Acta Biomaterialia 113 (2020) 1–22

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actbio

Advancing bioinks for 3D bioprinting using reactive fillers: A review

Susanne Heid, Aldo R. Boccaccini∗
Institute of Biomaterials, University of Erlangen-Nuremberg, Cauerstraße 6, 91058 Erlangen, Germany

a r t i c l e i n f o a b s t r a c t

Article history: The growing demand for personalized implants and tissue scaffolds requires advanced biomaterials and
Received 7 March 2020 processing strategies for the fabrication of three-dimensional (3D) structures mimicking the complexity
Revised 26 June 2020
of the extracellular matrix. During the last years, biofabrication approaches like 3D printing of cell-laden
Accepted 26 June 2020
(soft) hydrogels have been gaining increasing attention to design such 3D functional environments which
Available online 2 July 2020
resemble natural tissues (and organs). However, often these polymeric hydrogels show poor stability and
Keywords: low printing fidelity and hence various approaches in terms of multi-material mixtures are being devel-
Composite hydrogels oped to enhance pre- and post-printing features as well as cytocompatibility and post-printing cellular
Bioprinting development. Additionally, bioactive properties improve the binding to the surrounding (host) tissue at
Cell encapsulation the implantation site. In this review we focus on the state-of-the-art of a particular type of heterogeneous
Bioactive inorganic fillers bioinks, which are composed of polymeric hydrogels incorporating inorganic bioactive fillers. Such sys-
Bioactive glasses
tems include isotropic and anisotropic silicates like bioactive glasses and nanoclays or calcium-phosphates
Laponite
Hydroxyapatite
like hydroxyapatite (HAp), which provide in-situ crosslinking effects and add extra functionality to the
matrix, for example mineralization capability. The present review paper discusses in detail such bioac-
tive composite bioink systems based on the available literature, revealing that a great variety has been
developed with substantially improved bioprinting characteristics, in comparison to the pure hydrogel
counterparts, and enabling high viability of printed cells. The analysis of the results of the published
studies demonstrates that bioactive fillers are a promising addition to hydrogels to print stable 3D con-
structs for regeneration of tissues. Progress and challenges of the development and applications of such
composite bioink approaches are discussed and avenues for future research in the field are presented.

Statement of Significance

Biofabrication, involving the processing of biocompatible hydrogels including cells (bioinks), is being in-
creasingly applied for developing complex tissue and organ mimicking structures. A variety of multi-
material bioinks is being investigated to bioprint 3D constructs showing shape stability and long-term
biological performance. Composite hydrogel bioinks incorporating inorganic bioreactive fillers for 3D bio-
printing are the subject of this review paper. Results reported in the literature highlight the effect of
bioactive fillers on bioink properties, printability and on cell behavior during and after printing and pro-
vide important information for optimizing the design of future bioinks for biofabrication, exploiting the
extra functionalities provided by inorganic fillers. Further functionalization with drugs/growth factors can
target enhanced printability and local drug release for more specialized biomedical therapies.
© 2020 Acta Materialia Inc. Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license.
(http://creativecommons.org/licenses/by-nc-nd/4.0/)

1. Introduction
Biofabrication is gaining increasing attention to create com-
plex 3D structures mimicking tissues and organs in regenerative
∗
Corresponding author: Prof. Aldo R. Boccaccini: Cauerstraße 6, 91058 Erlangen,
Germany.
E-mail address: aldo.boccaccini@ww.uni-erlangen.de (A.R. Boccaccini).
medicine and tissue engineering [1,2]. As cells in the human body
are located within a three-dimensional matrix, this method allows
to resemble and mimic the original 3D structures and functions
of living tissues (and organs) [3]. Additionally, with this approach
a platform for drug testing and for the analysis of tissue mor-
phogenesis can be generated as cells are encapsulated in a suit-
able hydrogel matrix and subsequently processed to 3D structures,
for example by 3D bioprinting [3–5]. To differentiate between

https://doi.org/10.1016/j.actbio.2020.06.040
1742-7061/© 2020 Acta Materialia Inc. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license.
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
2 S. Heid and A.R. Boccaccini / Acta Biomaterialia 113 (2020) 1–22

Fig. 1. Distinction between a bioink (left side) and a biomaterial ink (right side). In a bioink, cells are a mandatory component of the printing formulation in the form
of single cells, coated cells or cell aggregates (of one or several cell types), or also in combination with materials (for example seeded onto microcarriers, embedded in
microgels, formulated in a physical hydrogel, or formulated with hydrogel precursors). In case of a biomaterial ink, in principle any biomaterial can be used for printing and
cell-seeding occurs post-fabrication. The images in this scheme are not displayed in scale. (Reproduced from ref. [13] according to the terms of the Creative Commons CC BY
license).

bioprinting and biofabrication the “International Society for Biofab-
rication” suggested a new definition of “Biofabrication” in the con-
text of tissue engineering and regenerative medicine within which
“Bioprinting” and “Bioassembly” are two complementary technolo-
gies followed by tissue maturation. Thus biofabrication is defined
as “the automated generation of biologically functional products with
structural organization from living cells, bioactive molecules, bioma-
terials, cell aggregates such as micro-tissues, or hybrid cell-material
constructs, through bioprinting or bioassembly and subsequent tissue
maturation processes” [6].
Using bioprinting, cells in combination with hydrogels (acting
as a temporary extracellular matrix) and active substances can be
precisely distributed hierarchically and spatially according to the
desired three-dimensional function [7]. Hence, bioprinting provides
free-shaping design in combination with controlled positioning of
materials and cells in one step by avoiding a post-cell-seeding pro-
cedure, which is commonly done in classical tissue engineering
[8,9]. Bioprinting additionally implicates an intimate connection
between cells and materials as well as a higher cell loading effi-
ciency with homogenous or tailored cell distribution which cannot
be achieved by conventional cell culture methods [10]. By choosing
the appropriate cell type and density, post-printing cell behavior
can be analyzed including favorable or impaired cell proliferation,
maintenance of cell integrity and morphology and preservation of
phenotype and genotype [11]. Furthermore, the selection of phys-
iologically relevant culture systems gives the opportunity to gain
insights into the fundamental biological processes of living cells in
3D architectures [12].
As suggested by Groll et al. [13] the term “Bioink“ should be
clearly defined and differentiated from the term “Biomaterial ink”
considering hydrogels for 3D printing, as displayed in Fig. 1. Bio-
material inks can be printed and if needed sterilized and sub-
sequently seeded with cells for applications as scaffold materials
or implants or for hybrid scaffolds providing mechanical support
in 3D printed constructs [14–16]. Bioinks on the other hand are
defined as materials which are capable to include cells and other
bioactive components for the use in biofabrication [13].
The need for processing ‘with cells’ has increased the challenges
that must be tackled in the development of suitable bioinks. To
fulfill as many requirements as possible (regarding bioinks and
bioprinted scaffolds), composite hydrogels consisting of a hydro-
gel matrix and inorganic fillers represent attractive systems, which
have been specially investigated for bone and cartilage tissue re-
construction so far [25–29]. This paper reviews polymeric hydro-
gels with incorporated bioactive inorganic fillers and encapsulated
cells for the use in bioprinting, here referred to as “composite
bioinks”, published in the last six years. In literature, there are
also other definitions for composite bioinks. Some authors have
used the expression for naming mixtures of two or more polymers
yielding blends or multi-phase materials [30–32], however in this
review only hydrogel + inorganic filler composites are considered
under the expression “composite bioink”. The current literature is
analyzed and discussed regarding printability, printing fidelity and
cell viability of composite bioinks incorporating bioreactive inor-
ganic fillers, e.g. fillers which are not ‘persistent’ but can change
their properties as function of time (for example by partially dis-
solving in the hydrogel matrix).
Overall, this review intends to present a comprehensive sum-
mary of the field of composite hydrogels for bioprinting, discussing
a broad variety of such composite bioinks and their properties, re-
cently reported for 3D bioprinting. Only such composite bioinks
were taken into account which consist of a polymeric matrix (hy-
drogel) and bioactive inorganic filler(s) and which are bioprinted
with embedded cells. The cited references found in the databases
(as listed below) were grouped by the type of inorganic filler.
Previous comprehensive review papers are available [9,33–36],
which have described the broad field of bioinks for bioprint-
ing/biofabrication, however with limited discussion of the type of
composite bioinks on which we focus the present review. Equally,
composite hydrogels with inorganic components have been
 S. Heid and A.R. Boccaccini / Acta Biomaterialia 113 (2020) 1–22
discussed in previous articles [16,37–40], but not with focus on
3D bioprinting approaches, e.g. such previous reviews have not
covered in detail composite hydrogels incorporating cells for bio-
printing. As such, a gap in the current literature is filled with the
present review article. In this literature review, we have focused on
the most relevant work published in the last six years regarding
the use of nanostructured silica, bioactive glasses, calcium phos-
phate or strontium-based bioactive materials and their applica-
tions in the field of 3D bioprinting. PRISMA guidelines for struc-
tured reviews were followed and the literature search was carried
out on platforms like Web of Science R
, Wiley database, Scopus
Pubmed, Google Scholar R
, Science Direct, as well as Springer,
Mendeley R
, IOP Science and science.gov databases. For the liter-
ature search, different combinations of the keywords “composite”,
“bioink”, “hydrogel”, “biofabrication”, “3D (bio)printing”, “cell encap-
sulation”, “bioactive”, “inorganic”, “filler”, “bioactive glass”, “laponite”,
“hydroxyapatite”, “tricalcium phosphate”, “clay”, “silica”, “silicate” and
“carbonate” were used. Publications that did not report cell incor-
porated composite hydrogels or where the word “composite” was
(erroneously) used to indicate blend hydrogels were not consid-
ered.
2. Composite bioinks
In the last years, the development and characterization of novel
bioinks have received increasing attention, as such systems are re-
quired to fabricate complex and highly customizable cell-laden bio-
printed scaffolds for various applications in personalized medicine,
tissue engineering, disease models or chips for high-throughput
screening [41]. Hydrogel composite bioinks are one of the most
suitable strategies for incorporating and combining various hydro-
gel and filler properties, which are not attainable by one-phase hy-
drogels alone. Indeed, the lack of suitable (bioactive) materials for
bioprinting has been one of the significant drawbacks for a fast
progress in this area [42,43]. Numerous new strategies are being
continuously developed to enhance the material features for 3D
bioprinting demands, which have been reviewed in recent articles
[30,35,42–52].
Facing a complex biological system in the human body, the re-
quirements for biomaterials which can be 3D printed incorporat-
ing cells to achieve complex geometrical designs are manifold and
challenging [20,21]. The demanding list of requirements of (com-
posite) bioinks which are used for bioprinting may be divided into
pre- and post-printing properties which on the one hand examine
the materialś characteristics necessary for bioprinting and on the
other hand focus on features of the bioprinted 3D constructs that
are required for maintaining cell viability and for inducing (or pre-
venting) a given behavior of encapsulated cells after the printing
process [17–19,41,46,53,54].
First of all, such biomaterials need to be biocompatible and
sterilizable, not eliciting an unresolved inflammatory response.
Usually, bioinks consisting of low-viscosity natural or synthetic
biocompatible hydrogels are used for bioprinting. Hydrogels are
hydrophilic, physically or chemically crosslinked polymers. They
possess a high-water content, which provides a suitable environ-
ment similar to the natural extracellular matrix (ECM) for cell
function and viability [55,56]. Nonetheless, pure hydrogels often
exhibit a lack of cell-binding sites (especially synthetic polymers),
relatively low mechanical and structural stability and they of-
ten degrade too fast or in an unpredictable manner, which nar-
rows their use as bioinks for long-term cell cultivation or for
the transfer to in vivo applications [57]. Hence, endeavors have
been made to design composite bioinks which combine the pos-
itive features of biocompatible, low-viscosity hydrogels with the
biofunctionality of inorganic fillers or higher-viscous hydrogels or
3
a combination of both [58]. New multi-material bioinks are cur-
rently being developed to not only improve the printability but
also to enhance features like mechanical resistance and stiffness,
to tune the degradation rate, or to add bioactive moieties which
show significant impact on cell behavior and tissue formation
[30,59–62]. One particular type of such multi-material bioinks are
hydrogel-inorganic phase composite systems (composite bioinks),
which are the subject of this review. By adding different nanopar-
ticles or anisotropic fillers, e.g. graphene, graphene oxide, carbon
nanotubes (CNTs), hydroxyapatite (HAp) and other calcium phos-
R
, phates, bioactive glasses, silica nanoparticles, and nanoclays, the
mechanical and biological characteristics of nanocomposite hydro-
gels can be improved [38,63–71]. For example, with the incorpo-
ration of such fillers physicochemical properties affecting bioprint-
ability can be tuned, like viscosity, stiffness, nonlinear viscoelas-
ticity (thixotropy/rheopexy), surface tension or density. Mechani-
cal properties of the bioinks should be adjusted to the used cell
type and the desired cell function. Additionally, due to the different
chemical compositions of the inorganic fillers and the release of
specific ions (upon dissolution, in case of bioreactive fillers), most
of these fillers are able to support cell adhesion and proliferation
and some of them also influence differentiation of stem cells, pro-
genitor cells or cell lines, leading to, for example, osteogenic (e.g.
bioceramics, HAp, nanoclays), chondrogenic (e.g. bioactive glasses),
angiogenic (e.g. some bioceramics, bioactive glasses), adipogenic
(e.g. silica nanoparticles) or neurogenic responses (e.g. carbon nan-
otubes) [72,73]. More detailed presentation and discussion of the
biological effects of the chemical and morphological properties of
different fillers are included further below in the respective sub-
sections.
In the reviewed literature, bioprinting of the designed cell-
laden composite bioinks has mostly taken place by using
extrusion-based 3D printers. This technique has a large printing
window ranging from roughly 101 –1013 Pa s [46]. Hence, a variety
of bioinks can be successfully printed. Such bioinks need to exhibit
shear-thinning and viscoelastic behavior and they must rapidly gel
and maintain their shape after printing. Considering shear-thinning
properties, bioinks are conventionally characterized by rheologi-
cal measurements (storage and loss moduli, tangent delta and re-
sponse to shear stress, for example). During printing, the bioink
matrix protects the cells from damage as too high shear forces
might disrupt the cell membrane (depending on the cell type). Not
only the bioink properties must be considered carefully, but also
technical issues like e.g. the nozzle size and geometry [58,74]. Em-
bedding inorganic fillers into bioinks changes the rheological prop-
erties, but, according to the review of the available literature, the
effect of such filler addition on the shear forces acting on cells and
on the respective cell viability after printing have not been yet fully
examined and understood.
After printing, cell containing constructs need to guide the
cellular response to develop a matured functional tissue. So far,
this task remains challenging as the time-dependent mechanical
properties of the native ECM must be matched as close as pos-
sible by the (degrading) 3D printed construct [46]. In this con-
text, it becomes a paramount requirement to carefully select the
hydrogel and the fillers to be used in composite bioink formula-
tions. Biodegradability, bioactivity, internal porosity, interconnected
porosity (for vascularization, ingrowth of surrounding tissue and
nutrients, waste and oxygen transport) are key characteristics of
3D bioprinted constructs which affect cell proliferation while the
biodegrading construct leaves space for the expansion of the de-
veloping ECM [22,23,75]. Gradient structures can furthermore help
to guide cell migration along the bioprinted scaffolds [24]. All such
time-dependent features of 3D bioprinted scaffolds are affected by
the chemical composition and physical properties of the chosen in-
organic fillers.
4 S. Heid and A.R. Boccaccini / Acta Biomaterialia 113 (2020) 1–22
In the following sections we review and discuss in detail the
current state-of the art in the field of composite bioink devel-
opment, focusing on hydrogels incorporating bioreactive inorganic
fillers, and discussing key involved issues such as printability and
cytocompatibility. The reviewed systems are grouped regarding the
inorganic fillers used in each case, as summarized in Table 1. The
chemical and physical properties of the used inorganic fillers are
described at the beginning of each respective subsection.
3. Silicate-based composite hydrogels
3.1. Silica nanoparticles as inorganic fillers
Silica-based bioceramics have received great attention in bone
tissue engineering, drug and gene delivery and theranostics, as
reviewed and summarized in previous studies [101–105]. Chemi-
cal modifications can change the properties of (mesoporous) silica
nanoparticles leading to bioinert, bioactive or biocompatible func-
tions [106]. The ionic degradation products of Si-containing con-
structs have been reported to lead to high osteoconductivity, while
possible cytotoxic side effects still need to be examined [105,106].
Silica nanoparticles have also found interest in the field of 3D bio-
printing. Wang et al. [76] investigated the effect of alginate/gelatin
hydrogels supplemented with silica, biosilica and polyphosphate
(polyP•Ca2+ ) on encapsulated SaOS-2 cells (human osteogenic sar-
coma cells), respectively. For this purpose, round gel cylinders
(13 mm diameter, 1.5 mm height) were printed with the composite
hydrogels using a sterile 3D-Bioplotter (Envisiontec, Gladbeck; Ger-
many). The addition of polyP•Ca2+ -complex and biosilica, but also
to a smaller extent ortho-silicate, resulted in a significant stimu-
lation of cell growth after 3 days of incubation in medium. Espe-
cially the bioinik with polyP and biosilica allowed the cells to pro-
liferate. Supplementing the incubation medium/ serum with addi-
tional bioactive glass nanoparticles (molar ratio of SiO2 :CaO:P2 O5 :
55:40:5) with a size of 55 nm led to a significant enhancement of
cell proliferation and biomineral formation during incubation for
3 days in standard cell culture medium and further 5 days in os-
teogenic differentiation medium. The growth potency of cells en-
capsulated in the composite bioink was not significantly affected
by incubation either in normal medium or medium with bioactive
glass particles.
Silica nanoparticles (NPs) of size < 100 nm can also be benefi-
cial for enhancing the physical and mechanical properties of hydro-
gels. Silica nanoparticles can effectively crosslink anionic polysac-
charides leading to improved printability and printing fidelity, as
shown in Fig. 2. Anionic polysaccharides, namely alginate (3%
(w/v)) and gellan gum (3% (w/v)) hydrogels, were printed with 6
wt% cationic silica (SiNP) nanoparticles or aminopropyl-modified
aminated cationic SiNPs (AmNPs) by Lee et al. [77]. Gellan gum
is reported to have shear thinning as well as fast recovery behav-
ior after printing [107]. After calcium crosslinking the pure poly-
mer hydrogel underwent shrinkage and hence the printing fidelity
was lowered immensely. The addition of silica particles greatly en-
hanced the shear viscosity (1062% increase). The storage modulus
was also increased by SiNPs (and even more with AmNPs) due to
a higher crosslinking density and vice versa a decreased pore size.
Subsequently, the composite bioink with AmNPs was printable into
stable and defined grid structures [77]. For both bioinks (with and
without AmNPs) the cell viability of printed bovine chondrocytes
was recovered to around 90% after one week of cultivation in dif-
ferentiation medium containing TGFβ -3. Immunostaining and col-
lagen staining confirmed the growth and extracellular matrix se-
cretion of the printed cells. Moreover, this approach could find ap-
plications for many other polymeric hydrogels, specially consider-
ing anionic polysaccharides such as alginate or hyaluronic acid.
3.2. Bioactive glasses – doped and undoped – as inorganic fillers
Hench et al. discovered that specific silicate compositions
demonstrated superior biocompatibility and the ability to bind to
bone [108]. In contact with biological fluids, bioactive glasses show
bioactivity through the formation of a carbonated hydroxyapatite
layer on the glass surface because of the release of distinct ions.
The basic constituents of bioactive glasses are SiO2 , Na2 O, CaO and
P2 O5 but several modifications in composition and doping with
(biologically active) metallic ions can be made for specific applica-
tions [109]. Most applications have considered bone regeneration
approaches, however applications in soft tissue repair are emerg-
ing [110].
In hydrogels, ions released from bioactive glass particles may
induce efficient osteogenic differentiation of encapsulated cells.
Human adipose stem cells (hASCs) incorporated in gellan gum or
collagen type I hydrogels, respectively, revealed significantly higher
cell numbers and higher osteogenic gene expression when cultured
in bioactive glass extract based osteogenic medium (BG-OM) [111].
The strong mineralization and stiffening behavior under these cul-
ture conditions showed great potential for bone-like grafts. In ad-
dition, gellan gum hydrogels crosslinked with BG extracts also
showed mineralization in pure osteogenic medium which could
not be found in the neat collagen hydrogels under the same condi-
tions. Nevertheless, collagen-hydrogels loaded but not printed with
hASCs under BG-OM conditions showed significantly higher os-
teogenic marker expression than the gellan gum counterparts with
an elongated and spread morphology as well as the highest miner-
alization and hydroxyapatite formation capability [111]. Based on
such results, bioactive glasses are considered attractive bioactive
fillers for developing composite hydrogels for bioprinting.
Often, bioactive glasses are used as pure scaffolds or in com-
bination with other materials to induce a hydroxyapatite coating
during immersion in simulated body fluid (SBF). This layer pro-
motes cell attachment, whereas cells in direct contact with bioac-
tive glass particles may behave differently [78]. Reakasame et al.
showed that the viability and growth of osteoblast-like MG-63
osteosarcoma cells encapsulated into ADA-keratin-45S5 bioactive
glass (0.5%) composite hydrogel microcapsules were drastically re-
duced during the first 7 days after encapsulation. However, dur-
ing the cultivation for 21 days the cell viability increased and be-
came comparable to that of alginate-keratin, but remained never-
theless permanently lower compared to the samples without BG
[78]. The outcomes were in agreement with the results reported
by Rottensteiner et al. [79]. In the latter study the addition of 45S5
BG nanoparticles into ADA-gelatin revealed slight cytotoxicity to
bone-marrow derived mesenchymal stem cells (MSCs) cultured on
the hydrogel films. Moreover, Reakasame et al. found that after 3
weeks of incubation under cell culture conditions the MG-63 cells
started to spread, which did not arise in the samples without BG
indicating a possible positive effect of the CaP formation induced
by the dissolution of the 45S5 BG particles [78].
The effect of stimulating osteogenesis was illustrated by a
comparison of 3D printed poly(ethylene glycol)dimethacrylate
(PEGDMA) incorporating bioactive glass particles (45S5; 20 μm),
hydroxyapatite (200 nm) or a co-print of both fillers, and bone
marrow-derived human mesenchymal stem cells, as reported by
Gao et al. [80]. The addition of bioactive fillers and cells resulted in
an overall decrease of mechanical properties which were recovered
over the course of 21 days of incubation in cell culture conditions
due to the possible ECM formation by encapsulated hMSCs under
osteogenic differentiation. This effect was not observed in PEG-BG
(bioactive glass containing cylinders) bioinks, which demonstrated
the lowest compressive modulus and highest mass swelling ratio
among the groups. Cell viability 24 h after printing was highest
in PEG-HAp samples. During the incubation period no significant
 S. Heid and A.R. Boccaccini / Acta Biomaterialia 113 (2020) 1–22 5

Table 1
Summary of hydrogel composites containing bioactive inorganic fillers for bioprinting and their potential applications.

Composite Bioink Cells Cell 3D Bioprinting Application Reference

Density/mL Technique (+ Figure)
Hydrogel Inorganic Bioactive Filler

5% (w/v) Alginate - 5% 50 μmoles/L silica/ 50 SaOS-2 (human Not reported Extrusion Bone [76]
(w/v) gelatin μmoles/L biosilica / 100 osteogenic
μmoles/L polyP∗ Ca2+ -> sarcoma cells)
silica

3% (w/v) Alginate - 3% 6 wt% Silica Bovine chondrocytes 1 × 108 Extrusion Tissue engineering and [77], Fig. 2
(w/v) gellan gum regenerative medicine
in general
2.5% (w/v) ADA (alginate 0.5% (w/v) Micro-bioactive MG-63 osteoblasts 1 × 106 Extrusion Bone-tendon interface [78]
dialdehyde) - 0.5% (w/v) glass (μBG, 45S5) regeneration
keratin
2.5% (w/v) ADA - 2.5% 0.1% (w/v) Nano-bioactive rMSCs (Rat bone 2 × 106 Extrusion Bone [79]
(w/v) gelatin glass (nBG, 45S5) marrow derived
mesenchymal stem
cells)
20% (w/v) PEGDMA 2% Bioactive glass/ 2% hMSCs (human 1 × 106 Modified Inket Cartilage/ osteochondral [80]
(poly(ethylene Hydroxyapatite/ 1% mesenchymal stem Printing interface
glycol)dimeth-acrylate BG + 1% HAp (all (w/v)) cells)
4% (w/w) Alginate - 5% 1% (w/w) Bioactive glass SaOS-2/ hBMSCs 5 × 105 Extrusion Bone [81]
(w/w) gelatin (+0.25% (human bone
(w/w) cellulose marrow stem cells)
nanofibers)
5% (w/v) ADA - 5% (w/v) 0.1/ 0.5 wt% Bioactive MG-63 osteoblasts Not reported Extrusion Bone [82], Fig. 3
GEL (ADA: alginate glass nanoparticles
dialdehyde) (BGNPs) or strontium
doped BGNPs
5–10% (w/v) Gelatin 0.01% (w/v) Silicate HUVECs + hMSCs 2 × 106 Extrusion Bone, Vascularization [83], Fig. 4
methacrylate nanoplatelets
(GelMA)-VEGF
10% (w/v) PEG:PEGDTT 4% (w/v) Nanosilicates, HUVECs Not reported Extrusion Drug delivery [84], Fig. 5
(poly(ethylene Laponite
R
XLG
glycol)-dithiothreitol)
4% (w/v) Alginate - 2% 4% (w/v) Montmorillonite BxPC3 (human 2 × 106 Extrusion Large scale bioprinted [85]
(w/v) clay pancreatic cancer scaffolds
carboxymethylcellulose cells)
3% (w/v) Alginate - 3% 3% (w/v) Laponite hTERTMSC (human 5 × 106 Extrusion Bone, drug delivery [86]
(w/v) methylcellulose mesenchymal stem
cell line expressing
hTERT (human
telomerase reverse
transcriptase))
20% (w/v) PEGDA 7% (w/v) Laponite
R
XLG ROBs (Primary rat 4 × 106 Extrusion Bone [87], Fig. 6
(polyethyleneglycol osteoblasts)
diacrylate)
2.0/ 2.5 wt% κ CA 3 - 6 wt% Nanosilicates MC3T3-E1 (Mouse Not reported Extrusion Tissue engineering (TE) [88]
(Laponite R
XLG) preosteoblasts) in general
10% (w/v) GelMA - 1% 2% (w/v) Nanosilicates MC3T3-E1 (Murine 1 × 106 Extrusion Large human tissues, [89], Fig. 7
(w/v) κ CA (laponite) 3T3 preosteoblasts, Bioactive 3D structures
(Kappa-carrageenan) Subclone 4)
7 wt% GelMA 0/ 0.01/ 0.05/ 0.5 wt% hMSCs 3 × 106 Micropatterning Bone, Cranial defects [90]
Nanosilicates
2% (w/v) Gellan Gum 1% (w/v) Laponite C2C12 (mouse 1 × 106 Extrusion in Bone, critical bone [91]
myoblasts) agarose fluid defect filling, skeletal
bed tissue engineering
5/ 7.5/ 10 wt% GelMA 0.5/ 0.75/ 1/ 2 wt% hBMSCs 5 × 106 Extrusion Hard and soft tissue [92]
Laponite R
XLG reparation, drug
delivery,
vascularization
2% (w/v) Chitosan 2% (w/v) MC3T3-E1 3 × 107 Extrusion Bone [93], Fig. 8, 9
compared to 3% (w/v) Nano-Hydroxyapatite
alginate (nHAp)
GelMA - 1 wt% 5% (w/v) hASCs (human 1 × 106 Microextrusion Bone [94], Fig. 10
methacrylated Nano-Hydroxyapatite adipose-derived and
hyaluronic acid stem cells) encapsulation
"by hand"
2 wt% Sodium alginate - 8 1 wt% hASCs 3 × 106 Dispense plotting Bone [95]
wt% gelatin (A2G8) Nano-Hydroxyapatite
2% (w/v) Alginate - 10% 0/ 4/ 8% (w/v) hMSCs 5 × 106 Extrusion Drug delivery, [18], Fig. 11
(w/v) gelatin Hydroxyapatite Microsphere
deposition, Soft tissue
engineering
(continued on next page)
6 S. Heid and A.R. Boccaccini / Acta Biomaterialia 113 (2020) 1–22

Table 1 (continued)

Composite Bioink Cells Cell 3D Bioprinting Application Reference

Density/mL Technique (+ Figure)
Hydrogel Inorganic Bioactive Filler

2% (w/v) Alginate −2% 0.5% (w/v) hMSCs 2 × 107 Fused deposition Bone [60]
(w/v) gelatin Nano-Hydroxyapatite modeling,
Injection
Gelatin - alginate (8:2 0/ 9.1/ 30 wt% Mouse chondrocytes 2 × 105 Extrusion Cartilage [96]
(w/w) Hydroxyapatite
2/2.5% (w/v) Alginate - 1% 2.5% (w/v) Hydroxyapatite MC3T3-E1 (Mouse 2.5 × 105 Extrusion Bone [97]
(w/v) polyvinyl alcohol calvaria 3T3-E1)
(PVA)
1% (w/v) RGD∗ -alginate 50% (v/v) nHAp-pDNA∗∗ Bone 1 × 106 Extrusion Bone, muscoskeletal [98]
marrow-derived tissue engineering
MSCs
5 wt% Collagen compared 20 wt% β -TCP (tricalcium MC3T3-E1 compared 1 × 107 Extrusion Bone [99]
to 5 wt% alginate phosphate) to hASCs
5 wt% GelMA 0.05/ 0.15/ 0.3/ 0.5% (w/v) hMSCs 5 × 106 Extrusion Bone [100], Fig. 12
SrCO3
∗
RGD – Arginine-glycine aspartic acid modified alginate.
∗∗
pDNA – plasmid DNA.

Fig. 2. A) Schematic presentation of 3D printing with AmNP-based nanocomposite inks. B) Bioprinted bovine chondrocytes in Alg/gellan or Alg/gellan/AmNP bioink were
cultured over 21d and their viability was assessed according to culture time. ∗ P-value < 0.05; ∗ ∗ P-value < 0.01; ns: not significantly different. C) Side view of 3D printing 10
layers using Alg/gellan inks with/without AmNPs. Red arrows indicate just extruded composite hydrogel that was not properly deposited on the underlying printed layer due
to collapse. Top view of the grids (2 × 2 cm) before and after crosslinking, showing shrinkage as the red boxes have the same size. D) Images and 3D scans of 3D printed
ear models after crosslinking, scale bar = 5 mm. E) Digitalized images of crosslinked ear models. The color indicates dimension difference between the printed, subsequently
crosslinked construct and the original 3D digital model. The gray background is the outline of the used 3D design. (Reproduced with permission from ref. [77]. Copyright
2018, American Chemical Society). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

cell proliferation could be observed in all samples but gene ex-
pression, measured by quantitative PCR, revealed that HAp in bio-
printed PEG-scaffolds was more effective with respect to stimula-
tion of hMSCs osteogenic differentiation and ECM production com-
pared to 3D printed PEG-BG constructs.
Furthermore, different cell types seem to have different sen-
sitivities to tolerate 3D bioprinting including the induced shear-
stress and the materialś viscosity for short- and long-term cul-
ture [112]. Ojansivu et al. prepared alginate-gelatin based bioinks
incorporating wood-based oxidized cellulose nanofibers (CNF) and
bioactive glasses (47.12 SiO2 - 6.73 B2 O3 - 6.77 CaO-22.66 Na2 O-1.72
P2 O5 - 5 MgO-10 SrO (mol%)) to tailor the rheological properties of
the composite hydrogel as well as the response of two different
bone cells (SaOS-2 and hBMSCs (human bone marrow stem cells))
embedded in these composite hydrogels [81]. The 4-layered rect-
angular grids were stable in cell culture medium over the incuba-
tion of 21 d but showed different cell number, viability and pro-
liferation. It was found that the incorporation of BG resulted in
death of SaOS-2 cells immediately after printing, which is assumed
to be the consequence of high shear forces induced in the bioink
 S. Heid and A.R. Boccaccini / Acta Biomaterialia 113 (2020) 1–22 7

Fig. 3. Bright-field microscopy images and life/ dead fluorescent staining (green: live cells, magenta: dead cells) of MG-63 cells after incubation for 24 h in various ADA-
GEL-BG composite hydrogels [82]. The cell viability of respective samples normalized to ADA-GEL without BGNPS (control) is shown on the left. Results are presented
as arithmetic mean ± standard deviation and the pairwise comparison of the means was performed with the Bonferroni’s test (post hoc comparison). (Reproduced with
permission from ref. [82]. Copyright 2016, IOP Publishing Ltd.). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version
of this article.)

through the addition of rigid bioactive fillers. Mixing of the hy-
drogel with small amounts of CNF enhanced the printability re-
markably whereas BG may enhance osteogenic capacity of encap-
sulated cells, as shown via alkaline phosphatase (ALP) activity in-
crease. Unlike the behavior of SaOS-2 cells, hBMSCs incorporated
in hydrogel containing BG and CNF maintained good viability but
also limited proliferation over 14 d [81].
Leite et al. [82] focused on the integration of MG-63 cells in
alginate-dialdehyde/ gelatin (ADA-GEL) hydrogel combined with
bioactive glass nanoparticles. In previous studies, ADA-GEL already
had been proven to be cytocompatible in combination with cell
seeding, encapsulation or as 3D printed constructs [69,113–117].
As ADA-GEL itself lacks bioactive behavior (mineralization abil-
ity), bioactive glass nanoparticles (BGNPs) were integrated to pro-
mote the deposition of a calcium phosphate layer, which should
lead to enhanced osseointegration of the construct [118]. ADA-GEL
was compounded with ternary formulations of 0.1% and 0.5% (w/v)
BGNPs (SiO2 –CaO-P2 O5 ) or strontium doped BGNPs (SiO2 –CaO-
P2 O5 -SrO), respectively [82]. Strontium was used due to its favor-
able effects on osteogenic stimulation as well as in vivo bone for-
mation ability [119,120]. The addition of BGNPs led to a decreas-
ing crosslinking time and thus a smaller processing window com-
pared to ADA-GEL, possibly due to the release of divalent cations
and their interaction with the guluronic parts of ADA. The in vitro
biomineralization study of bioplotted composite grid structures
(15 mm edge length, 4 layers) showed bioactive behavior lead-
ing to the formation of an apatite layer on the surface during in-
cubation in SBF. Moreover, these composite bioinks revealed the
possibility to sustainably release pharmacological drugs and they
displayed similar cell viability of encapsulated MG-63 osteoblasts
compared to neat ADA-GEL after 24 h of incubation (Fig. 3). The
special characteristic of BG fillers, being dissolvable or bioreactive,
remains to be exploited in future bioink strategies, not only for
bone regeneration (bioprinted) scaffolds. An interesting approach
is the incorporation of mesoporous BG particles in the hydrogel,
where the particles can act as local carrier for growth factors or
therapeutic drugs, thus exploiting the dual effect of biologically ac-
tive ions and drug release in the proximity of cells [82].
3.3. Anisotropic nanosilicates as inorganic fillers
Nanosilicates (NS), including for example laponite (trademark of
the company BYK Additives Ltd.), halloysite, montmorillonite, or
hectorit, are ultrathin anisotropic nanomaterials with a high as-
pect ratio (disk-shaped geometry of 20 - 50 nm in diameter and
1 - 2 nm thickness) and a high degree of functionality for appli-
cations in biomedicine [70,121–124]. Due to their shape and ori-
entation under shear, these nanosilicates can act as inorganic ad-
ditives to improve the mechanical and biological properties of 3D
(bioprinted) scaffolds, or they may be used as therapeutic drug de-
livery agents [125,126]. Beyond these, laponite, having the struc-
tural formula Na+ 0.7 [(Mg5.5 Li0.3 )Si8 O20 − (OH)4 ]− 0.7 , is extensively
researched for the development of composite hydrogels for 3D bio-
printing [125]. Especially Laponite R
XLG is studied for biomedi-
cal applications due to its lower heavy metal content. As the hy-
drous silicate contains elements such as magnesium, zinc, lithium
or iron that are also found in natural bone, its nontoxic degrada-
tion products [Na+ , Mg2+ , Si(OH)4 , Li+ ] can easily be absorbed by
the body [127,128]. During 3D printing, the anisotropic clay ma-
terials align and thus significantly affect the microstructure and
the rheological properties of the bioink [86,129]. Hence, nanosil-
icates are readily used due to their shear thinning characteris-
tics which protect embedded cells in the hydrogel from too high
detrimental stresses during the printing process [123,124,130]. This
anisotropic interaction between the polymer and nanosilicates is
based on the presence of both positive and negative charges on the
NS surfaces, which allow the formation of a physically crosslinked
network with anionic, cationic or natural polymers [131–133]. Fur-
thermore, the optical transparency of nanosilicates in aqueous me-
dia can be beneficial for imaging the subsurface cellular behavior
8 S. Heid and A.R. Boccaccini / Acta Biomaterialia 113 (2020) 1–22
to design complex printed tissues [131]. Beside these advanta-
geous properties, toxicological studies have shown that high con-
centrations of nanosilicates can reduce cell proliferation in vitro
[134]. Consequently, only low concentrations of nanosilicates (0.1
– 7%(w/v)) are generally used when applied as inorganic fillers in
hydrogel bioinks.
Xavier et al. [135] examined a gelatin-based hydrogel contain-
ing bioactive nanosilicate for enhanced matrix mineralization for
the regeneration of bone defects. In vitro encapsulation of NIH
MC3T3 preosteoblasts for 4 days showed cytocompatible proper-
ties which supported cell adhesion, growth and osteogenic dif-
ferentiation without the usage of growth factors. The composite
hydrogel could be bioprinted to precisely designed scaffolds with
a stable porous structure [135]. This result is in good agreement
with previous studies which had found that silicate nanoplatelets
significantly led to osteogenic differentiation of encapsulated hM-
SCs in GelMA hydrogels in growth-factor free medium [90,121].
A different approach to mimic the complex architecture of tis-
sues with multiscale vascular networks was carried out by Byam-
baa et al. [83]. They developed a functional vascular structure
within 3D printed bone tissue-like fibers consisting of GelMA hy-
drogels with different amounts of vascular endothelial growth fac-
tor (VEGF) and 100 μg/mL silicate nanoplatelets. A pyramidal ar-
rangement of printed cylinders was designed to represent differ-
ent cellular and chemical compositions to fabricate a gradient ma-
terial for vascularization. The central core cylinder having a fast
degradation rate and lower stiffness created the internal lumen
and consisted of 5% (w/v) GelMALOW . A co-culture of endothelial
cells (GFP-HUVECs) and human mesenchymal stem cells was car-
ried out. The other cylindrical fibers were printed from a stiffer
hydrogel made of 10% (w/v) GelMAHIGH , osteoinductive silicate
nanoplatelets and embedded hMSCs. During 7 days of static cul-
ture the inner core degraded and HUVECs and hMSCs formed a
densely packed network along the lumen-like hollow structure dis-
played in Fig. 4. Further maturation and osteogenic differentiation
were achieved by medium perfusion for 5 more days and another
9 days of static culture. Real-time polymerase chain reaction dis-
played an enhanced expression of angiogenesis-related gene CD31,
so that an organized, mature vascular niche was formed within
the printed model. The approach of Byambaa et al. [83] resulted
in a material that supported vasculogenesis and coincidently os-
teogenesis. As can be seen in Fig. 5, Peak et al. [84] combined
2D silicate nanoplatelets with a poly(ethylene glycol)-dithiothreitol
(PEGDTT) bioink with the goal to receive a shear-thinning bioink
that has the capability to load and sustainably release therapeutic
proteins from a 3D printed structure. The inclusion of PEGDA into
the hydrogels slowed down the degradation of the printed scaf-
folds. In agreement with previous studies, the interactions between
laponite and the polymer network prevented swelling of the con-
struct [136]. Within this 3D composite material, the nanosilicate
particles acted as a therapeutic delivery platform. Due to its nega-
tive and positive charges laponite can interact with a huge range of
biomolecules [137], like e.g. vascular endothelial growth factor, fi-
broblast growth factor (FGF), bone morphogenic protein 2 (BMP-2)
or platelet-derived growth factor [126,138,139]. To analyze the bi-
ological activity of the released VEGF and FGF from biocompatible
3D printed composites, the migration of HUVECs across a transwell
membrane was measured. The PEGDTT/nSi scaffold with encapsu-
lated growth factors showed similar behavior compared to the pos-
itive control of exogenously delivered growth factor.
As mentioned above, biofabrication approaches involving 3D
bioprinting are considered to develop tissue-like functional con-
structs by layer-by-layer deposition of hydrogels with aligned
and homogenously distributed living cells. The incorporation of
anisotropic fillers is being put forward for supporting not only the
printing process but also to promote cell alignment. Habib et al.
[85] blended a novel hybrid ink by mixing alginate, carboxymethyl-
cellulose and montmorillonite clay. The printability of this com-
position was tested with various complex 3D geometries and dif-
fering patterns. The bioink showed reproducible printability and
shape fidelity. Moreover, 84% of printed human pancreatic can-
cer cells were alive after 7 days of culture. In comparison, Ahlfeld
et al. [86] used laponite instead of montmorillonite clay for
extrusion-based printing at physiological conditions. Printed scaf-
folds demonstrated excellent shape fidelity even in scaffolds with a
height of 2 cm. More complex structures were plotted by changing
the layer orientation between 20° and 90°. Scanning electron mi-
croscopy images showed dense, smooth surfaces which remained
predominantly unaltered after 21 days of culture. Mechanical test-
ing revealed that non-porous, solid scaffolds had a higher compres-
sive strain and Young’s modulus than porous ones. However, after
21 days the mechanical properties of both scaffold types drasti-
cally decreased. Cell-laden constructs preserved their morphologi-
cal properties and showed a cell viability of 70% to 75% at the end
of the culture period. Furthermore, laponite could enhance the BSA
(bovine serum albumin) and VEGF release kinetics of the hydrogel
blend and by designing an appropriate inner porosity of the bio-
printed scaffolds the release was sustained.
Hong et al. [140] printed a PEG-alginate-nanoclay (Laponite R
XLG) hydrogel and infiltrated the porous structure with HEK (hu-
man embryonic kidney) cells encapsulated in a collagen matrix. Af-
ter 7 days the cell viability still remained very high (≈ 95%) within
this environment. Furthermore, the printed mesh was highly de-
formable. After stretching to 300% of its original length, relaxation
led to almost complete recovery of the initial shape. The unique
nature and the ability of nanoclays to improve the structure,
properties and performance of polymeric matrices have encour-
aged research groups to further develop this material for 3D bio-
printing with encapsulated cells. A polyethylene glycol diacrylate
(PEGDA)/Laponite R
XLG nanoclay bioink was recently co-printed
by Zhai et al. [87] in combination with hyaluronic acid sodium salt
by extrusion printing. The rectangular scaffolds showed suitable
print fidelity as a result of the increased nanocompositeś strength
and flow characteristics [87]. Within bioprinted structures, nan-
oclays have additionally been shown to directly influence cell be-
havior, which is depicted in Fig. 6. Primary rat osteoblasts (ROBs)
encapsulated in a PEG-clay composite hydrogel revealed a high cell
viability of more than 95% up to 7 days of culture [87]. Using a
two-channel printing setup, efficient delivery of nutrients and oxy-
gen to the growing cells, uniform cell distribution and deposition
efficiency were achieved. Both in vitro and in vivo, the printed rat
osteoblasts differentiated, which was considered to be enhanced
by the release of bioactive ions from the laponite clay, especially
Mg2+ and Si4+ ions [87].
Wilson et al. [88] designed new self-supporting composite
hydrogels from natural κ CA (Kappa-carrageenan; 2.5 wt%) stiff-
ened with 3 - 6 wt% nanosilicate addition (Laponite R
XLS) and
crosslinked with potassium ions. The resulting mechanical prop-
erties of the stable constructs were in the same order of mag-
nitude as cartilage tissue or blood vessels which undergo con-
stant mechanical load. Complex structures were printed, like 30-
layered cylinders, demonstrating a high shape stability of the
shear-thinning bioink at physiological temperatures, fine lattice
networks with high resolution or anatomical-scale models of noses
and ears. Bioprinting using mouse preosteoblasts MC3T3-E1 led to
an even distribution of cells in the printed strands as shown by
CellTracker red or green, respectively, using fluorescent microscopy.
As κ CA does not have cell binding moieties, printed cells showed
limited spreading while maintaining their metabolic activity dur-
ing 7 days incubation in cell culture medium in humidified atmo-
sphere. Thus, the κ CA-nanosilicate bioink could either be used for
3D printing non-adherent cells like chondrocytes or it could be
 S. Heid and A.R. Boccaccini / Acta Biomaterialia 113 (2020) 1–22 9

Fig. 4. Bioprinting of bone mimetic 3D architecture containing osteogenic and vasculogenic niches as well as formation of HUVECs/hMSCs-lined perfusable hollow lu-
men structure. The construct, containing both GFP-HUVECs and hMSCs, was co-cultured for 7 days, followed by medium perfusion for 5 days. A) Complex bone tissue
architecture consists of a perfusable vascular lumen lined with HUVECs which can be fabricated within a pyramidal bioprinted construct by arranging individual rods of
VEGF-functionalized GelMA bioinks with different mechanical strengths. Silicate nanoparticles were embedded in the hMSCs-laden three outer layers of cylinders to induce
osteogenic differentiation of hMSCs into bone tissue. The VEGF was covalently conjugated into the three outer layers of the cylindrical hydrogels with concentrations of
17.1, 34.2, and 68.5 ng mL−1 as determined with ELISA. B) Schematic 3D printing of independent cell-laden cylinders using the NovoGen MMX Bioprinter (Organovo). C)
Cross-sectional view of the whole bioprinted construct with Live/Dead staining shown from cross- and top-view of the encapsulated cells. D) DAPI and α -SMA stainings of
a HUVEC-lined vessel-like lumen within the bioprinted construct with cross- and top-sectioned confocal micrographs of central vessel at day 12 post culture. Encapsulated
endothelial cells lined the vascular walls (green fluorescence) and hMSCs were differentiated into pericytes (red fluorescence). E) Formation and lining of endothelial cells
inside the central channel. F) Immunostaining of endothelial cells and α -SMA-expressing hMSCs in the inner part of the lumen. G) Vascular hollow lumen network before
and after perfusion with fluorescent microbeads at day 7 post culture. (Reproduced with permission from ref. [83]. Copyright 2017, WILEY-VCH). (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)

modified with RGD-containing sequences to enhance cell migra-
tion, spreading and proliferation [88].
Taking this issue into account, a follow up work was done by
Chimene et al. [89], who developed a novel and unique nanoengi-
neered ionic-covalent entanglement (NICE) bioink with outstand-
ing printability and mechanical properties by taking advantage of
a novel reinforcement technique, which is schematically shown
in Fig. 7. The NICE bioink is composed of 10% w/v GelMA, 1%
w/v κ CA, 2% w/v nanosilicates and 0.25% w/v Irgacure2959 pho-
toinitiator and was demonstrated to be printable to freestanding,
high aspect ratio scaffolds of 3 cm in diameter and 150 layers in
height with an excellent shape fidelity without the need of addi-
tional support materials. After UV-crosslinking, the constructs had
improved mechanical properties, flexibility, elasticity, toughness,
bioactivity and flow properties, which are the result of the syner-
gistic combination of nanocomposite and ionic covalent entangle-
ments. Moreover, a Herschel-Bulkley fluid behavior model showed
the effect of the nanoclay altering the flow behavior of the NICE
bioink. A relatively low influence of stresses on cells during ex-
trusion bioprinting was demonstrated. Subsequently, encapsulated
3D printed mouse preosteoblasts proliferated well and maintained
high cell viability (≈90%) over a long-term incubation of 120 days.
NICE bioinks are thus an attractive composite system for replica-
tion of human tissues and for developing bioactive 3D structures,
where the presence of the inorganic filler (nanosilicate) confers the
bioink outstanding properties.
10 S. Heid and A.R. Boccaccini / Acta Biomaterialia 113 (2020) 1–22

Fig. 5. Printing and release of therapeutics in 3D. a) The high surface area and charged characteristics of nanosilicates are able to sequester protein therapeutics within the
3D printed structure. Therapeutics will be released during degradation of printed networks. b) The release of fluorescently labeled protein from 3D printed structure was
monitored over 28 d in PEGDTT (10% wt vol−1 )/nSi (4% wt vol−1 ) hydrogels (no PEGDA in formulation). Printed structure dimensions are ≈ 10 mm in diameter and 15 mm in
height (significantly larger mass than used in degradation studies). c) Sustained release of therapeutics was observed during initial time period, while complete protein was
released within 28 d in PEGDTT (10% wt vol−1 )/nSi (4% wt vol−1 ) hydrogels (n = 3, ∗ p < 0.05, one-way ANOVA with Tukey post-hoc testing). (Reproduced with permission
from ref. [84]. Copyright 2019, WILEY-VCH). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Another method to create (ECM)-mimicking GelMA hydrogels
reinforced with nanosilicates is the micropatterning technique
[141–144]. For preparing microchannel units (150 μm height) en-
capsulating hMSCs, the composite material was exposed to UV
light while using a photomask with arrays of linear microchannels.
The resultant pattern showed high interconnected porosity for all
compositions, a reduced degradation rate and an increasing com-
pressive modulus with increasing nanosilicate concentrations (from
0% to 0.5%). In vitro cytocompatibility studies with the GelMA-nSi
composite material showed uniform spreading except for the group
with 0.5% nSi. At concentrations of 0.01% and 0.05% nSi the sam-
ples did not show a difference of in vitro cytocompatibility com-
pared to the control. The same concentrations could demonstrate a
positive effect on osteogenic differentiation of hMSCs in a 3D ma-
trix. Subcutaneous implantation of the bioprinted scaffold in im-
munocompetent rats revealed the compositeś biocompatibility and
low localized immune responses indicating application potential in
the clinic, for e.g. to support bone repair [90].
Novel techniques to print complex functionalized 3D scaffolds
in a one-step procedure seem to be attractive for applications in
skeletal tissue engineering. Cidonio et al. [91] combined Laponite
R
XLG with gellan gum (GG) to receive highly stable constructs with
tunable swelling capacity printed in an agarose fluid bed as tem-
porary support before crosslinking. In vitro, printed C2C12 promy-
oblasts with sustained viability of around 80% yielded functional
capacity, as demonstrated by ALP expression, and proliferation over
an incubation period of 21 d. For the first time, the fluid bed
was used as functional loading platform for the localization of
drugs during 3D printing. Compared to pure GG, printed laponite-
GG showed promising localization and sustained release of lyso-
some and bovine serum albumin, which were used as analogues
for BMP-2 and VEGF, respectively. With a chick chorioallantoic
membrane (CAM) assay the potential of angiogenesis and integra-
tion of VEGF-containing laponite-GG printed scaffolds was evalu-
ated. Significant vascular infiltration was stimulated and guided by
laponite-containing hydrogels. Moreover, they analyzed similar is-
sues with laponite incorporated GelMA bioinks and printed them
with hBMSCs [92]. The incorporation of laponite nanoclay influ-
enced the visible-light mediated crosslinking of the 3D fabricated
constructs as demonstrated by a faster degradation compared to
pure GelMA. After parameter optimization, a shear-thinning, bio-
compatible bioink was developed with increased shape fidelity
post-crosslinking and exhibiting cell viability over a 21 d period
of incubation under cell culture conditions. Adsorption and release
of BSA and lysozyme in GelMA-laponite hydrogels revealed charge
dependent kinetics of the growth factors in digestive environment
due to interaction with the dipolar charging of laponite. Finally,
these bioinks showed promising results for stimulating angiogene-
sis, vascular network penetration and osteogenic differentiation in
the CAM assay [92].
The available studies in the literature clearly indicate the high
potential of bioinks incorporating nanosilicate fillers for bioprint-
ing and applications are expected to expand in the future. Explor-
ing different types of nanosilicates, also loaded with biomolecules,
incorporated in printable hydrogels appears to be a very promis-
ing avenue for future research. Nevertheless, long-term studies like
degradation or swelling kinetics as well as shape fidelity charac-
terizations or ion/ drug release were often done with casted disks
made from composite hydrogels so far. As 3D printed scaffolds
mostly have a more complex structure, their architecture, topol-
ogy and interconnected porosity may play an important role when
determining these properties and when designing more complex,
patient-specific tissue implants.
4. Calcium phosphate-based nanocomposite hydrogels
Calcium phosphates, especially HAp, have been intensively
investigated and used as inorganic fillers in bioprinting for
bone regeneration, since around 60 wt% of bone is made of
HAp (Ca10 (PO4 )6 (OH)2 ) [145]. Such composite hydrogels have
 S. Heid and A.R. Boccaccini / Acta Biomaterialia 113 (2020) 1–22 11

Fig. 6. A) 3D-bioprinted scaffold prepared by the two-channel method with gentian violet stained PEG-Clay and Rhodamine stained HAp. B) Live/dead assay of primary rat
osteoblasts (ROBs) 1 d after printing within PEG4K-Clay scaffolds, I) viable ROBs (green, calcein AM), II) dead ROBs (red, EthD-1), and III) merged. C) Fluorescently stained
ROBs after 3D-biofabrication (PEG4K-Clay-P) and traditional seeding (PEG4K-Clay-S) on PEG4K-Clay scaffolds after culturing for 0, 1, 3, 5, and 7 d. D) SEM image of 3D-
bioprinted ROBs in PEG4K–Clay scaffolds after 7 d of culture. The red rectangle shows ROBs that were marked with yellow color. E, I) Live/dead assay and II) cytoskeleton
staining of 3D-biofabricated ROBs within PEG4K-Clay scaffolds after 7 d of culture. F) Cell counting Kit-8 (CCK-8) analysis of ROBs after 3D-biofabrication (PEG-Clay-P) or
traditionally seeding on PEG-Clay scaffolds (PEG-Clay-S) on days 1, 3, 5, and 7 d of culture. G) ALP activity of biofabricated ROB containing PEG-Clay scaffolds after culturing
for 4, 7, 14, and 21 d. The insert images show respective ALP stainings of ROBs on I) PEG4K-Clay-P and II) PEG10K-Clay-P. Asterisks (∗ ) denote significant differences (∗ p <
0.05, ∗ ∗ p < 0.01). (Reproduced from ref. [87] according to the terms of the Creative Commons Attribution CC BY license). (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)

demonstrated very good integration with cells and with the sur-
rounding tissue, exhibiting bioactivity, osteoconductive properties
and osteogenic ability [146,147]. The strong chemical and struc-
tural similarity to the composition of the inorganic phase of bone
allows HAp to form a strong bond with surrounding bone [148].
Moreover, HAp can be used as drug delivery system or to stimu-
late the endogenous expression of osteogenic growth factors like
bone morphogenetic protein. HAp can also enhance ALP activity in
mesenchymal stem cells, which is important as ALP activity plays
a central role in the early mineralization process associated with
bone formation [57,149]. Nevertheless, the relatively slow degra-
dation and low mechanical strength limit the use of HAp as sin-
gle component material in bone tissue engineering, especially for
load-bearing applications. Consequently, HAp particles are used as
12 S. Heid and A.R. Boccaccini / Acta Biomaterialia 113 (2020) 1–22

Fig. 7. NICE bioinks combine nanocomposite reinforcement and ionic-covalent entanglement reinforcement mechanisms to create a tough, elastic, and highly printable bioink.
(a) NICE bioinks made from GelMA, κ CA and nanosilicates create a dually reinforced hydrogel network that behaves as a solid at low shear stresses and improves shear
thinning characteristics during bioprinting. Post-crosslinking, ICE and nanosilicate reinforcement synergistically improve the bioink’s mechanical strength. In TEM images
two-dimensional nanosilicate particles show a uniform morphology. (b) Encapsulated cells align parallel to the 3D printed NICE scaffolds after 30 days in culture. (c) 3D
printing of NICE bioink enables freestanding structures which are mechanically (film) and physiological (bifurcated vessel) stable and have high structural fidelity (3D printed
ear). (d) Crosslinked stiff and elastomeric structures can support more than 50-times their own weight (scale bar = 1 mm). (Modified from [89]. Copyright 2018, American
Chemical Society).

reinforcing agent in tough and flexible polymer matrices [150]. Me-
chanical properties are of interest since it has been shown that
cells differentiate according to matrix stiffness [23,151].
Nanoparticle-reinforced bioactive hydrogels often show
beneficial mechanical and biological properties compared to
microparticle-reinforced ones. To study this effect, Demirtaş
et al. [93] prepared four different bioinks – alginate, alginate-
hydroxyapatite (20 mg/ml), chitosan and chitosan-hydroxyapatite,
as shown in Fig. 8. 3% (w/v) alginate-based hydrogels were mixed
with 1% (w/v) CaSO4 to start the internal gelation for enhanced
printability. With the addition of nano-HAp the viscosity of both
bioink types increased. Mechanical properties were shown to be
higher for chitosan-HAp compared to alginate-HAp. The addition
of HAp resulted in a 3- to 6-fold increased Young’s modulus for
both hydrogels, respectively. HAp particles were also found to be
homogeneously distributed within the polymer matrix and the
bioprinted scaffolds showed overall pore structures (Fig. 9). Cell
studies with the four groups resulted in a general high viability
(average 89–93% for all samples on day 3, 90–95% after 9 days
incubation) and MC3T3-E1 pre-osteoblasts proliferated during
the 21 days of cultivation. It could be clearly seen that bone-like
nanostructured HAp increased the cell proliferation over time and
chitosan-bioinks were advantageous over alginate inks. Printed
chitosan-HAp hydrogels had peak expression levels for early and
late stage osteogenic markers and pre-osteoblasts were shown to
mineralize and differentiate osteogenically after 21 days of culture.
Adding HAp particles to polymer-based hydrogels enlarges the
range of applications, leading specifically to new approaches for
bone tissue engineering. Wenz et al. [94] added 5 wt% HAp with a
size of 50 nm - 1 μm to a GelMA/ methacrylated hyaluronic acid
and used their composite hydrogel to encapsulate primary human
adipose-derived stem cells (hADSCs). Simple grid structures with
an edge length of 10 mm were printed with hydrogels contain-
ing HAp particles (referred to as HAp-inks) and without particles
(GelMA-inks) and they were incubated under cell culture condi-
tions for 28 days. HAp-inks revealed a significant upregulation of
osteogenic markers and an extensive matrix production which ad-
ditionally led to an increase of elastic and viscous composite hy-
drogel properties. These effects were further enhanced by cultur-
ing the different inks in osteogenic differentiation medium, as dis-
played in Fig. 10. Furthermore, it was found that the way of mixing
(stirring, sonication and iterated stirring) varied the size distribu-
tion of the HAp particles strongly, ranging from a broad bimodal
distribution to distinct peaks which correlated to particle sizes of
40 nm and 1 μm.
Wang et al. [95] reported similar outcomes for sodium algi-
nate/gelatin/ nHAp composite hydrogels. In their study on the
osteogenic effects of nano-hydroxyapatite in 3D constructs, they
found that nano-hydroxyapatite could promote human adipose de-
rived stem cells (hASCs) to osteogenic differentiation in the 3D-
printed environment, which is consistent with current and pre-
vious studies [146,152,153]. With a 3D Bioplotter reticular scaf-
folds were fabricated, intended for the repair of bone tissue de-
fects. hASCs were encapsulated prior to printing as they still have
proliferation and osteogenic differentiation potential after bioprint-
ing and subsequently the 3D constructs were incubated in two
 S. Heid and A.R. Boccaccini / Acta Biomaterialia 113 (2020) 1–22 13

Fig. 8. Schematic diagrams of alginate and chitosan hydrogels as bioinks for 3D bioprinting for bone tissue engineering applications. (a) Mixing of alginate-nHAp solution
with CaSO4 solution results in a printable form of alginate solution. (b) MC3T3-E1 preosteoblast cells and nHAp are incorporated in a printable alginate-nHAp solution and
placed in 3D printer syringe. Disk-shaped scaffolds (6 mm diameter × 1 mm thickness) are printed and subsequently placed in a CaCl2 solution for fast ionic gelation. (c)
Chitosan-nHAp solution (pH: 4.0) is mixed with GP salt to generate a printable form of chitosan solution (pH: 6.95–7.0). (d) MC3T3-E1 pre-osteoblast cells and nHAp are
mixed with a printable form of chitosan-nHAp solution and placed in a syringe. A printed disk-shaped hydrogel is placed to 37 °C and 5% CO2 for thermal ionic gelation.
(Reproduced with permission from ref. [93]. Copyright 2017, IOP Publishing Ltd).

Fig. 9. SEM micrographs of hydrogels without cells and with MC3T3-E1 cells at different times of culture; alginate hydrogels (a) without cells, (e) day 7, (i) day 14 × 10 0 0
and × 2500, respectively; alginate-HAp hydrogels (b) without cells, (f) day 7, (j) day 14 × 10 0 0 and × 2500, respectively; chitosan hydrogels (c) without cells, (g) day 7, (k)
day 14 × 10 0 0 and × 2500, respectively; chitosan-HAp hydrogels (d) without cells, (h) day 7, (l) day 14 × 10 0 0 and × 2500, respectively. (Scale bars indicate 20 μm, inset
figures scale bars indicate 10 μm). (Reproduced with permission from ref. [93]. Copyright 2017, IOP Publishing Ltd).

different media. In comparison to proliferation medium, HAp par-
ticles could promote the osteogenic differentiation of hASC cells
incorporated in 3D bioprinted scaffolds when cultivated in os-
teogenic medium for 14 days. Cell viabilities after 1 and 7 days
of culture were around 88–90% and were not significantly dif-
ferent between hydrogels with and without HAp. In vivo stud-
ies in the back sub-cutaneous area of nude mice following Hall
et al. [154] demonstrated ectopic bone formation after 8 weeks
of implantation for scaffolds printed with cells. The printed
3D constructs revealed regular pores for vascularization as well
as interconnected pores for cell growth and proliferation. Nev-
ertheless, limitations for alginate/gelatin/hydroxyapatite used in
14 S. Heid and A.R. Boccaccini / Acta Biomaterialia 113 (2020) 1–22

Fig. 10. Evaluation of bone matrix formation in printed grid constructs according to ref. [94]. Hydrogel sections on days 1 and 14 or 28 of culture under osteogenic (osteo)
or control conditions (ctrl) were stained for the expression of the matrix proteins fibronectin and collagen I (day 28), as well as the bone-specific markers ALP and OPN
(day 14). (a) Scale: 200 μm. The antibody-labeled proteins are shown in red, the DAPI-stained nuclei are shown in blue. (b) Tile scans made of 4 × 5 single pictures. Scale:
20 0 0 μm. (Reproduced with permission from ref. [94]. Copyright 2017, IOP Publishing Ltd). (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

3D bio-printing technology have been noted, which mainly were
related to the relatively low mechanical properties of the hydro-
gels [155,156].
Inorganic fillers may also help to enhance the visibility and ac-
cessibility of printed and implanted hydrogel scaffolds using med-
ical detection methods. Wüst et al. [18] for example prepared 2%
(w/v) alginate/ 10% (w/v) gelatin hydrogels with final HAp concen-
trations of 8, 4% (w/v) for applications in bone tissue engineer-
ing, as shown in Fig. 11. Gelatin was added due to its thermo-
sensitive physical crosslinking behavior. When printing the com-
posite ink with a temperature-controlled printhead at 40 °C onto a
cooled surface, gelatin provided instantaneous stability, initial in-
creased viscosity and mechanical stability which led to superior
structural stability and a more accurate structure of the printed
constructs. In this approach, the long-term stability of the printed
scaffolds is provided by the slower ionic crosslinking of the al-
ginate with CaCl2 . Against the expectations, the incorporation of
varying HAp concentrations did not result in large differences of
the Youngś modulus. An additional benefit was that hydrogels con-
taining HAp showed superior radiopacity and thus visibility of the
printed scaffolds in μCT images. With respect to bioprinting and
the two-step post-processing treatment, the authors also investi-
gated the cell compatibility of their new composite hydrogel with
encapsulated human mesenchymal stem cells (hMSCs). They fabri-
cated simple cubic structures using a modified open-source two-
syringe 3D printer. The comparison of the three compositions did
not reveal any differences or reverse cytocompatibility effects with
an average cell viability of 84–85% over 3 days of in vitro cell cul-
ture.
Using the same alginate/ gelatin/ nHAp composite hydrogel,
Hernandez et al. [60] designed a hybrid system of printed poly-
caprolactone (PCL) gyroid scaffolds with infiltrated composite ma-
terial including encapsulated hMSCs for the reconstruction of large
bone defects. One scaffold could be printed in less than 10 min
and possessed a highly porous structure with pore sizes of around
400 μm and strut diameters in the range of 320 μm. Incubation in
SBF showed nucleation and growth of apatite crystals on the sur-
face after 12 days which confirmed the scaffoldś bioactivity. The
apatite formation is considered essential to support the osteogenic
differentiation of hMSCs [157,158] and hence an expedited osseoin-
tegration for in vivo applications may be expected from these scaf-
folds [159].
Related research using gelatin-alginate hydrogel with nHAp and
mouse chondrocytes for bioprinting was performed by Fan et al.
[96]. Relatively high amounts of nHAp (up to 30 wt%) resulted in
a cell viability of around 70% post-printing. Interestingly, it rose up
to 99% after 10 days of incubation in standard cell culture condi-
tions. The mechanism behind such favorable cell behavior and the
interaction between filler and cells were not further explained. Me-
chanical properties were found to be higher correlating with the
filler content and degradation could be adjusted with the addition
of bioactive fillers.
Another study which combines hydrogels and synthetic poly-
mers was conducted by Bendtsen et al. [97]. An alginate-polyvinyl
alcohol (PVA)-hydroxyapatite hydrogel for 3D bioprinting bone tis-
sue engineering constructs was designed with improved rheologi-
cal features. CaCl2 crosslinking led to shrinkage of around 24% due
to further crosslinking of the alginate chains. Degradation studies
in cell culture medium resulted in a swelling of around 32% com-
pared to nearly no swelling observed in the control (2.5% (w/v) al-
ginate with gelation controlling agents 0.15% (w/v) Na2 HPO4 and
0.20% (w/v) CaSO4 but without PVA-HAp). Swelling was caused by
an ion exchange between calcium ions crosslinked with the algi-
nate G-blocks in the egg-box structure and the sodium ions in the
 S. Heid and A.R. Boccaccini / Acta Biomaterialia 113 (2020) 1–22 15

Fig. 11. Schematic diagram of the two-step hydrogel gelation mechanism based on reversible thermal gelation of gelatin and irreversible chemical gelation of alginate.
(A) Biofabrication of heated hydrogel precursor including living cells onto a cold substrate. (B) Instantaneous gelation and primary strand stability take place due to the
temperature drop, which leads to solidification of the gelatin. (C) Post-printing immersion of the whole construct in a CaCl2 bath to crosslink the alginate present in the
hydrogel precursor. Chemical crosslinking is conducted in a cold environment to maintain construct stability until the procedure is completed. (D) Long-term stability is
ensured by the crosslinked alginate and the cooling plate can be removed. (Reproduced with permission from ref. [18]. Copyright 2014, Elsevier Ltd). (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version of this article.)

surrounding medium [160–162]. All bioprinted scaffolds remained
intact for 14 days of incubation. The cell viability of MC3T3-E1
pre-osteoblasts was assessed directly after printing (~95%) and af-
ter crosslinking inside the calcium bath (~77%) for the composite
bioink and for the control (~60% and 22.5%, respectively). Reasons
for the low viability in the control could be attributed to lacking
porosity, fast degradation or to physical forces exerted on cells.
A different approach to the ones described above is presented
in the research of Cunniffe et al. [98]. Nano-hydroxyapatite com-
plexes were used as a vector for plasmid DNA (nHAp-pDNA) de-
livery within an alginate hydrogel that was co-printed with poly-
caprolactone into a grid like structure. Bone marrow-derived MSCs
were encapsulated in the gene-activated bioink and the osteogenic
capability was analyzed over the course of 28 days in vitro as
well as during subcutaneous implantation in nude mice for 4 and
12 weeks post-printing, respectively. The polycaprolactone mesh
around the bioink enhanced the mechanical stability of the bio-
printed construct. In vitro results demonstrated sustained pro-
tein expression for up to 14 days post-printing. While nHAp has
osteoinducing properties, it effectively delivered the therapeutic
genes BMP-2 and transforming growth factor (TGF-β 3) addition-
ally to pDNA, enhancing local osteogenesis of MSCs in vitro and in-
ducing homogeneous matrix mineralization. Vascularized and min-
eralized tissues were also achieved after in vivo implantation (12
weeks) with an increased effect observed on cellularized gene-
activated bioprinted constructs compared to cell-free controls. Due
to their good printability and high cell viabilities in vitro (close to
100%) with cell spreading and osteogenic differentiation of MSCs,
these novel bioinks based on gene-activated compounds might be
superior to classical therapeutic drug or protein delivery matri-
ces and they could induce the expression of biologically functional
proteins. Hence, toxic doses or the dispersion of the drugs due to
fast degradation of the proteins could be prevented in future mus-
culoskeletal applications [98].
If the viscosity of the prepared bioink is lower compared to the
one in extrusion-based printing, Guillemot et al. [163] suggested
biological laser printing as a technique with high precision and a
good combination of different biological materials. In his studies
solutions of biopolymers, nHAp or cells were sprayed on a target
with the advantage of enabling higher fractions of cells per droplet
compared to ink-jet printing.
Besides HAp, tricalcium phosphates (TCP) were recently re-
searched in collagen and alginate bioinks as they can trigger the
differentiation of adipose stem cells without using an osteogenic
medium [99,164]. For bioprinting of mechanically stable 3D porous
structures with encapsulated MC3T3-E1 or hASCs the temperature
during bioprinting was set below the gelation temperature, which
reduced damage to the encapsulated cells resulting from high wall
shear stress in the microsized nozzle. Addition of up to 20 wt%
β -TCP revealed initial cell viabilities of more than 90% before and
after crosslinking. Pore size shrinkage correlated with an increase
of β -TCP. Compared to a control which was cultured in osteogenic
16 S. Heid and A.R. Boccaccini / Acta Biomaterialia 113 (2020) 1–22

Fig. 12. Printability of a) 5 wt% GelMA and b) Sr-GelMA bioinks (scale bars: 1 mm). Latter one can be printed into stable 3D grid structures with interconnected porosity
as optically analyzed by c) LM and d) SEM, respectively. e) Imaging of scaffold geometry throughout 28 days of in vitro culture displayed fiber shape retention of Sr-GelMA.
f) Cell viability was shown to be similar comparing hMSC-laden Sr-GelMA cast disks and bioprinted Sr-GelMA constructs which is also supported by g) live/dead staining
of bioprinted hMSCs. Osteogenic differentiation was evaluated by: h, i) Alizarin Red (red) and j) OCN (green), Col I (red) and cell nuclei (DAPI; blue) staining. Dashed white
lines indicate the edge of the Sr-GelMA filament. (Mean ± SD, n = 3 ∗ (p<0.05); Scale bars: a, b, c, e, g): 1 mm, d, h): 400 μm, I, j): 200 μm). (Modified from [100]. Copyright
2019, Elsevier Ltd). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

medium but did not contain the bioceramic inclusions, compos-
ite constructs demonstrated significant osteogenic gene expression.
It was therefore concluded that dissolution products from β -TCP,
which were released into the cell culture medium during the 21
day culture period, could promote osteogenic differentiation of the
cells present in the bioprinted composite structure [99].
The available studies on composite hydrogels incorporating CaP
particles for bioprinting anticipate that these bioinks will continue
to attract the attention of researchers, particularly for bone tissue
regeneration. Nevertheless, issues like specific ion release from the
CaP fillers and their influence on cell viability, proliferation and mi-
gration have to be examined in future studies to investigate the ef-
fect of the direct contact between reactive fillers and encapsulated
cells on the overall (time-dependent) cell compatibility of the con-
structs, which has not yet been considered in detail in the pub-
lished literature.
5. Strontium carbonate containing composite hydrogels
The alkaline earth metal strontium can normally be found in
the human skeleton, because it is a “bone-seeking element” and to
a low extent it is also present in the human plasma [165]. As ex-
amined in previous studies, low doses of strontium ranelate hinder
osteoclast activity and favor the proliferation of osteoblast cells,
leading to a dose-dependent increase in bone-volume and mechan-
ical properties [166,167]. Thus, Sr is already used as a treatment
of osteoporosis as it can integrate with bone hydroxyapatite and
reduce bone resorption by increasing the number of sites of os-
seous formation and decreasing the number of active osteoclasts
[148,168,169]. Strontium-doped bioactive glass, hydroxyapatite, and
calcium phosphate (scaffolds) therefore show great promise as
bone graft substitute materials [119,120,170–175].
Given these properties of Sr, strontium carbonate has been con-
sidered as inorganic filler in bioinks intended for bone regener-
ating constructs. Alcala-Orozco et al. recently designed a novel
bioink consisting of 5 wt% GelMA and 1.5 mg/mL crystalline stron-
tium carbonate (Sr-CO3 ) having the potential for guiding vascu-
lar ingrowth and serving as an adequate 3D matrix for cell func-
tionality (Fig. 12) [100]. Strontium carbonate was synthesized in
a self-assembly process resulting in multi-rod star-like structures
that were incorporated into GelMA precursor solution. It could be
shown that Sr did not affect the crosslinking reaction of GelMA
 S. Heid and A.R. Boccaccini / Acta Biomaterialia 113 (2020) 1–22
yielding a soft bioink with tailorable rheological properties. More-
over, holding times of 30–60 min prior to bioprinting did not
significantly affect cell viability which is important when print-
ing large-scale implants for clinical purpose. 3D bioprinted 10-
layered grid structures preserved their shape, geometry and pore
integrity during 28 days static culture in vitro with cell viabilities
above 90%. Culturing 3D bioprinted scaffolds in osteogenic medium
demonstrated that encapsulated hMSCs were able to differenti-
ate osteogenically with an increase of deposition of bone minerals
in the hydrogel matrix. For future research, the translation of Sr-
GelMA hydrogels for load bearing applications is of special inter-
est in bone tissue engineering. In this case the designed composite
inks could be combined with thermoplastic polymers or bioceram-
ics [100].
6. Discussion
By using 3D bioprinting it is in principle possible to develop
cell-laden tissue scaffolds of suitable mechanical stability, biolog-
ical compatibility and degradability, exhibiting 3D structural and
biochemical complexity to closely mimic natural tissues. In this re-
view, bioinks based on hydrogel - rigid inorganic filler composites
with bioactive properties were discussed based on the available
recent literature. Owing to their similar mineral phase to that of
the human body, certain inorganic materials such as hydroxyap-
atite, β -TCP, and bioactive glasses are known to have osteogenic
and mineralization effects and hence most reviewed studies have
focused on the development of bone tissue-like constructs. These
materials react with physiological fluids and form tenacious bonds
to hard (and in some cases soft) tissues through cellular activity
and they are therefore known as „bioactive“ [99]. In the reviewed
studies, encapsulated cells were in general not harmed neither by
the printing process itself nor through encapsulation into the 3D
composite hydrogel matrices. In such bioinks, cells showed high
viabilities after cultivation under normal cell culture conditions, as
reported in most of the reviewed studies. Nevertheless, other as-
pects specifically related to this type of hydrogel-inorganic filler
systems must be considered in more detail in future studies. For
example, the release of specific ions from the inorganic fillers must
be quantitatively studied in detail as some of them may have toxic,
antibacterial or pH enhancing effects in relation to their concentra-
tion and thus, direct contact with inorganic components in a hy-
drogel matrix may be harmful to cells. Such issues should be ad-
dressed in further studies. Indeed, bioreactive fillers behave differ-
ently to “persistent” fillers (e.g. carbon nanotubes) as they dissolve
during the time of contact with the hydrogel matrix and release
ionic dissolution products. Moreover, bioreactive inorganic particles
can be doped with therapeutic drugs or certain biologically active
ions like e.g. B3+ , Zn2+ , Sr2+ , Ag+ , Cu2+ or Mg2+ to adjust the com-
posite hydrogels to specific needs for tissue regeneration, for ex-
ample to induce angiogenesis, trigger specific cellular responses or
simply to act as crosslinking ions.
Additionally, the inorganic components are suitable to enhance
various properties of bioinks in terms of post-printing stability,
mechanical properties, printing fidelity, osteogenic differentiation
(when considered for bone tissue engineering) and vascularization.
Regarding the mechanical stability, in general, the addition of in-
organic fillers resulted in higher strength, shape recovery and en-
ergy dissipation under compressive stresses. Here, structural char-
acteristics like the particle size and the respective filler distribu-
tion in the hydrogel matrix have been recognized as important pa-
rameters affecting the mechanical behavior of the composite ink.
Moreover, the release of e.g. bivalent ions from some of the fillers
may lead to a (partial) crosslinking of respective composite bioinks
(e.g. alginate, ADA or gellan gum) over time. Nevertheless, a lo-
cal increase in pH, which might arise for example from the re-
17
lease of alkaline ions, needs to be prevented to assure sufficient
cell viability post-printing. The mechanical properties usually ex-
hibit a proportional increase with filler concentration. Moreover, a
too high concentration of inorganic particles strongly increases the
viscosity and hence leads to enhanced printing pressures as well
as high shear forces on the cells. So far, the exact mechanisms of
particle interactions with the surrounding hydrogel matrix are still
unknown. Probably, filler surface charges may interact with oppo-
sitely charged functional groups on polymer chains. Thereby, the
network and crosslinking density might be affected, leading to im-
proved printing characteristics.
The differentiation of various cell types in composite hydro-
gels is complex and depends on the filler concentration, as higher
amounts of inorganic particles lead to an increase of the stiffness
of the printed hydrogel making likely the formation of hard tissue
while low viscosity hydrogels usually promote the differentiation
of encapsulated cells to form soft tissues. Furthermore, the release
of a multitude of specific ions from the bioactive fillers like for ex-
ample silicon or calcium will have an (usually desired) influence on
cell behavior. However they could accumulate in the environment
causing local deviations from the physiological pH and potentially
significant variations in the ECM chemical composition. Dissolution
of ionic products from the surface of the bioactive filler usually
leads to mineralization of the hydrogel matrix [176]. The formation
of hydroxyapatite, which is based on the creation of silanol bonds
followed by the attachment of calcium and phosphate ions, pro-
motes protein adsorption, leading to anchorage, proliferation, and
subsequent cell differentiation [176,177]. Silicon has been shown to
enhance the formation of vacuoles in osteoblast cells [178]. Addi-
tionally, calcium phosphate-based materials can either release or
take up calcium ions from the environment. At the cellular level,
when cells attach to bioactive particles their surface characteristics
change. This approach, identified also as “cells grab on particles”,
has been recently discussed by Abalymov et al. [179]. As an exam-
ple, it is possible that cells directly attached onto inorganic fillers
will be able to get more calcium locally, compared to the calcium
concentration measured in the surrounding cell culture medium.
This process competes with the crosslinking of certain hydrogels,
like alginate, which also take up calcium. Following such complex
interactions, it is not trivial to measure the concentration of cal-
cium in the bioink, especially the free ion concentration which is
not bonded by the alginate chains and is available for interaction
with cells. Future research needs to look deeper into the inter-
action of released ions from bioreactive fillers and cell differenti-
ation in the bioink, as the process is not completely understood
so far.
Also the shape of the nanofiller is highly relevant as it has a
profound effect on mechanical properties and printability of such
composite bioinks, as clearly observed when considering nanosili-
cates/ clays. Current research on composite bioinks lacks a distinct
answer regarding the mechanisms by which filler shape and mor-
phology affect mechanical and rheological properties. Most studies
have assumed the formation of a house-of-cards structure, how-
ever this effect does not generally arise at the low concentrations
usually considered in the reviewed studies. It is possible to state
that by varying filler surface charges, orientation of particles and
release of (crosslinking) ions, the mechanical properties of the hy-
drogel network can be tuned to match the requirement of particu-
lar applications. Clearly, dedicated research is necessary to investi-
gate these complex interactions in composite bioinks.
Moreover, the design of composite materials offers an ex-
ceptional opportunity to combine biodegradability and bioactiv-
ity in optimized bioprinted tissue-engineering scaffolds. Composite
bioinks incorporating bioreactive fillers allow the creation of biore-
sorbable and bioactive structures with tailored time-dependent
physical and mechanical properties, which can be engineered in
18 S. Heid and A.R. Boccaccini / Acta Biomaterialia 113 (2020) 1–22
such a way that their resorption rate in the body matches the for-
mation rate of new tissue (exploiting the interaction of the hydro-
gel matrix and the intrinsic reactivity of the inorganic filler). As the
filler changes in shape and composition as function of time post-
printing, the overall properties of the construct vary as well and
a time-dependent behavior of the constructs is expected, which
can be tuned, for example, by considering the requirement of the
growth of new tissue.
In general, bioinks for 3D bioprinting should be adjustable to
the specific application, the operation site as well as the cho-
sen cell types to guarantee optimal tissue formation and regen-
eration. Using composite bioinks consisting of bioactive inorganic
fillers incorporated in hydrogels provides an extra degree of free-
dom to approach the complexity of natural tissues and thus leads
to promising steps towards the development of more advanced
patient-specific 3D matrices.
7. Final remarks and conclusions
We have reviewed the recent literature in the field of 3D bio-
printing of composite bioinks containing bioreactive fillers. The
main features which are required for such bioinks, consisting of
polymeric hydrogels and bioactive inorganic fillers of different
chemical composition, were presented and the impact of the filler
characteristics on various bioink properties were discussed. Exam-
ples from recent literature (the past six years) were displayed, con-
firming the great potential of such composite bioinks for bone and
cartilage repair/ regeneration, skeletal tissue engineering or drug
delivery, while more research is needed for soft tissue engineer-
ing applications. It was shown that the incorporation of bioac-
tive fillers often resulted in superior mechanical, rheological or
functional properties. Overall, bioprinted composite hydrogels have
been shown to lead to high cell viability exhibiting advantageous
impact on cell differentiation post-printing. Given the complexity
of composite bioinks, it is very important to understand the corre-
lation between the bioactive inorganic filler characteristics (e.g. ion
release, morphology) and the bioink properties as well as cell be-
havior, where deeper insights and broader knowledge need to be
gained. Such in-depth knowledge of composite bioinks and their
biological performance will allow a better comparison and tuning
of different bioink systems, with the aim to highlight the most
promising compositions and applications for 3D bioprinted con-
structs in the future.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
The authors thank the German Research Foundation (DFG); Col-
laborative Research Center SFB/TRR225 ’From the fundamentals
of Biofabrication to functional tissue models’) – project number
326998133 - TRR 225 (subproject B03) for financial support.
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