807108

research-article2019
JBF0010.1177/2280800018807108Journal of Applied Biomaterials & Functional MaterialsStealey et al.

JABFM Journal of Applied

Biomaterials &
Functional
Original Article Materials

Journal of Applied Biomaterials &

Stability improvement and

Functional Materials
January-March: 1­–5
© The Author(s) 2019
characterization of bioprinted Article reuse guidelines:
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pectin-based scaffold DOI: 10.1177/2280800018807108

https://doi.org/10.1177/2280800018807108
journals.sagepub.com/home/jbf

Samuel Stealey1, Xiaoru Guo2, Lixia Ren3, Elizabeth Bryant4,

Matey Kaltchev1, Junhong Chen2, Subha Kumpaty5,
Xiaolin Hua6 and Wujie Zhang1

Abstract
Purpose: Bioprinting is an alternative method for constructing tissues/organs for transplantation. This study investigated
the cross-linker influence and post-printing modification using oligochitosan and chitosan for stability improvement.
Methods: Oligochitosan was tested as a novel cross-linker to replace Ca2+ for pectin-based bio-ink. Oligochitosan (2
kD) and different molecular weight of chitosan were used to modify the bioprinted scaffold. Fourier transform infrared
(FTIR) spectroscopy and scanning electron microscopy (SEM) were used to characterize the scaffolds.
Results: Oligochitosan failed to serve as a viable cross-linker. Successful post-printing modification was confirmed by
FTIR and SEM analyses.
Conclusion: Regarding post-modification, chitosan-treated scaffolds showed enhanced stability compared to untreated
scaffolds. In particular, scaffolds modified with 150 kD chitosan exhibited the highest stability.

Keywords
Bioprinting, bio-ink, pectin, hydrogel, scaffolds, oligochitosan, chitosan

Date received: 17 November 2017; revised: 20 March 2018; accepted: 4 September 2018

Introduction oligochitosan, which was previously discovered.10,11 Calcium

According to the United States Department of Health and
Human Services, over 116,000 people are on the organ
transplant waiting list, and each day 20 people die while
waiting for a transplant.1 Bioprinting of artificial tissues
and organs provides a method for stemming this discrep-
ancy between organ donors and those waiting for a trans-
plant.2,3 These tissues and organs can be engineered using
ions can be toxic at elevated levels within the body,12
1Physics and Chemistry Department, Milwaukee School of Engineering,
Milwaukee, WI, USA
2Mechanical Engineering Department, University of Wisconsin-
Milwaukee, Milwaukee, WI, USA
3School of Materials Science and Engineering, Tianjin University, Tianjin, China
4Computer Science and Engineering Department, LeTourneau

a bio-ink made of biopolymers for scaffolding incorpo- University, Longview, TX, USA
rated with the patient’s own cells.4,5 5Mechanical Engineering Department, Milwaukee School of Engineering,

During a previous study, a novel pectin-based bio-ink and
corresponding printing method was developed.6 Pectin shows
similar properties to alginate, a common polysaccharide used
for constructing scaffolds, but is preferable to alginate, which
may lead to allergic reactions.7 Pectin is a natural biopolymer
found in citrus rinds and is composed of galacturonic acid with
carboxyl groups. Because of these negatively-charged carboxyl
groups, pectin can be cross-linked with divalent cations to cre-
ate hydrogel scaffolds. Ca2+ has commonly been used for such
cross-linking purposes.8,9 Another potential cross-linker is
Milwaukee, WI, USA
6Department of Obstetrics and Gynecology Xinhua Hospital, Shanghai
Jiao Tong University School of Medicine, Shanghai China
Corresponding authors:
Wujie Zhang, Department of Physics and Chemistry, Biomolecular
Engineering Program, Milwaukee School of Engineering, 1025 N
Broadway, Milwaukee, WI 53202, USA.
Email: zhang@msoe.edu
Xiaolin Hua, Department of Obstetrics and Gynecology, Shanghai Jiao
Tong University School of Medicine, Shanghai, China.
Email: huaxiaolin@xinhuamed.com.cn

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2 Journal of Applied Biomaterials & Functional Materials 00(0)
Figure 1.  (a) Top and side view of pectin/Pluronic F-127 extrusion-based bioprinted scaffolds: cross-linked with calcium (left
panel), cross-linked with calcium and further treated with oligochitosan (middle panel), and cross-linked with oligochitosan (right
panel). (b) Top view of Pectin/Pluronic F-127 bioprinted kidney-shaped scaffolds cross-linked with calcium.
while oligochitosan has been demonstrated to show excellent
biocompatibility.13,14 In addition to cross-linking, post-printing
modification is necessary to further stabilize scaffolds. One
such modification is treating the scaffold with oligochitosan/
chitosan. Chitosan is the only naturally-occurring positively-
charged biopolymer with good biocompatibility and biodegra-
dability.15,16 Modification using polycationic polymers is
critical for tissue engineering applications, since a positively-
charged structure is preferred for cell adhesion.17–19
Materials and methods
Materials
Low-methoxyl pectin and Pluronic® F-127 were obtained
from WillPowder (Miami Beach, FL) and Sigma-Aldrich
(St. Louis, MO), separately. Pharmaceutical grade (>90%
deacetylation) oligochitosan (2–3 kD) and chitosan (50,150,
and 1000 kD) were purchased from Zhejiang Golden-Shell
Pharmaceutical Co. (Zhejiang, China). A BioBot1 bioprinter
(Philadelphia, PA) was used for bioprinting scaffolds.
Dulbecco’s Modified Eagle’s Media (DMEM) was obtained
from American Type Culture Collection (Manassas, VA).
Scaffold bioprinting process
An STL file of an object was first opened by the Repetier-
Host software. Object size and position adjustments were
made to optimize printing parameters. The object was then
sliced with Slicr software and converted into a G-Code
file. The G-Code file was then imported to the BioBot soft-
ware that controlled the bioprinter.
The bio-ink used was composed of 3% (w/v) pectin,
20% (w/v) Pluronic® F-127, and a red food coloring dye,
which was used to aid visualization.6 The bio-ink was
extruded through a 27 gauge blunt needle tip to begin the
bioprinting process with a strand diameter of approximately
0.4 mm. A printing pressure of 35 psi was used with an axis
movement velocity of 0.5 mm/s.
The bio-ink was extruded via a single strand onto a petri
dish on top of an AmScope microscope temperature control
stage slide warmer at 37°C (TCS-100; AmScope, Irvine, CA)
to induce gelation of the Pluronic® F-127 in the first few lay-
ers of the scaffold.6 Warm (~37°C) 150 mM CaCl2 was added
around the base of the scaffold during the printing process to
cross-link the pectin, further gelling the structure and provid-
ing a more permanent foundation for the remaining layers.
Post-printing modification
Once printing was complete, scaffolds were immersed in
37°C 150 mM CaCl2 for 5 min. The CaCl2 was then
removed. The scaffolds were further treated with 2 kD, 50
kD, 150 kD, and 1000 kD chitosan solution, respectively,
at a concentration of 0.25% (w/v) for an additional 5 min.
Scaffold characterization
For scanning electron microscopy (SEM) imaging, scaf-
folds were vacuum-dried and then mounted onto an alu-
minum stub. Samples were then examined under a Hitachi
S-4800 ultrahigh resolution cold cathode field emission
scanning electron microscope (FE-SEM). Dried scaffolds
were also used for Fourier transform infrared (FTIR) anal-
ysis. FTIR spectra of the samples were recorded at room
temperature using a NicoletTM 6700 spectrometer (Thermo
Fisher Scientific, Waltham, MA).
Stability testing
Scaffolds were immersed in DMEM and incubated at
37°C, with their integrity and weight being monitored at
different time intervals for a period of three days.
Results and discussion
Bioprinted pectin/Pluronic F-127 scaffolds were not
stable if no cross-linking occurred (Figure 1(a)).
Cylinder-shaped scaffolds were used to demonstrate the
Stealey et al. 3

Scheme 1.  A schematic illustration of the bioprinting process and subsequent post-printing modification.

cross-linking effectiveness, though other geometries

could be printed using the extrusion-based method
(Figure 1(b)). The initial gelation was due to heating of
the Pluronic F-127 from the heat stage on which the
petri dish was placed.6,13 However, once the scaffold
was removed from this heat the scaffold lost its defined
shape, becoming a viscous liquid rather than a solid gel.
Permanent hydrogel scaffolds can be created using
divalent cations that serve as cross-linkers for the nega-
tively-charged pectin molecules. Calcium chloride was
added around the edges of the scaffold mid-print, as
shown in Scheme 1, creating more stability than only
heat-induced gelation of Pluronic F-127. Heat-induced
gelation of Pluronic F-127 only occurred in the first few
layers of the printed scaffold; the rest of the scaffold
was gelled with calcium.6 Using oligochitosan as a
novel cross-linker was found to be ineffective, unfortu-
nately, as scaffolds quickly reverted to viscous liquid
state (Figure 1(a)). It is thought that oligochitosan did
not cross-link the pectin in the same manner as Ca2+.11,20
Pectin cross-linking with Ca2+ results in an “egg-box”
like structure caused by intermolecular chelate binding,
while pectin cross-linking with oligochitosan results in
a polyelectrolyte complex.11 Figure 2.  Fourier transform infrared (FTIR) spectra of
As shown in Figure 2, all post-printing modified sam- bioprinted scaffolds.
ples showed similar FTIR spectra due to the strong hydro-
gen interaction of pectin and chitosan both intramolecularly 1680 cm-1 represent the free amines (NH2), which come
and intermolecularly. For all samples, the carboxylic acid from chitosan. FTIR results confirmed pectin was further
groups from pectin were ionized to carboxylate groups in cross-linked by adding chitosan, leading to higher scaffold
the presence of Ca2+ or chitosan. The signals at 1600 cm−1 stability.10,15
and 1413 cm−1 represent the carboxylate group. However, The SEM images shown in Figure 3 provide additional
compared with the control sample, a signal at 1620 cm−1 is insight into the surface and internal morphology of the sam-
much more pronounced in the samples with oligochitosan ples discussed above. It is clearly seen that the 150 kD scaf-
or chitosan. This is caused by the protonated amine (NH3+) folds have a less layered structure as compared to the other
group from chitosan due to the neutralization of carboxylic samples both internally and at the surface—there are rela-
acid (pectin) by amine (chitosan). In addition, signals at tively large, uniform, monolithic areas. This morphology
4 Journal of Applied Biomaterials & Functional Materials 00(0)
Figure 3.  Scanning electron microscopy (SEM) images of
bioprinted scaffolds cross-linked with Ca2+ and then further
treated with 2 kD oligochitosan, 50 kD chitosan, 150 kD
chitosan, or 1000 kD chitosan. Left panels depict surface
structure, while right panels show internal structure. Scale
bar: 20 µm.
Figure 4.  (a) Stability testing of bioprinted scaffolds cross-linked with calcium and further treated with oligochitosan or various
molecular weights of chitosan based on percentage of initial scaffold mass. Scaffolds incubated in DMEM at 37°C for three days. (b)
Prolonged stability testing of bioprinted scaffolds incubated in DMEM at 37°C for seven days. Scaffolds tested were cross-linked
with calcium and either modified with 150 kD chitosan or unmodified.
can explain the increased stability by providing higher
resistance to the structural deterioration.
Calcium cross-linking and subsequent chitosan modifi-
cation of scaffolds was found to be more effective than only
Calcium cross-linking (Figure 4). All scaffolds showed a
substantial increase in mass in the first few hours of incuba-
tion in DMEM, which can be attributed to liquid uptake by
the hydrogel. Sodium bicarbonate and phosphate ions in
DMEM can also negatively affect scaffold stability by
“grabbing” calcium ions away from the pectin.21
Among chitosan-coated scaffolds, 150 kD chitosan was
the most effective, while 50 kD and 1000 kD were rather
ineffective (Figure 4(a)). Oligochitosan-treated scaffolds
were extremely brittle after a period of only three hours,
falling apart while being weighed (data not shown). It is
thought that oligochitosan became competitive with Ca2+
for carboxylic acid group binding, which led to deconstruc-
tion of the scaffold.11 Chitosan-coated and calcium-treated
scaffolds maintained their shape through 24 hours. However,
after this period 50 kD-coated scaffolds fell apart more eas-
ily than other scaffolds. After the incubation period of three
days, 150 kD-modified, 1000 kD-modified, and Ca2+ scaf-
folds had maintained their shape and remained above their
initial mass, a measure of stability. The 150 kD scaffolds
resisted breakage substantially more than other scaffolds.
Furthermore, 150 kD-modified scaffolds were more stable
than unmodified Ca2+-treated scaffolds after incubation in
DMEM for seven days (Figure 4(b)). Most likely, 50 kD
chitosan was too small to effectively complement the
Calcium cross-linking, and, instead, competed for binding
sites. On the other hand, 1000 kD chitosan was too large to
penetrate deep enough to effectively stabilize the scaffolds.
Thus, post-printing coating with chitosan, specifically 150
kD chitosan, shows great promise for creating stable pectin-
based scaffolds and further implantation of cells to engineer
artificial tissues and organs.
Stealey et al. 5
Conclusion
Bioprinted pectin-based scaffolds were found to be most
stable when cross-liked with Ca2+ and then modified with
150 kD chitosan. Oligochitosan was determined to be an
ineffective cross-linker and modifier for the scaffolds.
Scaffolds modified with chitosan remained relatively sta-
ble compared to unmodified scaffolds after incubation in
DMEM during the testing period, showing great promise
for potential inclusion of cells to engineer artificial tissues
and organs.
Acknowledgements
The authors would like to graciously thank Mason Degeneffe and
Devon McCune for their technical assistance and Mark Hoelzer
for the schematic diagram.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
Funding
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article:
This work was partially supported by the National Science
Foundation (Grant Number EEC-1460183) and a Milwaukee
School of Engineering Summer Faculty Development Grant.
References
1. Organ donation statistics. https://optn.transplant.hrsa.gov/
2. Mandrycky C, Wang Z, Kim K, et al. 3D bioprinting for
engineering complex tissues. Biotechnol Adv 2016; 34:
422–434.
3. Murphy SV and Atala A. 3D bioprinting of tissues and
organs. Nat Biotechnol 2014; 32: 773–785.
4. Hospodiuk M, Dey M, Sosnoski D, et al. The bioink: a com-
prehensive review on bioprintable materials. Biotechnol
Adv 2017; 35: 217–239.
5. Wang X, Ao Q, Tian X, et al. Gelatin-based hydrogels for
organ 3D bioprinting. Polymers 2017; 9: 401.
6. Banks A, Guo X, Chen J, et al. Novel bioprinting method
using a pectin based bioink. Technol Health Care 2017;
25: 651–655.
7. Gangemi S, Spagnolo EV, Cardia G, et al. Fatal anaphylactic
shock due to a dental impression material. Int J Prosthodont
2009; 22: 33–34.
8. Bruchet M and Melman A. Fabrication of patterned cal-
cium cross-linked alginate hydrogel films and coatings
through reductive cation exchange. Carbohydr Polym 2015;
131(Suppl C): 57–64.
9. Kamimura W, Hattori R, Koyama H, et al. A calcium-cross-
linked hydrogel based on alginate-modified atelocollagen
functions as a scaffold material. J Biomater Sci Polym Ed
2012; 23: 609–628.
10. Crouse JZ, Mahuta KM, Mikulski BA, et al. Development
of a microscale red blood cell-shaped pectin-oligochitosan
hydrogel system using an electrospray-vibration method:
preparation and characterization. J Appl Biomater Funct
Mater 2015; 13: 326–331.
11. Zhang W, Mahuta KM, Mikulski BA, et al. Novel pectin-
based carriers for colonic drug delivery. Pharm Dev Technol
2016; 21: 127–130.
12. Ross AC, Manson JE, Abrams SA, et al. The 2011 dietary
reference intakes for calcium and vitamin D: what dietet-
ics practitioners need to know. J Am Diet Assoc 2011;
111: 524–527.
13. Zhang W, Gilstrap K, Wu L, et al. Synthesis and charac-
terization of thermally responsive Pluronic F127-chitosan
nanocapsules for controlled release and intracellular deliv-
ery of small molecules. ACS Nano 2010; 4: 6747–6759.
14. Yoo D and Lee D. Oligochitosan-stabilized photolumines-
cent gold nanoconstructs for optical bioimaging. Biomater
Res 2017; 21: 20.
15. Zhang W, Zhao S, Rao W, et al. A novel core-shell micro-
capsule for encapsulation and 3D culture of embryonic
stem cells. J Mater Chem B Mater Biol Med 2013; 2013:
1002–1009.
16. Ahsan SM, Thomas M, Reddy KK, et al Chitosan as bio-
material in drug delivery and tissue engineering. Int J Biol
Macromol 2018; 110: 97–109.
17. Ko JH, Kim YH, Jeong SH, et al. Collagen esterifica-
tion enhances the function and survival of pancreatic beta
cells in 2D and 3D culture systems. Biochem Biophys Res
Commun 2015; 463: 1084–1090.
18. Suga T, Osada S, Narita T, et al. Promotion of cell adhesion
by low-molecular-weight hydrogel by Lys based amphiph-
ile. Mater Sci Eng C Mater Biol Appl 2015; 47: 345–350.
19. Zhang W, Choi JK and He X. Engineering microvascular-
ized 3D tissue using alginate-chitosan microcapsules. J
Biomater Tissue Eng 2017; 7: 170–173.
20. Feng Y, Kopplin G, Sato K, et al. Alginate gels with a
combination of calcium and chitosan oligomer mixtures as
crosslinkers. Carbohydr Polym 2017; 156: 490–497.
21. Drury JL, Dennis RG and Mooney DJ. The tensile prop-
erties of alginate hydrogels. Biomaterials 2014; 25:
3187–3199.