(624b) An Antibacterial and Photocurable Hyaluronic Acid/Elastin like Polypeptide Hybrid Hydrogel for Cartilage Repair

Authors: 
Shirzaei Sani, E., Northeastern University
Annabi, N., Northeastern University

An antibacterial and photocurable hyaluronic acid/elastin like polypeptide
hybrid hydrogel for cartilage repair

Ehsan Shirzaei
Sani1, Iman Noshadi1,2,3, Roberto Portillo Lara1,Wendy
Yu1, Benjamin Geilich1, Thomas J. Webster1,4,5,and Nasim Annabi1,2,3

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

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

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

4Center of Excellence for
Advanced Materials Research, King Abdulaziz
University, Jeddah, Saudi Arabia.

5Wenzhou Institute of Biomaterials and Engineering,
Wenzhou Medical University, Wenzhou, China.



Introduction

Cartilaginous
tissues play an important role in supporting mechanical loads and energy
dissipation in joints of the musculoskeletal system. They are composed of specialized
cells called chondrocytes, and a dense extracellular matrix (ECM) comprised
primarily of type II collagen and proteoglycans. These tissues are
characteristically avascular and alymphatic, and exhibit
low cell densities, which limits their ability for self-repair after injury. Hyaluronic
acid and elastin are also key components of the ECM in connective tissues, and play
important mechanical and biological roles in cartilage repair. Elastin fibers
are major ECM macromolecules that are critical in maintaining the integrity,
elasticity, and the mechanical properties of articular cartilage. Hyaluronic
acid on the other hand is an important component of cartilage and synovial
fluid in the joints, and is involved in cell migration, differentiation, and
proliferation, as well as regulation of ECM organization and metabolism [1, 2].

In this work, hybrid hydrogels
containing methacrylated hyaluronic acid (MeHA) and an elastin-like polypeptide (ELP) were engineered
for cartilage repair, by photocrosslinking different
ratios of MeHA and ELP. ELPs are thermoresponsive,
elastic artificial proteins, whose macromolecular structure can be
tailored for different biomedical applications, by modifying their aminoacidic sequence through DNA recombinant techniques.
The ELP sequence used in this study contained cysteine residues, in which the
thiol groups were able to form disulfide bonds upon exposure to visible or UV
light [3]. Antimicrobial ZnO nanoparticles were incorporated
into the engineered hybrid hydrogels to impart antibacterial properties. The antimicrobial
and mechanical properties of the engineered hybrid hydrogel, as well as pore sizes
and swelling ratios, could be fine-tuned based on the ratio of MeHA/ELP, final polymer concentration, and crosslinking
conditions. Furthermore, the biocompatibility of the engineered MeHA/ELP hydrogels was investigated in vitro.

Materials
and Methods

All chemicals were purchased from
Sigma-Aldrich and used without further purification. MeHA
and ELP were prepared according to procedures described in our previous works [3,
4]. Briefly, MeHA/ELP prepolymers
at different compositions were added to a 0.5% (w/v) photoinitiator
solution. The ELP concentration varied from 0 to 20 % (w/v) and the
concentration of MeHA varied from 0% to 2% (w/v). The
solutions were then mixed with various concentrations of antibacterial ZnO, ranging from 0.1-0.3% (w/v). The mixtures were then
sonicated for 60 min and photopolymerized for 120 sec
under UV light (intensity: 6.9 mW/cm2).
The compression modulus, swelling ratio, and pore size characterization of the
hydrogels as well as cell studies were performed based on procedures described previously
[3]. Cell viability tests were performed using 3T3 fibroblast cells (ATCC®
CRL-1658™) and cells were cultured in DMEM 1X medium with 10% FBS and 1% penicillin/streptomycin
and incubated at 37 °C with 5% CO2 (details described in [3]). Bacterial
growth on hydrogel samples was evaluated using a colony forming unit (CFU)
assay with methicillin-resistant Staphyloccocus aureus (MRSA, ATCC® 43300™). The plate
colony-counting method was employed as described previously [5]. In order to
perform statistical analysis, GraphPad Prism 6
software package was used. A two-way ANOVA test was performed to characterize
statistical differences between mean ± standard deviation from every
measurement.

Results
and Discussion

The results of compression tests showed
that by increasing the ELP concentrations from 0% to 15% in a 2% MeHA prepolymer, the compressive
modulus of the resulting hydrogel was increased 3-fold from 13.9 kPa to 39.9 kPa, respectively (p
< 0.0001) which shows a good improvement compared to previous studies. Although,
the compressive modulus of cartilage is 0.45–0.80 MPa, the engineered hydrogels
showed sufficient mechanical strength to support cell encapsulation and growth
[6]. In addition, the swelling ratios of MeHA/ELP
hydrogels decreased 9-fold from 5900% to 630% by increasing the ELP
concentration from 0% to 15% (w/v), respectively (p < 0.001). This result
suggests that by changing the composition of hybrid hydrogels, the swelling
properties of the engineering hydrogels are tunable. Therefore, it is possible
to control the pore sizes and swelling ratio of the hydrogel which are key
properties for cell spreading and differentiation [7]. Based on the in vitro studies, MeHA/ELP
hydrogels with 2% w/v MeHA and 10% w/v ELP showed more
than 90% cell viability at days 1, 3, and 5 post-seeding (p < 0.05). The
antibacterial results showed that after addition of ZnO
nanoparticles, the number of MRSA CFUs decreased consistently after 24 hours of
culture. In particular, the optimal formulation corresponded to a ZnO concentration of 0.2%, where the number of CFUs on the
hybrid hydrogels decreased significantly from 40 to 25 per cm2 compared
to the sample without ZnO as a control (p < 0.05).

Conclusions

In this study, we developed an
antimicrobial photocrosslinkable hybrid hydrogel
based on MeHA/ELP/ZnO with tunable
physical properties. The engineered hydrogels were biocompatible in vitro and also prevented bacteria
growth at optimal ZnO concentrations. In summary,
this study showed that the presently engineered hydrogels have the potential to
be used in biomedical applications especially as an antibacterial hydrogel for
cartilage tissue repair.

References

[1] G. C. Yeo et al., Adv
Healthc Mater. 2015, 4, 16, 2530-56.

[2] J.
Yu et al, J
Anat. 2010, 216, 4, 533–541.

[3] Y. Zhang et al., Adv. Funct. Mater. 2015, 25, 4814–4826.

[4] G. Camci-Unal
et al., Biomacromolecules, 2013, 14, 4, 1085-1092.

[5] N. C. Verissimo
et al. J Biomed Mater Res Part A 2015:103A:3757–3763.

[6] I. L. Moss et al., Spine, 2011,
36, 13, 1022-1029.

[7] H. Park et al., Biomacromolecules. 2009, 9; 10, 3, 541–546.

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