Strength-tunable printing of xanthan gum hydrogel: Via enzymatic polymerization and amide bioconjugation

Article abstract of DOI:10.1039/d0cc00326c

Amide bioconjugation and interfacial enzyme polymerization are designed to provide a general strategy for regulating the mechanical strength (storage modulus from 3 kPa to 100 kPa) of printable hydrogel inks.

Full text of DOI:10.1039/d0cc00326c

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Strength-tunable Printing of Xanthan gum Hydrogel via Enzymatic
Polymerization and Amide Bioconjugation†
Hui Pan#a, Bolin Zheng#b, Hongdou Shena, Meiyuan Qia, Yinghui Shanga, Chu Wua, Rongrong Zhub,
Received 00th January 20xx,
Accepted 00th January 20xx
Liming Chengb and Qigang Wang *a b
DOI: 10.1039/x0xx00000x
Amide bioconjugation and interfacial enzyme polymerization are
designed to provide general strategy for regulating the to avoid the mutual effect between polymerization and
mechanical strength (storage modulus from 3kPa to 100kPa) of extrusion. The natural polysaccharide xanthan gum is utilized as
Herein, a dual-nozzle extrusive printing model was employed
a
printable hydrogel inks.
the stable 3D printing framework in both nozzles. The interfacial
enzyme polymerization is triggered by the separated loading of
enzymes and substrates within different nozzle. In general,
enzymatic polymerization is usually nontoxic31-33. The selected
Glucose oxidase (GOx)/glucose/N-Hydroxy sulfosuccinimide
(Sulfo-NHS) system can quickly produce nitrogen radical and
trigger polymerization in the absence of oxygen. Enzymatic
polymerization can be regulated by the interface diffusion of
enzymes and substrates. In our dual-nozzle extrusive mode, the
interfacial polymerization can be achieved through mutual
penetration after printing. Amide-bond bioconjugation by
Sulfo-NHS and EDC⋅HCl is another widely used biological
modification technique34, which can interact slowly and
regulatably between the amino and carboxyl group. At the same
time, due to the presence of carboxyl or amino groups, natural
polymers, like xanthan gum, biologically active guest bodies,
and even cells are readily coupled to poly (N-acryloyl-L-lysine)
without additional modification. As a result, our printable
polymer hydrogel platforms offer advantages such as adjustable
strength, no modification was required for most bio-inks and a
comprehensive biological approach that can be potentially
Hydrogels are soft-wet materials made up of three-
dimensional hydrophilic macromolecular networks. Due to their
water-rich microenvironment and tuneable strength, polymer
hydrogels with hydrophilic network have attracted emerging
attention as scaffolds in biocatalysis1-5, biomedicine6-7 and
tissue engineering fields 8-12. Materials used for the preparation
of polymer hydrogel can be either from natural or synthetic
sources13-17. Natural protein/polysaccharide hydrogels with
reversible physical interactions are shear-thinning and thus
printable18-19, but lack of mechanical strength20-21. The typical
polymer hydrogels formed by the copolymerization of
monomers and multivinyl crosslinker are tough, but non-
processable due to their significantly viscosity increasement
around gel point22-26. Therefore, the combination of the
strength performance of synthetic polymer and the printable
ability of non-covalent system is a reasonable approach for
fabricating tough and printable polymer-composite hydrogels27-
28. Liu and co-workers have demonstrated that poly (N-acryloyl
glycinamide) hydrogels with remarkable supramolecular
hydrogen bonds can be printed via the reversible sol-gel
transition at high temperature29. Zhao and co-workers have
utilized the viscous clay as printable matrix to execute photo-
polymerization for strength regulation30. As deduced from
these pioneered literatures, we know that the contradiction of
in-situ polymerization and stable extrusion is the constraining
factor for ensuring the stable viscosity and successful printing.
a. School of Chemical Science and Engineering, Tongji University, Shanghai 200092,
China. E-mail: wangqg66@tongji.edu.cn.
b.Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry
of Education, Orthopaedic Department of Tongji Hospital, School of Medicine, Tongji
University, 389 Xincun Road, 200065 Shanghai, P. R. China
# These authors contributed equally to this work and should be considered co-first
authors.
Fig 1. Schematic illustration of the decoupled enzymatic polymerization initiated by GOx
Electronic Supplementary Information (ESI) available: See DOI: 10.1039/x0xx00000x
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/ glucose / Sulfo-NHS, the gelation by cross-linking of EDC⋅HCl and Sulfo-NHS and dual-
nozzle extrusion printing.
without N-acryloyl-L-lysine demonstrated a sextetVsieiwgnAratilclwe Oitnhlinge
value of 2.0058, aN = 1.95G, aH =15.4G, DwOhIi:c1h0.1w03e9r/eDc0oCiCn0c0id32e6nCt
with the values of a DMPO trapped nitrogen centred radical.
The signal of the propagating carbon radical (Fig. S2, ESI†) with
g value of 2.0061 was also observed in the enzyme-initiated
system containing with N-acryloyl-L-lysine. Therefore, our bio-
initiated system could be used to mildly initate radical
polymerization with high cytocompatibility compared to
traditional heat or photo polymerization. In normal conditions,
GOx catalyzes the production of glucono-lactone by glucose, the
Sulfo-NHS was reduced as the hydrogen acceptor, which
produces nitrogen free radicals under anaerobic conditions (Fig.
2a). Then these free radicals were transferred to form carbon
radicals to initiate polymerization.
The carboxyl group in the polysaccharide could be biocoupled
with the amino group on the N-acryloyl-L-lysine to crosslink.
EDC⋅HCl can react with the carboxyl group on the
polysaccharide to form an unstable urea derivative, and the
stability of products crosslinked by EDC⋅HCl, was enhanced by
Sulfo-NHS by the forming more stable ester (Fig. 2c). As shown
in Fig. 2d, the conjugation process was observed by infrared
spectroscopy, the vibration peak of amide-Ⅱ at 1530 cm-1 and
N-H at 1620 cm-1 were increasing as the reaction progress.
These indicate that the bioconjugation reaction of N-acryloyl-L-
lysine with Xanthan gum continues to occur during gel
formation. The polymerization process can also be monitored
by nuclear magnetic resonance (NMR) characterization (Fig. S3,
ESI†), the conversion test revealed that almost 90% of
conversion of N-acryloyl-L-lysine was completed in one hour.
The gelation process of composite hydrogels was monitored by
dynamic time sweep test (Fig. 2e), the storage modulus G’ and
loss modulus G” increased rapidly within 900 seconds due to the
Fig. 2 (a) Schematic diagram of the anaerobic catalysis mechanism of GOx in our gelation
system. (b) EPR spectrums of radical catalyzed by GOx/glucose with DMPO as the spin
trap. (c) Schematic diagram of EDC⋅HCl /sulfo-NHS crosslink mechanism amino and
carboxyl groups (d) Infrared spectra of hydrogel at different times. (e) Dynamic time
sweep tests of composite hydrogel. (f) SEM image of the hydrogel.
combined with biologically active guests.
All components were illustrated in Fig. 1, xanthan gum was quick enzymatic polymerization. The morphology of the
used as the backbone and supporting material for 3D printing, hydrogel was investigated by scanning electron microscopy
the dual-nozzle extrusion process was used to regulate the (SEM), as shown in Fig. 2f, the hydrogel had a uniform three-
gelation process of enzymatic polymerization. Our bio-printable dimensional porous structure with pore sizes of 3-4 micron in
hydrogel contains three functional components: gelling agent the hydrogel matrix.
solution, enzyme initiation system, amide bioconjugate
Since mechanical properties affect the formability of
reagent. Xanthan gum with a mass concentration of 5% was materials and the living environment of cells, proper
selected as the printable skeleton, and 2-6wt% concentration of mechanical strength is necessary for materials. Rheological
N-acryloyl-L-lysine solution was used as a polymerizable studies of Xanthan gum, poly (N-acryloyl-L-lysine) hydrogel and
monomer, enzymatic polymerization was initiated by mutual composite hydrogel are shown in Fig. 3a, xanthan gum shows
penetration of GOx and glucose. In the bio-crosslinking system, the lowest storage modulus G’ and loss modulus G”, which
the amount of EDC⋅HCl and N-acryloyl-L-lysine are varied to makes it easy to print, and the composite hydrogels exhibited
control the final degree of crosslinking, and the hydrogel was the highest storage modulus G’ and loss modulus G”, this
finally formed at room temperature.
indicates that the composite hydrogels have gel-like rheological
To verify the mechanism of enzymatic polymerization, properties. Furthermore, the composite hydrogel with 0.5%
electron paramagnetic resonance (EPR) spectroscopy was EDC⋅HCl and 4% N-acryloyl-L-lysine showed good compression
performed to detect the existing radicals within the bio- toughness. At 40% compression strain, the compression curve
initiated system of GOx (100U/ml)/glucose (50mg/ml)/Sulfo- had almost no change after 10 times (Fig. 3b), and the
NHS (10mg/ml). Similar to the reported GOx/glucose/NHS compression modulus was about 23kPa. A series of composite
system35, the anaerobic metabolism of glucose and Sulfo-NHS hydrogels with various monomer content were prepared by the
catalyzed by glucose oxidase can produce nitrogen radicals by enzymatic
polymerization
(GOx
(50U/ml)/glucose
hydroxyl reduction. Dimethyl pyridine N-oxide (DMPO) was (10mg/ml)/Sulfo-NHS (5mg/ml).). As indicated in Fig. 3c, the
employed to trap the unstable and short-life time free radicals compression modulus of composite hydrogels increased from
to form a long-lived nitroxide for EPR characterization. As 27.9 kPa to 52.1 kPa as the amount of monomer is increased
shown in Fig. 2b, the EPR spectrum of the initiated system from 2 wt% to 6 wt% with 0.5 wt% EDC⋅HCl. It can be
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using two printing head, through the observation
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DOI: 10.1039/D0CC00326C
comparison of SEM images, we could find
understood that the addition of the monomer increased the
Fig. 4 (a) Printing method showing. (b) Cyclic compression experiment on the cylinder
crosslinking density. The compression
after printing. (c-e) 3D printing of composite hydrogels.
Fig. 3 (a) Rheological assessment of xanthan gum, poly (N-acryloyl-L-lysine) hydrogel and
composite hydrogel. (b) Cyclic compression test of Composite hydrogels (c) Compressive
modulus of composite hydrogels with different N-acryloyl-L-lysine amounts. (d)
Compressive modulus of hydrogels with different EDC⋅HCl amounts.
that the structure obtained by using ink A and ink B was slightly
larger than that of a single ink, and the surface was smoother
(Fig. S8, ESI†). The cylinder after printing underwent cyclic
compression experiments showed good toughness (Fig 4b). At
40% compression strain, it cycles 10 times and the compression
curve hardly changes. The printed round tables retained its
structural integrity and the stacking of layers could be seen
clearly (Fig. 4c). Furthermore, we also printed love
model(25×20×5mm) and ears model (26×40×4 mm) (Fig. 4d,4e),
the printed construct was stable and there has no deformation
or collapse during the printing process. After we have finished
printing for 60 minutes, the printed materials could be easily
and completely removed. Through the Fig. 4e, the complete
structure and obvious pores of the ear can be seen. These
results show that the printing of double extrusion nozzle is very
suitable for hydrogel formed by enzyme catalysis, xanthan gum
provides appropriate viscosity for extrusion printing. The
printed object shows a high precision, complete structure and
obvious pores.
In vitro cytotoxicity assay and 3D cell culture were conducted
to examine the biocompatibility of composite hydrogel.
Composite hydrogels could load cells for bioprinting and is low-
toxic to cells. Enzyme-catalyzed free radical polymerization is
often mild and low-toxic. The hydrogels which were constructed
with monomer obtained from natural lysine modified by acrylic
acid, and crosslinked by amide bonds induced by low-toxic
EDC⋅HCl and sulfo-NHS, exhibited high biocompatibility. NIH-
3T3 cells from a mouse embryonic fibroblast cell line are chosen
as the cell model. As shown in Fig. 5a, cell viability basically
unchanged after co-incubation with gel powder, suggesting that
this enzyme-initiated gelation system has relatively low
cytotoxicity. Cell viability was assessed by fluorescent staining.
After co-culture with different concentrations of gel powders (0,
1, 10 and 100 μg/mL) for 24h and 48h, living cells were bright
green fluorescence while dead cells were stained with red
fluorescence on fluorescent inverted microscope views. To our
delight, few dead cells are observed. Our results indicate that
modulus of composite hydrogels increased from 14.5 kPa to
50.8 kPa as the amount of EDC⋅HCl increased from 0.25 wt% to
0.75 wt% with 4wt% N-acryloyl-L-lysine (Fig. 3d), and the tensile
modulus of composite hydrogels increased from 8.03kPa to
35.4kPa (Fig. S4, ESI†). This can also be attributed to the
increase in crosslinking density. Moreover, special cross-linking
allows N-acryloyl-L-lysine to cross-link many other natural
polymer containing amino or carboxyl groups. Various natural
proteins or polysaccharides could be used to build hydrogel
networks (Fig. S5, ESI†), and different natural polymer doping,
which exhibits different mechanic strength. For example, the
compression modulus increased from 1.0kPa to 4.4kPa as the
amount of bovine serum albumin (BSA) increased from 4wt% to
10wt% with 4% N-acryloyl-L-lysine and 0.5% EDC⋅HCl (Fig. S6,
ESI†). The same situation happens when carboxymethyl
cellulose (CMC) is added (Fig. S7, ESI†). In summary ， our
hydrogels exhibited excellent mechanical properties and
tunable strength after coupling with natural molecules.
Extrusion type 3D printing requires stable viscosity. However,
enzyme-catalyzed free radical polymerization tends to cause a
sharp rise in viscosity. In order to address these limitations, we
have developed
a new printing strategy. Our strategy
incorporates two inks: Ink A prepared from glucose oxidase, N-
acryloyl-L-lysine, EDC⋅HCl / Sulfo-NHS and xanthan gum, Ink B
prepared from glucose, N-acryloyl-L-lysine, EDC⋅HCl / Sulfo-NHS
and xanthan gum. As shown in Fig. 4a, when the one printhead
prints a layer of ink A, the other printhead immediately prints
the ink B on the pattern printed previously. Xanthan gum
provides the suitable viscosity and printed skeleton for
extrusion printing, subsequent infiltration of glucose and
glucose oxidase initiates free radical polymerization to reinforce
the printed skeleton. This printing method could accurately
place interpenetrating hydrogel bundles and builds complex
geometries. The dual-inks were compared with single ink by
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NIH-3T3 cells maintained high survival rates after co-culturing
Conflicts of interest
View Article Online
DOI: 10.1039/D0CC00326C
There are no conflicts of interest to declare.
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enhanced owing to polymerization and the bioconjugate
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for hydrogel reinforcement——various natural polymer bio-
inks containing amino or carboxyl groups could be cross-linked
with the N-acryloyl-L-lysine without additional modification.
The free radical polymerization mechanism and the amide
cross-linking process were verified by EPR and IR. The final
composite hydrogel showed adjustable mechanical strength.
The double extrusion nozzle printed structure exhibited
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35 T. Su, Z. Tang, H. He, W. Li, X. Wang, C. Liao, Y. Sun and Q.
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