(67e) Using Magnesium Oxide Nanoparticles to Improve Inhomogeneous Soft and Hard Tissue Growth

Introduction: Where ligaments meet bone, there
exists a transitional region of inhomogeneous tissue that is graded from hard,
highly mineralized fibrocartilage at the bone interface to un-mineralized soft
tissue at the ligament. This structure, called the enthesis, disperses stress
concentrations that arise due to the vastly different mechanical properties of
bone and ligaments. However, in the event that a ligament is injured and
requires surgery, the tendon-to-bone insertion site (TBI) must be destroyed and
cannot easily be restored due to avascularity in the region and because of consequent
slow healing times within the inhomogeneous tissue compared to healing within
uniform tissues. It is believed that this loss of enthesis functionality
following joint reconstructive surgery is a leading cause of high failure rates
for such surgeries (5-25% failure rates for ACL surgery) [1]. Therefore, there
is considerable interest in the development of a nanostructured biomaterial
that is capable of regenerating the TBI.

The
current study developed and characterized for the first time poly (l-lactic
acid) (PLLA) mineralized with nanoparticles of hydroxyapatite (HA) and
magnesium oxide (MgO) as a tissue scaffold to direct the regeneration of the
enthesis. HA constitutes about 60% of the dry mass of bone and has been used
previously as a successful material for bone grafts. Magnesium is an essential
mineral that regulates HA crystal size and density, and Weng and Webster have
shown that nano-rough magnesium increased bone cell density by up to three-fold
when compared to bulk magnesium [2]. Presently, the ability of materials to
promote tissue growth at the TBI was characterized via cell adhesion and
proliferation experiments with fibroblasts and osteoblasts. Further
characterization of materials was performed using scanning electron microscopy
(SEM), contact angle tests, and mechanical load testing.

Methods: Hydroxyapatite (HA) precipitates
were synthesized by a wet chemistry process and then treated hydrothermally to
give nanoparticles of sizes consistent with HA crystals observed in natural
bone, as reported by Zhang et al. [3]. Poly (l-lactic acid) (PLLA)
(MW=50,000), (Polysciences, Warrington, PA) was placed in 20 mL scintillation
vials and dissolved in 10 mL of chloroform (Sigma Aldrich, St. Louis, MO) to
reach 3 wt% PLLA in chloroform. HA nanoparticles and MgO nanoparticles
(particle diameters of 20 nm, US Research Nanomaterials Inc., Houston, TX) were
added to separate vials in concentrations ranging from 5-20 wt% in PLLA. Samples
were heated to 55 °C and sonicated for 1 hour, then poured into 60 mm diameter
pyrex petri dishes (Sigma Aldrich, St. Louis, MO). Samples were heated at 60 °C
for 1.5 hours to evaporate excess chloroform, producing polymer films about 0.2
mm thick which were then allowed to sit overnight before being 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 material analysis software (ADMET, Norwood, MA). A
10 lb. load cell was used and small pieces of paper towel were attached to the
tensile grips to prevent the polymer composites from breaking from the force of
the grips. This set-up was used to obtain the elastic modulus, material
elongation, and maximum load endured for each sample. Cell adhesion and
proliferation tests were performed by seeding 3500 cells/cm² of
fibroblasts and osteoblasts (American Type Culture Collection, Manassas, VA)
onto 1-cm² samples and culturing for 4, 24, 72, and 120 hours under
standard conditions. Cell numbers were quantified using MTT assays (Sigma
Aldrich, St. Louis, MO). Experiments were conducted in quadruplet and repeated
three times. Data was analyzed using Student's t-tests.

Results: Results indicated for the first time
that MgO nanoparticles in plain PLLA or PLLA/HA composites significantly
increased osteoblast and fibroblast adhesion on PLLA (Figure 1). SEM images
showed considerable differences in nanoscale surface topography between
samples. However, preliminary contact angle results demonstrated no differences
in wettability among all samples. Mechanical tensile testing revealed that the
addition of HA nanoparticles to plain PLLA hardened the polymer, reducing the
material elongation and increasing its elastic modulus. These results were
exaggerated for PLLA mineralized with MgO nanoparticles. The maximum load that
the polymer was capable of withstanding was slightly increased for MgO
mineralized samples.

Figure 1.
Adhesion of fibroblasts and osteoblasts on pure PLLA and PLLA mineralized with
nanoparticles (20% HA, 20% MgO, and 10% HA/10% MgO). Control is empty cell
culture well.  Data represents mean ± SD. n = 4. % = weight%. *P<0.05,
**P<0.005 compared to controls.

Conclusions: Here, MgO nanoparticles have been
shown to increase osteoblast and fibroblast cell adhesion on PLLA, and should
therefore be further investigated as a material to promote bone tissue growth
at one end of the tendon-to-bone insertion site, and fibrous tissue growth at
the other end of the TBI.

Acknowledgements:
The authors
thank Robert Eagan, William Fowle, Scott McNamara, David McKee, the
Northeastern University Department of Chemical Engineering for facilities and
funding, and the NSF-IGERT grant #0965843 for funding.

References

[1]   Smith L,
Thomopoulos S, US Muscoskel. Rev., 6 (2011), 11-5.

[2]   Weng L, Webster
TJ, Nanotechnology. 23 (2012), 485105.

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