(256d) Aqueous Based Hydrogel/Apatite Nanocomposite Scaffolds for Guided Bone Regeneration

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.

Bone matrix is a
composite material consisting of aqueous and inorganic phases. The inorganic
component contributes approximately 65% of the wet weight of the bone. The
organic component usually contributes a little more than 20% of the wet weight
and water can contribute as much as 15% by wet weight in cortical bone. The
aqueous component gives bone its form and contributes to its ability to resist
tension, while the inorganic, or mineral, component primarily resists
compression. Bone that has been demineralized is flexible, pliable, and
resistant to fracture. Bone that its aqueous phase has been removed is rigid
and brittle and a slight deformation factures it. The bone mineral crystals are
classified as apatite and contain both carbonate ions and acid phosphate
groups. The carbonate and acid phosphate groups of the mineral crystals are
very labile and play important roles in the interaction of the crystals with
the surrounding extracellular fluid and with the aqueous components of the
matrix. Recent studies demonstrate that there is a significant physical and
chemical interaction between the aqueous and mineral phases of the bone matrix.

The aqueous phase of the
bone matrix, even though it constitutes only 15% of the matrix, plays a central
role in regulation of collagen fibril mineralization, modulation and control of
cell division, cell migration, cell differentiation, cell maturation,
maintenance of matrix integrity, growth factor modulation, signaling from the
cell to the nucleus, and the extent of mineral-collagen interactions. A
plethora of noncollagenous proteins reside within the aqueous phase which
control the cellular function and the rate of bone turnover. These include
glycoproteins, small integrin-binding ligands, and matrix extracellular
proteins. We hypothesize that hydrogel/apatite nanocomposites are the ideal
biomaterial to mimic the physio-chemical and biologic properties of the bone
matrix and to fabricate scaffolds for bone regeneration. In this work, we
describe synthesis, characterization, and fabrication of hydrogel/apatite
nanocomposite scaffolds for bone regeneration.

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). 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. 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 thermogravimetric analyzer was used to measure the amount of grafting on
the HA surface. When sonication was used to disperse the nanoparticles during
the grafting reaction, grafting as high as 40% by weight was measured. The
morphology of the HA nanoparticles were examined with TEM. 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.

Poly(lactide-ethylene
oxide-fumarate) (PLEOF) unsaturated terpolymer was synthesized by condensation
polymerization of low MW PLA and poly(ethylene glycol) (PEG) with fumaryl
chloride (FuCl) and triethylamine (TEA) as the catalyst. PLEOF macromers were
synthesized using PEG with Mn ranging from 1 to 5 kD and PLAF with Mn ranging
from 1 to 7 kD. The weight ratio of PEG to PLA was varied from 100/0 to 85/15
to produce hydrophilic water-soluble terpolymers. The structure of PLEOF
macromer was characterized by ¹H-NMR and FTIR.

Hydrogel/apatite porous
scaffolds were prepared using PLEOF as the degradable macromer,
methylenebisacrylamide (MBIS) as the crosslinking agent, a neutral redox
initiation system, and sodium chloride crystals as the porogen. The redox
system consisted of ammonium persulfate (APS) and tetramethylethylenediamine
(TMEDA), respectively. Salt crystals were sieved and the fraction retained on
the 300 um mesh sieve was used for scaffold fabrication. Grafted-HA based on
total weight of the hydrogel (sum of the weight of PLEOF, PBS, MBIS, initiator
and accelerator solutions, and the graft weight of HA) was added to the
polymerizing mixture, transferred to a 5 mm diameter x 3 mm height Teflon mold
and pressed manually to maximize packing. The mold was placed in a conduction
oven to facilitate crosslinking and the porogen was leached out by soaking the
scaffolds in distilled water for 2 days, during which time water changes occurred
every 8 h. The scaffolds were dried in vacuum for at least 12 h before use. The
pore morphology was studied with an environmental scanning electron microscope
(ESEM) equipped with an electron backscattered detector and an integrated x-ray
energy dispersive analysis system. Macropores created by the porogen and
micropores created by the partial phase separation of hydrophilic (PEG) and
hydrophobic (PLA) domains of PLEOF hydrogel were observed in the ESEM
micrographs.

Scaffolds were
sterilized with ethanol, washed with PBS and seeded with neonatal heart
fibroblast cells, and incubated for 48 h to study cell attachment to these
composite surfaces. Attached cells were fixed, rinsed with PBS, and
permeabilized by soaking in PBS with 0.1% triton x-100 and 0.1M glycine. Cell
nucleus was stained with 4',6-Diamidino-2-phenylindole or SYTOX Green and
counterstained with Texas Red-X Phalloidin. Since the PLEOF hydrogel is an
inert non-interacting macromer, cells did not attach to this model surface and
had rounded morphology. However, when the nanocomposite scaffold was treated
with collagen type I, having integrin binding RGD domains, cells adhered to the
surface and had extended morphology with focal point adhesion. The cell
morphology was further examined by SEM, demonstrating strong attachment of the
fibroblast cells to the scaffold surface mediated by the integrin binding
sequences on the collagen fibrils. Our results demonstrate that
hydrogel/apatite nanocomposites are an attractive alternative as a biomaterial
for hard tissue regeneration.