471521 An Antibacterial and Photocurable Hyaluronic Acid/Elastin like Polypeptide Hybrid Hydrogel for Cartilage Repair
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 Womens 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.
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 . 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 . 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 ). 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 . 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 0.450.80 MPa, the engineered hydrogels showed sufficient mechanical strength to support cell encapsulation and growth . 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 . 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).
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.
 G. C. Yeo et al., Adv Healthc Mater. 2015, 4, 16, 2530-56.
 J. Yu et al, J Anat. 2010, 216, 4, 533541.
 Y. Zhang et al., Adv. Funct. Mater. 2015, 25, 48144826.
 G. Camci-Unal et al., Biomacromolecules, 2013, 14, 4, 1085-1092.
 N. C. Verissimo et al. J Biomed Mater Res Part A 2015:103A:37573763.
 I. L. Moss et al., Spine, 2011, 36, 13, 1022-1029.
 H. Park et al., Biomacromolecules. 2009, 9; 10, 3, 541546.