(256c) Synthesis and Characterization of Apatite Nanoparticles Grafted with Unsaturated Hydrophilic Macromers

Tissue engineering
strategies for the regeneration of damaged orthopedic tissues involve the use
of highly porous and interconnected scaffolds with acceptable mechanical
properties to serve as a substrate for adhesion, spreading, migration,
proliferation, and differentiation of osteoblastic cells. One approach to bone
replacement involves the use of prefabricated scaffolds for cell
transplantation to promote three dimensional tissue growth, nutrient diffusion,
matrix production, and vascularization. These prefabricated scaffolds present a
large surface area for cell growth and reduce the diffusional barriers for
material transport.

Calcium phosphate
ceramics such as hydroxyapatite (HA) and tricalcium phosphate (TCP) have shown
to promote bone ingrowth, are biocompatible and osteoconductive. The major
drawback of ceramics based on HA, TCP, or their combination is that the initial
mechanical properties are less than cancellous bone which leads to difficulty
in maintaining the composite within the defect during surgery. In particulate
form, they offer increased mechanical strength to polymeric composite materials
primarily in compression, but are less effective in enhancing resistance to
torsional and bending forces. To improve shear and tensile strength and fracture
toughness of ceramics, composite biomaterials based on natural or synthetic
polymers and ceramics in particulate form have been developed. The use of calcium
phosphate ceramics reinforced with natural or synthetic polymers has improved the
fracture toughness of these composites and has enabled faster and more
aggressive rehabilitation. We hypothesize that surface grafting of apatite
nanoparticles (with large surface to volume ratio) with hydrophilic unsaturated
macromers will improve their dispersion in the aqueous phase and enhance
bonding at the interface between the filler and the aqueous matrix in
polymer/ceramic composites and subsequently increase the resistance of the
composite biomaterial to torsional and bending forces. In this work, we describe
a novel method for grafting unsaturated hydrophilic macromers to the surface of
apatite nanoparticles for use as filler in synthetic bone substitute.

HA nanoparticles were
grafted with hydrophilic unsaturated poly(ethylene glycol) oligomers to improve
their suspension stability and interfacial bonding in the aqueous hydrogel
solution. The grafting reaction was carried out in two steps. In the first
step, poly(ethylene glycol) methacrylate (PEGMA) was condensed with
3-isocyanatopropyltrimethoxysilane (iCPTMS) to form a PEGMA-PTMS urethane with
unsaturated methacrylate and trimethoxysilane end-groups. In the second step,
the trimethoxysilane end of the urethane was reacted with reactive phosphate
and carbonate groups on the HA surface using ammonium hydroxide and methanol as
the catalysts to produce HA with grafted PEGMA oligomers (gHA). In a typical
reaction, PEGMA was dried by azeotropic distillation and hydroquinone was added
as a radical scavenger to prevent free radical polymerization of methacrylate
groups of PEGMA. DMF and iCPTMS was added dropwise with stirring to the reactor
and the condensation reaction was allowed to proceed under reflux conditions
and with excess PEGMA. The reaction was allowed to cool to ambient conditions, HA
was added to the reactor with stirring, and the reaction mixture was sonicated
to break up the aggregate of HA nanoparticles using a probe sonicator. The
suspension was sonicated for 30 min with an Ultrasonic Processor with a power
and frequency of 10 Watts and 20 kHz, respectively, in the continuous mode. Ammonium
hydroxide and methanol were added to the reaction, the mixture was sonicated
for an additional 30 min, and grafting reaction was allowed to continue under
reflux conditions. The grafted HA was washed with methylene chloride,
centrifuged, and re-dissolved at least 5 times to remove all unreacted components and dried
under vacuum.

gHA was characterized
with FTIR, TGA, and TEM. A Thermo Nicolet FTIR Nexus 470 was used to measure
the absorption spectrum of gHA powder in the IR region. Samples were dried
under vacuum for at least 12 h at 50°C
before acquisition of FTIR spectrum. The unmodified and grafted HA powders were
packed and pressed firmly in the reference and sample compartments of the FTIR
cell and spectrum was collected under a dry nitrogen atmosphere with 16
averaged scans and a resolution of 4 cm^-1. The broad absorption band
with peak position at 2880 cm^-1 was due to symmetric and asymmetric
C-H vibrations of the ?CH₂- group of PEG in PEGMA and propyl group
in iCPTMS. The shoulder at 2950 cm^-1 was due to symmetric and
asymmetric C-H stretching vibration of the ?CH₃ groups of
methacrylate and methoxy silane. These absorption bands were absent in the
spectrum of untreated HA. The absorption with peak position at 1720 cm^-1
and a shoulder at 1740 cm^-1 were due to the C=O stretching vibration
of the ester group of methacrylate in PEGMA. This absorption was absent in the
spectrum of HA grafted with PEG terminated with methoxy (mPEG) in place of
methacrylate group. The absorption with peak location at 1670 cm^-1
was assigned to carbonyl absorption band of the urethane group (amide I) and
the broad absorption with peak location at 1580 cm^-1 was
collectively attributed to N-H bending and C-N vibration (amide II) of the urethane
group. These peaks were absent in the spectrum of untreated HA. The absorption
with peak location at 1360 cm^-1 was assigned to the C-H bending
vibration of the alkene group of methacrylate in PEGMA which was absent in the
spectrum of HA grafted with PEG terminated with methoxy group. The absorptions
with peak locations at 1300 cm^-1 and 1250 cm^-1 were due
to the C-N and N-H bending vibrations and to C-O bending vibration of the
urethane group, respectively, formed by the reaction of PEGMA and iCPTMS. The
absorptions with peak locations at 1420 and 1460 cm^-1 were
attributed to vibrations of the phosphorous/oxygen/silicon complex (P-O-Si) on
the surface of HA by the reaction of methoxysilane with phosphate groups. The
absorptions in the FTIR spectrum of gHA with untreated HA as the reference
confirmed the grafting of PEGMA-PTMS urethane on the surface of HA.

A Perkin Elmer thermogravimetric
analyzer was used to measure the amount of grafting on the HA surface. The
experiments were carried out under helium atmosphere. The sample was heated to
120°C, allowed to
equilibrate for 15 min to remove water content, and heated to 900°C at a rate of 10°C/min. Samples included untreated HA
(HA), HA grafted with PEGMA-PTMS urethane without sonication (gHA-WOS), and HA grafted
with PEGMA-PTMS urethane with sonication (gHA-WS). The weight loss of HA,
gHA-WOS, and gHA-WS after reaching 900°C
was 5.0%, 8.0%, and 46.0%, respectively. After subtracting the weight loss of
5.0% due to bound water, the percent grafting of gHA-WOS and gHA-WS was 3.0%
and 41.0%, respectively. These results demonstrates that sonication of the HA
nanoparticles during the grafting reaction reduces the aggregate size and
substantially increases the extent of grafting and that the extent of grafting
can be controlled by sonication.

The morphology of the HA
nanoparticles were examined using a JEOL 100 CXII transmission electron
microscope at an accelerating voltage of 100 kV. The nanoparticles in the gHA
sample without sonication had whisker like morphology, similar to untreated HA,
with long and short axis of 100 and 20 nm, respectively, while those in the gHA
sample with sonication had a more rounded morphology with long and short axis
of approximately 20 nm. Our results demonstrate that sonication of the
suspension during the grafting reaction breaks the nanoparticles along the long
axis, forming rounded nanoparticles, and creating new surface area for
grafting. This explains the substantially higher extent of grafting for gHA
samples sonicated during the grafting reaction.