Cartilage Tissue Engineering by the 3D Bioprinting of iPS Cells in a Nanocellulose/Alginate Bioink

Abstract

Cartilage lesions can progress into secondary osteoarthritis and cause severe clinical problems in numerous patients. As a prospective treatment of such lesions, human-derived induced pluripotent stem cells (iPSCs) were shown to be 3D bioprinted into cartilage mimics using a nanofibrillated cellulose (NFC) composite bioink when co-printed with irradiated human chondrocytes. Two bioinks were investigated: NFC with alginate (NFC/A) or hyaluronic acid (NFC/HA). Low proliferation and phenotypic changes away from pluripotency were seen in the case of NFC/HA. However, in the case of the 3D-bioprinted NFC/A (60/40, dry weight % ratio) constructs, pluripotency was initially maintained, and after five weeks, hyaline-like cartilaginous tissue with collagen type II expression and lacking tumorigenic Oct4 expression was observed in 3D -bioprinted NFC/A (60/40, dry weight % relation) constructs. Moreover, a marked increase in cell number within the cartilaginous tissue was detected by 2-photon fluorescence microscopy, indicating the importance of high cell densities in the pursuit of achieving good survival after printing. We conclude that NFC/A bioink is suitable for bioprinting iPSCs to support cartilage production in co-cultures with irradiated chondrocytes.

Full text

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Scientific RepoRts | 7: 658 | DOI:10.1038/s41598-017-00690-y
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Cartilage Tissue Engineering by
the 3D Bioprinting of iPS Cells in a
Nanocellulose/Alginate Bioink
Duong Nguyen2,3, Daniel A. Hägg1, Alma Forsman3, Josene Ekholm3, Puwapong
Nimkingratana3, Camilla Brantsing3, Theodoros Kalogeropoulos1, Samantha Zaunz1,
Sebastian Concaro4, Mats Brittberg4, Anders Lindahl3, Paul Gatenholm1,5, Annika Enejder2 &
Stina Simonsson3
Cartilage lesions can progress into secondary osteoarthritis and cause severe clinical problems in
numerous patients. As a prospective treatment of such lesions, human-derived induced pluripotent
stem cells (iPSCs) were shown to be 3D bioprinted into cartilage mimics using a nanobrillated
cellulose (NFC) composite bioink when co-printed with irradiated human chondrocytes. Two bioinks
were investigated: NFC with alginate (NFC/A) or hyaluronic acid (NFC/HA). Low proliferation and
phenotypic changes away from pluripotency were seen in the case of NFC/HA. However, in the case of
the 3D-bioprinted NFC/A (60/40, dry weight % ratio) constructs, pluripotency was initially maintained,
and after ve weeks, hyaline-like cartilaginous tissue with collagen type II expression and lacking
tumorigenic Oct4 expression was observed in 3D -bioprinted NFC/A (60/40, dry weight % relation)
constructs. Moreover, a marked increase in cell number within the cartilaginous tissue was detected
by 2-photon uorescence microscopy, indicating the importance of high cell densities in the pursuit of
achieving good survival after printing. We conclude that NFC/A bioink is suitable for bioprinting iPSCs to
support cartilage production in co-cultures with irradiated chondrocytes.
ree-dimensional bioprinting technology is anticipated to radically change regenerative medicine because it
would enable tissues and organs to be printed on demand1, 2. ree-dimensional bioprinting allows the distri-
bution of dierent cells and supporting biomaterials (bioink) in sophisticated ways with high spatial resolution
in order to resemble the microarchitecture of dierent tissues. In particular, bioprinted cartilage replacements
for the treatment of secondary osteoarthritis (OA) and chondral and osteochondral injuries are believed to have
the potential to nd early clinical translation, as the need is substantial and many materials suitable for bioprint-
ing have been used in FDA-approved devices/systems. Putative cartilage gras have previously been bioprinted
with human mesenchymal stem cells3, 4. Currently, autologous chondrocyte implantation (ACI) is a cell-based
procedure with a clinically acceptable outcome; however, patients are subjected to two surgical procedures, and
healing is dependent on the quality and quantity of the patients’ autologous cells5–7. Since cartilage is immuno-
privileged, heterologous cells can be used in graing; thus, we investigated whether an established and dened
human-derived induced pluripotent stem cell (iPSC) line8 could be bioprinted, with the advantages that such a
technique reduces the need for multiple surgical procedures and oers coherent and controllable cell responses
as well as unlimited supplies. Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal multipotent
cells that can be isolated from bone marrow, adipose- and synovial tissue, Wharton’s jelly/umbilical cord and
many other connective tissues. MSCs can dierentiate into cells of the mesodermal lineage, giving rise to a range
of specialized connective tissues, including bone, adipose tissue and cartilage. However, transplanted MSCs pref-
erentially dierentiate into bone, in contrast to transplanted chondrocytes, which tend to mature into cartilage9.
Recently, the healing eects of MSCs have been explained by the ability of MSCs to interact with immune cells,
13D Bioprinting Center, Dept. of Chemistry and Chemical Engineering, Chalmers University of Technology,
Gothenburg, Sweden. 2Chemical Biology, Dept. of Biology and Biological Engineering, Chalmers University of
Technology, Gothenburg, Sweden. 3Institute of Biomedicine at Sahlgrenska Academy, Department of Clinical
Chemistry and Transfusion Medicine, University of Gothenburg, Gothenburg, Sweden. 4Cartilage Repair Unit,
University of Gothenburg, Region Halland Orthopaedics, Kungsbacka Hospital, Kungsbacka, Sweden. 5Wallenberg
Wood Science Center, Chalmers University of Technology, Gothenburg, Sweden. Correspondence and requests for
materials should be addressed to S.S. (email: stina.simonsson@gu.se)
Received: 11 November 2016
Accepted: 3 March 2017
Published: xx xx xxxx
OPEN

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Scientific RepoRts | 7: 658 | DOI:10.1038/s41598-017-00690-y
leading to the modulation of inammatory conditions such as OA. Allogenic MSCs have been used recently in
combination with autologous chondrons for the treatment of cartilage lesions10. Here, we used both iPSCs that
originated from chondrocytes and the iPS generation process to rejuvenate the cells into the blastula stage of
development, which means that they are pluripotent and can give rise to any cell type in the body, including nerve
cells11, MSCs or chondrocytes. Dierentiation protocols for directing pluripotent stem cells toward the chondro-
genic lineage are emerging, and the most robust protocol to date has been co-culturing with chondrocytes mitot-
ically inactivated by irradiation, which are called iChons here and which diminish with time12. Newer protocols
have emerged, but these include uorescence-activated cell sorting (FACS), which of course is impossible for
encapsulated cells aer 3D bioprinting.
Cell viability, as well as the ability to print bioinks and maintain 3D structures long term, were investigated
in two dierent nanobrillated cellulose (NFC) compositions with either alginate (A) or hyaluronic acid (HA)
hydrogels. NFC provides structural and mechanical support for forming the physiological mimetic environment.
In the case of cartilage, the NFC mimics the bulk collagen matrix, alginate simulates proteoglycans, and hyalu-
ronic hydrogel substitutes for the hyaluronic acid found in cartilage. Alginate and nanobrillated cellulose, both
of which are xeno-free and FDA-compliant materials, have previously been used in non-printed 3D cultures of
iPSCs for expansion and dierentiation towards the chondrogenic lineage13–15. Plant-derived NFCs have been
shown to successfully maintain iPSC pluripotency and clustering into spheroids13, while alginate maintains iPSCs
by its gentle encapsulation into microcapsules, forming clustered spheroids14. Hyaluronic acid-based hydrogels
represent another category of FDA-compliant materials, with HA being a major component in native cartilage.
ese hydrogels have been shown to encapsulate iPSCs well enough for injecting into structures with desired
architectures, sustaining stem cell pluripotency, and supporting dierentiation in 3D16. ese biomaterials were
all found to support spatial distribution and to promote the expansion and maturation of iPSC populations in
long-term cultures for up to 3–4 months17–19. In addition, combinations of these materials have viscoelastic prop-
erties that allow rapid prototyping via the inkjet printing of dened architectural constructs16, 20. To date, the
combinations of these hydrogels (NFC/A and NFC/HA) have not been used to bioprint iPSCs. In this study, we
3D bioprinted iPSCs to test these hydrogel combinations for cartilage regeneration.
Results
Protocols used to induce the dierentiation of the iPSCs in the bioprinted materials were inspired by current
strategies with the highest success rate, relying on co-culturing with irradiated mature chondrocytes (iChons)8.
Growth factors combined with a 3D environment are essential for directing iPSCs towards the chondrogenic
lineage. Factors such as TGFβ1, TGFβ3, GDF5, and BMP2 have been found to be crucial for the production of the
important hyaline cartilage matrix components collagen type II, IX, and XI and aggrecan21–23.
To ensure the formation of a functional mimic of cartilage tissue, visualization of the 3D arrangement of the
extracellular matrix (ECM) and living cells in native hydrated conditions is central. Hence, bright-eld micros-
copy was complemented with nonlinear microscopy to simultaneously acquire second-harmonic generation
(SHG) images of collagen and two-photon excited autouorescence (TPEF) images of living cells in unlabeled,
printed constructs24.
To ensure that the bioinks were compatible with the iPSC phenotypic properties, 2D monolayer culturing was
conducted on the bioprinted materials with dierent dry weights or volume percent ratios of the structural (NFC)
and cell-supporting (A or HA) components together with the crosslinking solutions (Fig.1A and Supplementary
Table1). Brighteld microscopy images conrmed no morphological or proliferative changes for the NFC/A
60/40 wt% and NFC/A 80/20 wt% bioinks with the CaCl2 crosslinking solution compared to unprinted controls.
Positive staining for iPSC marker OCT4 was indicative of cellular pluripotency for the cells in culture with the
NFC/A bioink treatment. When in contact with the NFC/HA bioinks, the cells were rounded, with low positive
OCT4 staining; hence, this material and the crosslinking conditions induced phenotypic changes of the cells away
from pluripotency (Fig.1A). e crosslinking agent, H2O2, which was used for NFC/HA, was not the cause of the
reduction of OCT4 since H2O2 exposure alone did not reduce OCT4 staining (Supplementary Fig.1).
The proliferation of iPSCs encapsulated in different bioink compositions and maintained in DEF-CS
medium was examined for one week to ensure that the material was compatible with supporting 3D expansion.
Observations made using bright-eld microscopy demonstrated the dierences in cell distribution and cluster
sizes (Fig.1B, Supplementary Table1, and Supplementary Fig.2). e NFC/HA bioink showed little to no prolif-
eration of the limited cell population remaining in the construct aer encapsulation. e NFC/A 80/20 wt% con-
structs contained an even distribution of cells that proliferated into smaller, elongated and more irregular-shaped
cell clusters. e greatest amount of growth and largest spherical clusters were observed within the NFC/A
60/40 wt% constructs by bright-eld microscopy as well as aer sectioning and staining with Alcian blue-van
Gieson (Fig.1B and C and Supplementary Table1).
Aer three weeks of growth factor-mediated dierentiation (with prior maintenance of iPSCs in the hydrogels
in DEF-CS for one week), RNA expression showed phenotypic increases in chondrogenic markers for all three
types of bioinks (Fig.1D). Hence, the NFC/HA bioink still seemed promising for use in further experiments if an
increase in cell number could be achieved through improved proliferation and delivery. e iPSCs printed with
the NFC/A 60/40 wt% bioink showed better survival than iPSCs printed in the NFC/A 80/20 wt% bioink (Fig.1E;
more live cells (stained green) are seen in the NFC/A 60/40 wt% bioink, and increased survival was observed with
time, Supplementary Fig.3). Hence, using a lower concentration (60 wt%) of NFC yielded a higher number of
viable cells, although a higher concentration (80 wt%) of NFC yielded more stable and dened printed constructs
(as can be seen in Fig.1E, lower; the printed grid lines using NFC/A 60/40 did not retain the same thickness and
homogeneity compared to those using 80/20, Supplementary Fig.4).
To direct iPSCs towards chondrocytes, we used a combination of growth factors (in short, Wnt 3a and Activin
A for 3 days followed by GDF5 + BMP2 + TGFβ1 for up to ve weeks) printed in the NFC/HA (Supplementary

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Figure 1. Material compatibility and cell pluripotency of dierent bioinks. (A) Bright-eld and uorescent
images at day 2 of the iPSCs being in contact with the three bioink compositions: (1) NFC/A 60/40 crosslinked
with 100 mM CaCl2 solution, (2) NFC/A 80/20 crosslinked with 100 mM CaCl2 solution, and (3) NFC/HA
crosslinked with 0.001% H2O2 solution (the scale bars represent 50 μm). Cell morphology and Oct4-positive
staining (orange) indicated the compatibility and inertness of both NFC/A treatments. However, NFC/
HA treatment changed the cell morphology to be spherical with less Oct4 staining and less cells. (B–D)
Encapsulation of iPSCs in bioinks for a three-week dierentiation period resulted in dissimilar cell distribution
and dierentiation phenotypes. (B) e NFC/A 60/40 bioink had a greater amount of clusters with larger
diameters (each black arrow points to one cluster) compared to the 80/20 bioink (each gray arrow points to a
cluster). By contrast, the NFC/HA bioink had minimal cell clusters (each white arrow points to one of the low-
density cell clusters) (the scale bar represents 500 μm). (C) Alcian blue-van Gieson-stained histological sections
revealed more rounded clusters in the NFC/A 60/40 bioink compared to the elongated clusters in the NFC/A
80/20 bioink and the lack of cells and clusters in the NFC/HA bioink. (D) Furthermore, the three bioinks
supported the phenotypic expression of SOX9, aggrecan, and collagen type 2A1. (NFC/HA samples at week 3
were lacking due to the small amount of RNA). (E) At day 5 aer printing, the NFC/A 60/40 bioink composition
was superior for cell viability compared to the NFC/A 80/20 bioink, as shown in the wide-eld uorescence
images with live staining (green, live) (the scale bars represent 200 μm). Confocal images (upper right corners)
show signs of cell proliferation in the NFC/A 60/40 bioink with multiple cells in a cluster (DAPI, blue; actin,
green) (the scale bar represents 50 μm).

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Fig.5) or NFC/A bioink or co-cultured with irradiated chondrocytes (iChons) using the growth factors GDF5,
BMP2, TGFβ1 and TGFβ3. First, the iPSCs and the iChons were printed in separate strands in an overlapping
grid structure (Supplementary Fig.6A). e production of GAGs was moderately increased in the intersec-
tions, indicating that crosstalk between chondrocytes and iPSCs was benecial (Supplementary Fig.6B and C).
erefore, iPSCs and iChons were mixed at a ratio of 1:1 and printed together. Clones of the cells appeared 1
week aer printing, suggesting cell proliferation and clonal expansion (Fig.2A, confocal microscopy images).
e co-cultured printed constructs intentionally had twice the cell density from the start compared to the control
constructs with only iPSCs or iChons to maintain a comparable constant iPSC density due to the mortality of
the iChons (Fig.2B). e iChons control constructs showed a decreasing number of visible cells over time when
analyzed by TPEF microscopy and were diminished aer 3 weeks in culture (Fig.2B, iChons, and Supplementary
Fig.6). In agreement with previous ndings that the use of irradiation to make chondrocyte replication incom-
petent also causes apoptosis, all cells were dead within 25 days of irradiation12. Fluorescence in situ hybridization
(FISH) analysis of the 3D-printed constructs aer 5 weeks (Fig.2C) further supported the disappearance of
iChons over time. is observation implies that the iPSCs are the most likely source for cartilage matrix forma-
tion. e co-cultured printed protocol gave rise to the appearance of hyaline-like cartilaginous tissue at week 5,
which was visualized by (i) Alcian blue-van Gieson staining of collagen, connective tissue and acidic polysac-
charides, such as glycosaminoglycans in cartilage (blue), and (ii) Safranin-O staining, which stains cartilage red
(Fig.3B). Furthermore, areas with dark staining indicated hyaline cartilage tissue generation that was similar
to stained native cartilage tissue and, in areas of increased Alcian blue-van Gieson staining, nuclei clusters with
lacunae were seen (Fig.3B, zoomed in, upper row). An increase in cell number was correlated with higher GAG
production (Fig.3B and C (TPEF, cells are yellow in 3C), corresponding areas in an unstained serial section are
marked with red and green squares). While no signicant dierence in the SHG signal could be detected between
the areas (Fig.3C), the production of cartilage-specic collagen type II was evident by immunohistochemistry
using a specic antibody for collagen type II (Fig.3D, green; and Supplementary Fig.7A) and was surprisingly
more intense than the human control cartilage (Supplementary Fig.7B), further suggesting that cartilage-like
tissue had been generated aer ve weeks of dierentiation. e immunohistochemistry results were important
since the prints containing alginate, even without cells, gave a background staining with Alcian blue-van Gieson.
e lack of signicant dierence in the SHG signal between the areas (Fig.3C) could result from the ne collagen
II brils generating such a weak SHG signal that their build-up disappears in the high background interference
from cellulose. e sensitivity of this technique could be improved by using polarization sensitive SHG micros-
copy25. We next wanted to determine whether we could generate cartilage mimic tissue from iPSCs without using
iChons. We succeeded in generating higher GAG production from iPSCs in an area (Fig.4A and B) when the
iPSC prints (60:40 NFC/A) were initially maintained in a conditioned DEF medium since single cell survival is
dependent on factors produced from other iPSCs (Fig.4C). Finally, we assessed the level of pluripotency protein
OCT4 prior to and aer printing (Fig.5). OCT4 could be detected even in the presence of iChons, which have
been shown to produce BMP212, aer 1 week of printing when maintained in the conditioned DEF medium.
OCT4 signals were not detected aer dierentiation for 3 or 5 weeks (Fig.5).
Discussion
Even when we varied the bioink composition of NFC/A, we showed that the iPSCs remained in a pluripotent state
(as determined by the OCT4 protein level). In particular, cells in the NFC/A 60/40 system were able to maintain
a pluripotent phenotype aer 3D bioprinting when using iPSC-conditioned medium, which probably helped the
cells to sense that they were not isolated. e OCT4 protein (encoded by Pou5f1) was undetectable in the prints
aer 5 weeks of dierentiation, which is important for implementing this technology in a clinical setting because
pluripotency increases the risk of potential tumor formation. Hyaline-like cartilaginous tissue expressing collagen
type II was observed in the histology sections of the 3D bioprints aer ve weeks when using the co-culturing
with irradiated chondrocytes protocol and bioink composed of NFC/A 60/40. Moreover, an increase in cell num-
ber was observed in the cartilaginous tissue within the 3D-bioprinted constructs, thus highlighting the impor-
tance of cell quantity for cartilage production. We concluded that NFC/A bioinks were suitable for bioprinting
iPSCs to support cartilage production in co-culture with irradiated chondrocytes. We noticed that aer printing,
viable cells increased in number over time, which was in agreement with previous observations with other cell
types that human chondrocytes bioprinted in noncytotoxic, nanocellulose-based bioink exhibited cell viabilities
of 73% and 86% aer 1 and 7 days of 3D culture, respectively26, 27. e positive co-culture eect supported earlier
ndings that irradiated chondrocytes would stimulate iPSC dierentiation by cell-to-cell contact combined with
local inherent growth factors8, 28. BMP2 has previously been determined to be one of the growth factors secreted
by irradiated chondrocytes12. By including this morphogene in the chondrogenic dierentiation medium, we
could enhance the co-culture protocol and detect the dierentiation of iPSCs without co-culture. GDF5, another
growth factor that we included in the dierentiation medium, also belongs to the bone morphogene protein
(BMP) family. Determining which growth factors to include or exclude, along with the timing and concentration
of growth factors, is key to further improving these types of protocols. In summary, the identication of a proto-
col using NFC/A that aer 3D bioprinting, directed iPSCs into chondrogenic commitment could permit further
delineation of the molecular pathway to optimize and improve articular cartilage tissue generation. We believe
that this research will help bring forward 3D bioprinting with iPSCs as a future treatment to repair damaged
cartilage in joints.
Methods
A bioink composed of nanocellulose and alginate was prepared for 3D bioprinting. Dierent fractions of nanocel-
lulose and alginate were prepared for cell encapsulation, and the viability of the cells was evaluated aer printing.
Cell morphology and distribution were analyzed by nonlinear microscopy. iPSCs were mixed with irradiated

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Figure 2. Co-culture increased iPSC density in the NFC/A 60/40 bioink aer printing. (A) Confocal
microscopy images of the NFC/A 60/40 co-culture samples at week 0 and week 1 stained for actin (green) and
nuclei (blue) show cells evenly distributed with cluster formation aer week 1, thus indicating proliferation (the
scale bar represents 50 μm). (B) Label-free nonlinear microscopy (two-photon excitation uorescence, with the
autouorescence of cells shown in yellow) images show similar distributions in the co-cultures compared to
the confocal images. However, a decrease in the cell number was seen over time for the irradiated chondrocyte
(iChons)-only prints as expected (co-culture prints were conducted with a 1:1 ratio of iPSC to iChons) (the
scale bar represents 50 μm). (C) Fluorescence in situ hybridization (FISH) stained only the X chromosomes (X
chromosomes, green; Y chromosomes, red) in the co-cultured cartilage-like tissue. Female line, iPSCs; male,
iChons (the scale bar represents 10 μm).

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Figure 3. ree-dimensional-bioprinted cartilage-like tissue. (A and B) Histology sections of the 3D-bioprinted
constructs. (A) At week 3 (blank - no cells), week 0, week 1, and week 2 of dierentiation, which followed 2
weeks of proliferation in the iPSC maintenance medium (stained with Alcian blue-van Gieson for proteoglycans/
glycosaminoglycans (GAGs) (blue) and nuclei (brown)) (the scale bar represents 100 μm). (B) e 3D-bioprinted
chondrocyte-derived iPSCs (printed together with iChons, which had been diminished) at week 5 of
dierentiation, zoomed in (upper row) and whole section (lower row) images of sections stained for GAGs,
Safranin O for cartilage (with nuclear counterstain), and hematoxylin and eosin (H&E) for extracellular matrix
(with nuclear counterstain) (the scale bar represents 100 μm or 500 μm). (C) Label-free images of unstained
sections (of areas corresponding to red and green boxes from the lower row of B) shows highly dense cell areas
(cell autouorescence, yellow) and collagen-like brils (second-harmonic generation, cyan). e highly dense cell
area in the red box corresponded to higher GAG staining (the scale bar represents 50 μm). e number of cells per
ml was calculated from the high-density (red square) and low-density (green square) areas. (D) Fluorescent image
of an immunohistochemistry section (from the same 3D printed sample as B and C) stained for collagen type II
(green) (with nuclear counterstain shown in blue), which shows the production of extracellular matrix collagen
type II in a representative cell cluster (the scale bar represents 10 μm).

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chondrocytes (to acquire a chondrocyte-free print, the chondrocytes were irradiated (25 Gray) prior to mixing)
and 3D bioprinted to direct dierentiation towards cartilage. Up to 6 weeks aer printing, the 3D bioprinted
constructs were sectioned and stained with Alcian blue-van Gieson.
Fabrication of bioinks. e bioinks were prepared as described previously26. Briey, nanobrillated cellu-
lose (NFC) was produced using mechanical renement and enzymatic treatment29. NFC was sterilized using elec-
tron irradiation at 25 kGy. Sterile alginate (150–250 kDa; FMC Biopolymers, Norway) with ≥60% α-1-guluronic
Figure 4. ree-dimensional-bioprinted cartilage-like tissue from iPSCs excluding iChons. (A and B)
Histology sections of 3D-bioprinted constructs at week 6 (week 1 proliferation in conditioned DEF medium for
the iPSCs plus 5 weeks of chondrogenic dierentiation with TGFβ1, TGFβ3, GDF5 and BMP2) (stained with
Alcian blue-van Gieson for proteoglycans/glycosaminoglycans (GAGs) (blue) and nuclei (brown)) (the scale
bars represent 500 μm in A and 100 μm in B). (C) Cell survival of single iPSCs in the DEF medium or in the
iPSC-conditioned DEF medium.
Figure 5. Pluripotency marker Oct4 was still expressed 1 week aer 3D bioprinting the iPSCs with iChons
in conditioned DEF medium, and the diminishing of Oct4 was seen aer chondrogenic dierentiation. (A)
Western blot of cells before printing in the primary chondrocytes passage 1 before irradiation, in hESCs (human
embryonic stem cell line SA121 passage 17), and in chondrocyte-derived iPSC line A2B in DEF xeno- and
feeder-free passage 31 or DEF feeder-free culture at passage 17. No expression of pluripotency markers was
detected in the chondrocyte cultures. β-Actin was used in the western blot analysis to show equal loading.
(B) Immunohistochemistry for Oct4 and nuclei DAPI 1, 4 and 6 weeks aer printing the iPSCs with iChons
and maintaining the samples in iPSC medium for the rst week, followed by the induction of chondrogenic
dierentiation (the scale bar represents 50 μm).

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acid was used for the bioink. e composition of the bioink was calculated by the weight percent of the complete
hydrogel. To obtain the physiological osmolarity, 4.6% mannitol was added to the hydrogel solution30.
Corgel® Biohydrogel, a hyaluronan-based hydrogel (HA) with 5% tyramine substitution, was purchased from
Lifecore Biomedical (MN, USA). A 5% (volume %) nal hydrogel of HA in nanocellulose was produced for the
encapsulation or printing of iPSCs and/or irradiated chondrocytes. Aer the constructs were formed or printed,
a solution of 0.001% (v/v) H2O2 in water was used for crosslinking for 5 minutes.
Culture of human iPSCs and irradiated chondrocytes. We previously generated iPSC lines from sur-
plus chondrocytes using mRNA-based reprogramming6. e A2B iPSC line was maintained under feeder-free
conditions in Cellartis DEF-CS™ (TaKaRa ClonTech, Sweden). is iPSC line was karyotype-tested, was normal
even at late passages, was pluripotent with regards to the expression of pluripotency markers and was able to
dierentiate into all germ layers6 (Supplementary Fig.8). is line was also shown to be superior in the dieren-
tiation protocol to generate articular cartilage matrix in 3D pellets and was used for 3D printing in subsequent
experiments. In addition, iPSC-conditioned DEF medium from conuent clone A2B iPSCs was used aer print-
ing since increased survival has been noticed for single cells in a conditioned medium. For co-culture conditions,
human surplus chondrocytes were irradiated (iChons) before being mixed with iPSCs to prevent the prolifer-
ation of the chondrocytes. e cell number was counted in a nucleocounter NC-200TM using Via1-CasettesTM
(ChemoMetec, Denmark).
Three-dimensional bioprinting. The final concentration of iPSCs and/or irradiated chondrocytes
(iChons) was 20 million cells per ml of bioink. A 3D Discovery (regenHu, Switzerland) 3D bioprinter was used
with a 300-µm nozzle. e grid construct was designed using the BioCAD soware (Biomedical Modeling Inc,
USA) and was printed using the 3D Discovery HMI soware. Printing parameters were set to 10–20 mm/s feed
rate, 20–30 kPa pressure, and 0.05–0.07 mm dosing distance. Prior to printing, the apparatus was sterilized
using 70% ethanol, and ltered air (0.22 µm) was used to reduce the risk of contamination. Printing was per-
formed at room temperature in an open area within a clean room. e printing occurred at ambient temperature
and humidity, and six-layer grids were printed into 24-well plates. e grids were 7 by 7 mm and 1.2 mm thick
(Supplementary Fig.4). Immediately aer printing, the NFC/A constructs were crosslinked in 100 mM CaCl2
solution for 5 minutes, and NFC/HA constructs were crosslinked with a water solution of 0.001% (v/v) H2O2 for
5 minutes (iPSCs controls, Supplementary Fig.1). e HA crosslinking occurred at the substituted functional
tyramine group, where a covalent bond at the tyramine carbon ring was formed. Aer crosslinking, the constructs
were briey rinsed in the culture medium, which was replaced with fresh medium. e constructs were thereaer
transferred to an incubator held at 37 °C and 5% CO2. e rheological properties, shape delity and mechanical
strength of the 60/40 and 80/20 bioinks have been previously tested26.
Directed chondrogenic differentiation of iPSCs in 3D-printed constructs. Equal numbers of
iPSCs were mixed with iChons to give a nal concentration of 20 million cells/ml in the bioink, and the printed
constructs were maintained at 37 °C and 90% humidity in 5% CO2. Aer 7 days of culture in the DEF CS™
(TaKaRa ClonTech, Sweden) pluripotent medium mixed with an equal volume of conditioned DEF-CS medium
(conditioned DEF medium was taken from 80% conuent pluripotent iPSCs aer 24 hours of culture, sterile
ltered, and fresh growth factors (GF1, GF2 and GF3; TaKaRa ClonTech, Sweden) were added), the medium
was changed every day. Cells were then initiated to dierentiate by changing the medium to a dened chon-
drogenic medium (high-glucose Dulbecco’s modied Eagle’s medium; PAA Laboratories) supplemented with
5.0 μg/ml linoleic acid solution (Sigma-Aldrich), 1x ITS-G premix (10 mg/l insulin, 5.5 mg/l transferrin, 6.7 μg/l
selenious acid; Life Technologies), 0.11g/l sodium pyruvate,1.0 mg/ml human serum albumin (Equitech-Bio,
TX, USA), 10 ng/ml TGFβ1 (R&D Systems, Abingdon, UK), 10 ng/ml GDF5, 10 ng/ml BMP2, 100 nM dexa-
methasone (Sigma-Aldrich), 80 μM L-ascorbic acid (Sigma-Aldrich), and 1x penicillin/streptomycin (PEST; PAA
Laboratories). e medium was changed three times a week. Relevant control cultures with only printed irradi-
ated chondrocytes were kept throughout the dierentiation protocol.
Histological preparations. Samples were rinsed twice with PBS containing CaCl2 before xation with
Histox (5% paraformaldehyde; HistoLab Products AB, Sweden) for 20 minutes. CaCl2 prevented the prints from
disintegrating. Aerwards, the samples were rinsed twice with PBS before being stored in 100 mM CaCl2 for
transport to HistoLab (Gothenburg, Sweden) for paran embedding, slicing (10-μm sections), and staining with
Alcian blue and van Gieson’s dye for glycosaminoglycans, Safranin O for cartilage-like extracellular matrix pro-
duction, and hematoxylin and eosin for nuclei and matrix components. An upright Nikon Eclipse 90i microscope
was used to obtain images of the histology slices.
FISH analyses for chromosomes X and Y were performed on histology sections at the Department of Clinical
Chemistry at Sahlgrenska University Hospital.
Collagen II immunohistochemical analysis. Histology sections of the 3D prints were deparanized,
rehydrated and treated with 8,000 U/ml of hyaluronidase (Sigma-Aldrich, St. Louis, MO) in PBS for 60 minutes
at 37 °C. e sections were blocked with 3% bovine serum albumin (Sigma-Aldrich) in PBS and incubated with
a primary mouse anti-human collagen II antibody, diluted 1:150 (MP Biomedicals Europe, Illkirch, France),
at 4 °C overnight, followed by a 2 hour incubation with a secondary antibody at room temperature in darkness
using goat anti-mouse Alexa Fluor 488 diluted 1:400 (A11017; Life Technologies). Samples were mounted using
ProLongGold Antifade that included DAPI (4,6-diamidino-2-phenylindole; Life Technologies) to visualize
nuclei. Samples were observed using a Nikon Eclipse Ti-U uorescence microscope.

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Scientific RepoRts | 7: 658 | DOI:10.1038/s41598-017-00690-y
Pou5f1 (Oct4) immunohistochemical analysis. Samples stained for Oct4 were (i) iPSCs seeded on coat
1 (TaKaRa ClonTech, Sweden) and exposed to NFC/A or NFC/HA for 2 days in the DEF medium (TaKaRa
ClonTech, Sweden), and (ii) serial sections from the 3D prints that were rst deparanized. e primary anti-
body used was rabbit anti-Oct4 diluted 1:400 (C30A3; Cell Signaling Technology, Danvers, MA, http://www.
cellsignal.com). e secondary antibody used was goat anti-rabbit Alexa Fluor 546 diluted 1:400 (A11071; Life
Technologies).
Microscopy. Microscopy images were taken of samples with (i) live/dead staining on a wide-eld uores-
cence microscope; (ii) actin/nuclear staining on a confocal microscope; (iii) no labels on a nonlinear optical
microscope, which has been described previously31; and (iv) bright-eld and uorescence images on a Nikon
Eclipse Ti-U with an attached Andor Zyla camera.
Label-free imaging of cellular intrinsic uorescence and matrix anisotropy was performed using two-photon
excitation uorescence (TPEF) and second-harmonic generation (SHG). A diode-pumped solid-state laser
(Nd:vanadate, 10 W) was used to generate two-picosecond pulsed beams: 1064 nm and 532 nm (7 ps, 76 MHz).
e 532 nm beam was guided into an optical parametric oscillator (Levante Emerald OPO, APE Berlin, 690–
900 nm) to generate a beam at 817 nm. Laser beams were directed onto samples mounted on an inverted micro-
scope (Eclipse TE2000-E with a C2 confocal microscope scanning head, Nikon) with a 40x oil-immersion
objective (Nikon Plan Fluor, NA 1.30). TPEF and SHG signals were obtained with 405 ± 10 nm and 609 ± 57 nm
optical-density lters, respectively. Single photon counters from Becker & Hickl Gmbh were used to detect TPEF
and SHG simultaneously. Image analysis and 3D rendering were performed with ImageJ (National Institutes of
Health, USA).
RNA extraction and RT PCR (Reverse transcription-polymerase chain reaction). Samples were
frozen at −80 °C at various time points for RNA extraction. Lysis of the construct was performed with RLT buer
from the Qiagen Mini-Kit and Matrix Lysis D (MP Biologics), which was shaken at 25 Hz for 2 minutes on a
Qiagen Tissue Lyser. e lysate was then used for RNA extraction following the standard protocol of a Qiagen
Mini Kit. e RNA concentration and quality were obtained immediately aer extraction using the NanoDrop
2000 (ermo Fisher). For cDNA synthesis and quantitative reverse transcriptionase polymerase chain reaction
(PCR), all reagents, instruments, and soware were purchased from Applied Biosystems (Life Technologies).
e cDNA was prepared from total RNA using a High-Capacity cDNA Reverse Transcriptase Kit with random
hexamers and RNase Inhibitor on a 2720 ermal Cycler. All samples were analyzed in duplicate on the 7900HT
instrument using TaqMan Gene Expression Master Mix. e following human TaqMan gene expression assays
were used: SOX9 (Hs00165814_m1), COL2A1, splice variant (Hs01064869_m1), and ACAN (Hs00153936_m1).
CREBBP (Hs00231733_m1) was used as a reference gene. All samples were treated with RNase-Free DNase
(Qiagen Gmbh, Germany) to avoid genomic DNA contamination. e fold change for each sample was calcu-
lated using the 2−ΔΔCT method32, 33, and the expression level was calculated relative to an in-house calibrator.
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Author Contributions
D.N. and S.S.: Conception and design, collection and assembly of data, data analysis and interpretation,
manuscript writing, and nal approval of the manuscript; D.H., A.F., J.E., P.N., C.B., S.Z. and T.K.: Collection
and assembly of data, data analysis and interpretation, and nal approval of the manuscript; S.C., A.L. and M.B.:
Conception and design, provision of study material or patients, and nal approval of the manuscript; M.B.,
A.L., P.G., A.E. and S.S.: Conception and design, data analysis and interpretation, nancial support, manuscript
writing, and nal approval of the manuscript.
Additional Information
Supplementary information accompanies this paper at doi:10.1038/s41598-017-00690-y
Competing Interests: e authors declare that they have no competing interests.
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