Supercritical Carbon-Dioxide Processed Resorbable Polymer ... · Supercritical Carbon-Dioxide...

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Supercritical Carbon-Dioxide Processed Resorbable Polymer Nanocomposites as Bone Graft Substitutes + 1,2 Baker, K C; 2 Bellair, R; 2 Manitiu, M; 1 Gratopp, C A; 1 Herkowitz, H N; 2 Kannan, R M + 1 William Beaumont Hospital, Royal Oak, MI 2 Wayne State University, Detroit, MI [email protected] 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 100 o C 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 2 o C 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 1x10 6 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 3.36 o to 4.41 o 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% Compressive Strength (MPa) 0 2 4 6 8 10 Compressive Modulus (MPa) 0 20 40 60 80 100 Compressive Strength 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 μm 200 μm Poster No. 1213 56th Annual Meeting of the Orthopaedic Research Society

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Page 1: Supercritical Carbon-Dioxide Processed Resorbable Polymer ... · Supercritical Carbon-Dioxide Processed Resorbable P olymer Nanocomposites as Bone Graft Substitutes +1,2 Baker, K

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

[email protected]

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%

Co

mp

res

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tre

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MP

a)

0

2

4

6

8

10

Co

mp

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siv

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MP

a)

0

20

40

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100

Compressive Strength

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