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Supercritical Carbon-Dioxide Processed Resorbable Polymer Nanocomposites as Bone Graft Substitutes
+1,2Baker, K C; 2Bellair, R; 2Manitiu, M; 1Gratopp, C A; 1Herkowitz, H N; 2Kannan, R M
+1William Beaumont Hospital, Royal Oak, MI 2Wayne State University, Detroit, MI
INTRODUCTION
Drawbacks associated with the use of autograft and allograft bone have
stimulated interest in the development of synthetic bone graft
substitutes. Resorbable polymers are candidate materials for this
application, as they possess predictable resorption kinetics and
biocompatibility. However, porous resorbable polymers do not possess
the strength necessary for load-bearing applications. Researchers have
reinforced polymer matrices with a variety of fillers, including
hydroxyapatite and phosphate glass to improve the strength of porous
constructs. Due to poor dispersion and weak polymer-filler interaction,
the resulting composite exhibit only modest gains in compressive
strength. Recently, Montmorillonite clays have demonstrated the ability
to enhance mechanical properties of polymeric materials. These
nanostructured clays (nanoclays) consist of a layered aluminosilicate
structure. Recent in vitro studies have demonstrated that these
nanoclays are also biocompatible.
Using a supercritical CO2 (scCO2) processing technique,
nanocomposite constructs consisting of nanoclays dispersed in a porous
resorbable polymer matrix, were created. The following study examined
the porous morphology and dispersion of nanoclay imparted by the
scCO2 technique. Polymer-clay interaction, compressive mechanical
properties and biocompatibility of the resorbable polymer
nanocomposites were also characterized.
METHODS Poly-d-lactide (PDLA, Lakeshore Biomaterials) was mixed with 0, 1 or
2.5wt% of nanoclay in a steel mold. The mold was placed in a pressure
vessel and saturated with CO2 at a temperature of 100oC and 13.8 MPa..
After 60 minutes, the vessel was depressurized at a rate of 0.2 – 0.4
MPa/sec. Constructs were removed from the mold and kept at 2oC for
24 hours.
Pore morphology, diameter and degree of connectivity was assessed by
examining fracture surfaces with scanning electron microscopy (SEM).
Nanoclay dispersion in the polymer matrix, as determined by changes in
the 001 spacing of the nanoclay, was characterized by small angle X-ray
diffraction (SAXD). Pure and nanocomposite constructs were subjected
to rheological testing to characterize polymer-nanoclay interaction. For
mechanical testing, 10 mm x 10 mm cylinders were preloaed to 10 N
and compressed to 50% strain at a displacement rate of 0.5 mm/min.
Cells were seeded directly onto nanocomposite constructs at a density
of 1x106 cells per construct. Controls were performed using the same
cell density on polystyrene culture plates. Alkaline phosphatase activity
of the cultured osteoblasts was assayed at 21 days. Seeded
nanocomposite constructs were also subjected to SEM to examine cell
morphology and mineralization.
RESULTS
Synthesis using the supercritical CO2 method resulted in pure polymer
constructs with mean pore diameters of 236.2 µm (+/- 4.8 µm),
respectively for 100PDLA. Similar pore diameters were noted for
nanocomposite constructs, as shown in Figure 1. Both pure and
nanocomposite constructs exhibited a high degree of pore
interconnectivity, as assessed by analysis of fracture surfaces by SEM.
Figure 1. SEM of pure (left) and nanocomposite (right) constructs.
SAXD of the nanocomposites showed a shift in the 001 peak of pure
nanoclay from 3.36o to 4.41o 2θ indicating an increase in platelet spacing
of 1.79 nm. Melt rheology of the constructs showed a shift in the
crossover frequency, characteristic of the polymer chain relaxation time,
from 0.23 to 0.10 rad/s. Results indicate excellent nanoclay dispersion
and significant polymer-clay interaction.
The addition of 1wt% and 2.5wt% nanoclay significantly increased the
compressive strength and moculus of the constructs, as shown in Figure
2. With as little as 2.5wt% nanoclay, compressive strength was
increased by a factor of 2.5.
100PDLA
100PDLA-1wt%
100PDLA-2.5wt%
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Compressive Modulus
Figure 2. Compressive strength and modulus of constructs.
Nanocomposite constructs yielded higher output of alkaline
phosphatase when compared to polystyrene culture plates. The
difference in ALP expression between the construct with 2.5wt%
nanoclay and polystyrene culture plates was statistically significant (p =
0.002). SEM of the seeded constructs demonstrated cellular infiltration
and mineralization deep within the porous structure, shown in Figure 3.
Figure 3. SEM images demonstrating mineralization of a
nanocomposite construct after 21 days in culture.
DISCUSSION
Supercritical CO2 processing of resorbable polymer/nanoclay mixtures
results in the formation of porous nanocomposite constructs, which
exhibit biocompatibility and significant improvements in mechanical
properties. Improvements in mechanical properties of the constructs are
related to the dispersion of nanoclay within the polymer matrix and
subsequent reduction in polymer chain mobility. The biocompatibility
of the constructs, as determined by the alkaline phosphatase activity of
cultured osteoblasts, was greater than the polystyrene culture plate
controls. Supercritical CO2-processed resorbable polymer
nanocomposites represent a potential alternative to autograft and
allograft in orthopaedic procedures requiring bone graft.
ACKNOWLEDGEMENTS This research was funded by a Seed/Starter grant from the Cervical
Spine Research Society. 500 µµµµm 200 µµµµm
Poster No. 1213 • 56th Annual Meeting of the Orthopaedic Research Society