(601h) Improved Cellular Functions and Reduced Bacterial Infection on MgO Nanocomposites

Improved Cellular Functions and Reduced Bacterial
Infection on MgO Nanocomposites

Daniel J. Hickey¹ and Thomas J. Webster^1,2

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. [1], 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 10⁶Staphylococcus 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.

References:

[1]   Zhang L et al.,
International Journal of Nanomedicine, 3 (2008), 323-34.