Improved Cellular Functions and Reduced Bacterial Infection on MgO Nanocomposites
Daniel J. Hickey1 and Thomas J. Webster1,2
1Department of Chemical Engineering, Northeastern University, Boston, MA, USA
2Center of Excellence for Advanced Materials Research, King Abdulaziz University, Jeddah, Saudi Arabia
Introduction: Regeneration of complex orthopedic tissues (such as ligaments, bones, and the tendon-to-bone insertion site) is problematic due to a lack of suitable biomaterials with the appropriate chemical and mechanical properties to elicit the formation of tissues with similar structure, organization, and functionality to natural tissues. Additionally, a non-trivial fraction of implanted biomaterials become infected by bacteria, which can lead to implant failure, secondary surgeries, and the spread of infection to other tissues throughout the body. To address these issues, the current study investigated novel magnesium oxide (MgO) nanomaterials to improve orthopedic tissue regeneration and reduce bacterial infection.
Materials and Methods: MgO nanoparticles (particle diameters of 20 nm, US Research Nanomaterials Inc., Houston, TX) and hydroxyapatite (HA) nanoparticles, synthesized following the method reported by Zhang et al. , were combined with poly (l-lactic acid) (PLLA, MW=50,000, Polysciences, Warrington, PA) and dissolved in chloroform with sonication. Polymer solutions were cast to glass petri dishes and heated to evaporate the excess solvent. The resulting polymer sheets were cut into strips for further study.
Samples were cut into 1 cm x 3 cm rectangular strips for tensile testing with a uniaxial tensile tester equipped with a 10-lb. load cell and material analysis software (ADMET, Norwood, MA). This arrangement was used to obtain the elastic modulus, material elongation, and maximum load endured for each sample. Cell tests were performed using osteoblasts and fibroblasts (American Type Culture Collection, Manassas, VA). Cell viability on each substrate was assessed at times from 4 hours to 5 days using an MTS assay (Promega, Madison, WI). The expression of several relevant genes including alkaline phosphatase (ALPL) and type-1 collagen (COL1A) were assessed by quantitative real-time PCR (qRT-PCR), and cellular actin networks and vinculin focal adhesions were visualized using a Zeiss confocal LSM 700 microscope.
Nanocomposite antibacterial efficacy was assessed by seeding approximately 106 Staphylococcus aureus (ATCC 12600) onto 1-cm2 nanocomposites and culturing for 24 and 48 hours under standard culture conditions. The adhered bacteria were then lysed (lysis buffer, Life Technologies, Carlsbad, CA) and the DNA stain Hoechst 33258 (Molecular Probes, Eugene, OR) was added to each solution. Fluorescence intensities were measured (ex: 350 nm, em: 450 nm) and compared to a standard curve to quantify cell numbers. Experiments were conducted in quadruplet and repeated three times. Data were analyzed using single-factor analysis of variance (ANOVA) followed by t-tests to establish statistical significance.
Results and Discussion: MgO-HA-PLLA nanocomposites supported the greatest adhesion and proliferation of osteoblasts and fibroblasts, and also exhibited the most suitable mechanical properties (highest Young's modulus and strength, with a ductile mode of failure) for application within cancellous bone. Supernatant from degraded samples containing MgO nanoparticles supported greater osteoblast proliferation compared to supernatant from non-MgO samples. The presence of MgO nanoparticles significantly increased the expression of alkaline phosphatase, and slightly decreased type-1 collagen expression. MgO nanocomposites were found to be highly antibacterial and bactericidal towards S. aureus, whereas HA nanoparticles did not affect bacterial functions. These results together indicate the promise of MgO nanoparticles as antibacterial materials for the fabrication of optimized scaffolds for orthopedic tissue engineering.
Conclusions: Here, MgO nanocomposites showed excellent bactericidal efficacy in addition to their ability to enhance the functions of fibroblasts and osteoblasts. Moreover, the addition of MgO nanoparticles allowed for the tailorability of PLLA mechanical properties for bone or ligament tissue. Therefore, MgO nanoparticles should be further investigated as an antibacterial material to promote orthopedic tissue regeneration.
Acknowledgements: This work was supported by NSF-IGERT Grant No. 0965843.
 Zhang L et al., International Journal of Nanomedicine, 3 (2008), 323-34.