
Will automated biofabrication enable
support the structure of gels during deposition, avoiding gel flow and enabling time for
high-throughput manufacture of 3D
crosslinking to occur, after which the fluid gel can be removed, leaving a solid crosslinked NVU models?
3D hydrogel construct (Figure 4E) [82].
Will 3D NVU models improve our
Outcome Measures for 3D NVU Models understanding of neurovascular
function?
To assess the functionality of 3D NVU models a range of outcomes can be measured. BBB
function can be measured by trans-endothelial electrical resistance (TEER), which uses a
How can the use of 3D NVU models
current between two electrodes as a measure of BBB permeability, or by movement of a help the discovery of new therapeutic
coloured or fluorescent dye (e.g., fluorescein isothiocyanate–dextran) [10,50,83]. Although and diagnostic agents for Alzheimer’s
TEER values measured in vivo in rat brain range between 1200 and 1900 V cm2[270_TD$IF], in cultured disease and other dementias with a
vascular component?
cells the permeability barrier is considered effective when the TEER value is above a threshold
(typically 250 V cm2) [10]. The integrity of cell–cell interactions can be measured by immu-
Will ‘humanized’ 3D NVU models
nofluorescent microscopy using antibodies against specific proteins, such as against VE- accelerate the translation of neurovas-
cadherin for endothelial cell adherens junctions; claudin, occludin and junctional adhesion cular and/or neurodegenerative dis-
molecule for tight junctions; N-cadherin for endothelial cell–pericyte adherin junctions and ease therapies from bench to
bedside?
Connexin 43 for gap junctions. If required, the biofabricated 3D NVU models can be subjected
to normal tissue histology approaches, such as paraffin embedding and tissue slicing, prior to
immunohistochemistry [50]. Specific proteins can be quantified using a variety of protein
quantification techniques, including western blot and ELISA [60,84,85], while mRNA can be
quantified by reverse transcription qPCR [24]. Single-cell analyses can be utilised to charac-
terise particular cell types within the NVU, by digesting the matrix to release the cells, purifying
an individual cell type from the rest by flow cytometry and then using RNA sequencing or
proteomics to identify changes of interest under different conditions (e.g., disease-specific
mutation versus non-disease control). Other outcome measures will depend on the question
being addressed of an NVU model. For example, the deposition of amyloid-b and phosphor-
ylation of tau, and the ability of drugs to reverse these Alzheimer’s-like pathologies [86,87],
could be measured in NVU models. The transport of amyloid-b, which was shown to be
facilitated by apolipoprotein E across bioengineered human cerebral vessels [50], could be
monitored across an NVU model.

Concluding Remarks and Future Perspectives

Biofabrication of an in vitro 3D NVU model through bioinks and bioprinting enables the
development of a multicellular model that more closely mimics the in vivo situation (see
Outstanding Questions), with the introduction of bioprinting enabling the production of inter-
active interfaces between the various cellular components. A tissue-engineered model that can

respectively, enabling the production of a layered 3D NVU construct with direct interaction through the layers of the
hydrogel. (D) By using direct bioprinting of cell-laden hydrogel and a sacrificial biomaterial, a channel can be manufactured
in the construct. Printing the sacrificial biomaterial within the bioprinted hydrogel allows for channel flushing once the gel
has set and subsequent seeding of vascular cells within the channel to create a blood–brain barrier; addition of a
microfluidic pump can introduce shear flow to the model. (E) Direct bioprinting of bioink into a self-healing fluid gel enables
suspended manufacture of 3D NVU models through an extended and controlled gelation of bioinks. Removal of fluid gel
leaves a solid postprint construct, ready for cell culture.

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 TIBTEC 1600 No. of Pages 16

replicate the 3D tissue microenvironment of the NVU is imperative to elucidating cell–cell and
neural–vascular interactions that are important to function and disease within an in vivo NVU.
Both direct and indirect printing techniques are able to create channels within the 3D construct,
with subsequent seeding of endothelial cells enabling the fabrication of a BBB or vascular
network. Central to the effective execution of bioprinting a 3D NVU are the bioinks selected to
support cells before, during and after bioprinting. By utilising bioinks and 3D bioprinting, the
quality of the NVU model is increased significantly, which will lead to a better understanding of
the molecular and cellular interactions between the different cell types in the NVU and how
these go wrong in diseases associated with neurovascular dysfunction. The availability of 3D
NVU models may also facilitate screening for drugs to correct the neurovascular dysfunction
underlying stroke, Alzheimer’s disease and other dementias.

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