(277b) Engineering an Electroactive Hydrogel for Tissue Engineering Applications

Authors: 
Spencer, A., Northeastern University

Engineering an
Electroactive Hydrogel for Tissue Engineering Applications

Andrew Spencer1
Hicham Fenniri1*, Nasim Annabi1,2,3**

1 Department
of Chemical Engineering, Northeastern University, Boston, MA, 02115-5000, USA

2Biomaterials
Innovation Research Center, Brigham and WomenÕs Hospital, Harvard Medical
School, Boston, MA, USA.

3Harvard-MIT
Division of Health Sciences and Technology, Massachusetts Institute of
Technology, Cambridge, MA, USA.

*h.fenniri@neu.edu;**n.annabi@neu.edu

INTRODUCTION

Hydrogels are hydrophilic three dimensional (3D)
polymeric networks with physical, chemical and biological properties that can
be designed to closely mimic the native extracellular matrix (ECM) of mammalian
tissues. Because of their biomimetic properties, hydrogels are often used in
various tissue engineering and regenerative medicine applications. For tissues whose
function is highly dependent on electrochemical signaling between cells and
that are responsive to an electric field, such as the heart, it is desired that
the hydrogels are electroactive [1].  However, most hydrogels are inherently non-conductive.
Numerous approaches have been taken to improve the conductivity of hydrogels using
materials that have limited biocompatibility and biodegradability.

There exists a library of conducting organic polymers
that offer an attractive alternative to other conductive materials for
biomedical applications, due to their ease of processing, versatility, and enhanced
biocompatibility both in vitro and in vivo [2]. Unlike many other
conductive materials, such as carbon nanotubes, these conductive polymers can
be easily dispersed in aqueous solutions Ð a requirement for biological applications.
In addition to their practical advantages, they actuate upon electrical stimulation
[3]. This enables simultaneous electrical and mechanical stimulation of cells,
which may help to maintain their phenotype and facilitate their growth.

By dispersing a well-established conducting polymer
complex, poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS), at
various concentrations within a photo-crosslinkable naturally derived polymer,
gelatin methacrylate (GelMA), we have synthesized an electroactive and flexible
hydrogel that can be used for 3D cell encapsulation. The engineered 3D
conductive hydrogel with tunable mechanical and electrical properties can be
used for various tissue engineering applications, including muscle tissue
regeneration and heart repair.

MATERIALS AND METHODS

Hydrogel formation and characterization: Gelatin methacrylate (GelMA) was synthesized
according to a previously reported protocol [4]. PEDOT:PSS was obtained from
Heraeus (Germany) in the form of an aqueous dispersion called CleviosTM PH 1000
(PEDOT:PSS ratio 1:2.5). The concentration of the dispersion in GelMA
prepolymer after filtering through a 0.22 mm filter was determined to be 0.95 wt% after drying
and weighing. Solutions of 7% GelMA at 0%, 0.1% and 0.3% PEDOT:PSS with 0.5%
photoinitiator (Irgacure 2959) were prepared for all experiments. The hydrogels
(thickness: 2 mm, diameter: 4 mm) were formed by exposing the GelMA/PEDOT:PSS
solutions to UV light at 6.9 mW/cm2 for 360 s. Then, the compressive properties of the
engineered hydrogels were tested using an Instron 5944 mechanical tester. Compressive
modulus was taken as the slope of the linear portion of the stress/strain curves
at 0-5% strain.

3D Cell Encapsulation: C2C12 myoblast cells (ATCC) were cultured in complete
media (DMEM + 10% FBS + 1% penicillin/streptomycin) in a humidified 5% CO2
incubator prior to use. Cell were trypsinized and resuspended in solutions of
GelMA/PEDOT:PSS precursor at concentration of 106 cells/mL of
prepolymer solution. 7 mL of the mixture was then sandwiched between a glass slide and a petri
dish with a 150 mm spacer and exposed to UV light for up to 30 s to crosslink to form a
cell-laden hydrogel. Live/Dead, Actin/DAPI and PrestoBlue assays were performed
on days 1, 3 and 5 to evaluate the cell viability, spreading, and proliferation
in 3D hydrogels with varying mechanical and electrical properties.

RESULTS AND DISCUSSION

Mechanical testing demonstrated that the compressive
modulus increases with increasing concentration of PEDOT:PSS. This might be due
the interaction between the negatively charged PSS and the positively charged
arginine or lysine groups in GelMA, adding physical crosslinking to the
composite hydrogel in addition to chemical bonds generated during
photopolymerization. The in vitro
data also showed that incorporation of PEDOT:PSS into the hydrogels did not
affect the metabolic activity of the cells, as confirmed by PrestoBlue cell
viability assay. Live/Dead assay also confirmed that the incorporation of
PEDOT:PSS into GelMA did not affect cell viability. In addition, the cells
start spreading inside the engineered hydrogels at day 3 of culture. These
results suggest that the incorporation of conductive polymer complex PEDOT:PSS created
a biocompatible conductive hydrogel that will enable uniform electrical
stimulation of electrically responsive tissues.

CONCLUSION

            An electroactive hydrogel was developed via the
incorporation of a conducting polymer complex into a naturally derived hydrogel.
Compressive modulus was improved via the addition of PEDOT:PSS into the
hydrogel. Cell viability, metabolic activity, and spreading were not affected
by the incorporation of the conductive material into the hydrogel network as shown
by PrestoBlue, Live/Dead and Actin/DAPI assays. These results suggest that the engineered
composite hydrogel is suitable for various tissue engineering applications
where the tissues are electrically responsive.

ACKNOWLEDGMENT

The authors would like to thank
Northeastern University for funding this research.

REFERENCE

[1] N. Annabi, A. Tamayol, J. Uquillas,
M. Akbari, L. Bertassoni, C. Cha, G. Camci_Unal, M.
Dokmeci, N. Peppas, A. Khademhosseini, Adv Mater 2014.

[2] R. Balint, N. Cassidy, S.
Cartmell, Acta Biomaterialia 2014

[3] M. R. Abidian, D. _H. Kim, D. C. Martin, Adv Mater 2006

[4] W. Nichol, S. T. Koshy, H.
Bae, C. M. Hwang, S. Yamanlar, A. Khademhosseini, Biomaterials 2010