Download A Biomimetic Cementeous Material: Gelatinous Hydroxyapatite in

● A Biomimetic Cementeous Material: Gelatinous Hydroxyapatite in Organosilica Polymeric Matrix
+1Ko, C-C; 2An, K-N
+1University of North Carolina, Chapel Hill, NC, 2Mayo Clinic College of Medicine, Rochester, MN
[email protected]
Methods and Materials
The gelatinous hydroxyapatite nanocomposite (HAP-Gel) was
synthesized through the biomimetic procedures based on coprecipitation principles1. The HAP-Gel slurry was washed with
methanol and condensed via centrifugation (5000 rpm for 15
minutes). The condensed HAP-Gel was then freeze-dried and
pulverized.
A putty formula was developed using the silanized HAP-Gel
powders and 1X PBS (phosphate buffer saline). The silanization
used organically modified animosilane (bis[3-(trimethoxysilyl)propyl]ethylenediamine [enTMOS]). The ionic strength of PBS
triggered sol gel reaction with the enTMOS coating on the HAP-Gel
particles and hydrate gelatin molecules, which provide putty
property of the cement. The setting time for various amounts (10
wt%, 20 wt%, and 27 wt%) of enTMOS used was characterized by
the rate of weight loss and linear shrinkage within the first 120
hours after mixing. The compressive strength was measured using
the dried cylindrical samples (3.5 mm diameter and 7 mm length)
with n=5. An Instron machine (model 4411, Instron Co., Norwood,
MA) was used at the speed 0.5 mm/min to determine ultimate failure
strength for the cement. Thermal graphic analysis (TGA) was used to
estimate the weight percentage of individual components.
Osteoblasts, MC3T3-E1, were cultured in 96-well plates (Falcon,
Becton, Dickinson Labware, Frankin Lakes, NJ, USA) in which all
wells were coated with gelatinous hydroxyapatite silica cement.
Cells were seeded at a density of 1× 104 per milliliter using αMEM
medium supplemented with 10 % of FBS and 1%
penicillin/streptomycin under 37 °C, 5% CO2 atmosphere. Cell
proliferation was measured at 1, 4, 7, 10, and 13 days after seeding.
At the end of the designed cultivation period, CellTiter reagent
(CellTiter 96® Aqueous One Solution Proliferation Assay, Promega
Co, Madison, WI) was added to each well to quantify the number of
living cells in culture. The control used the 96-well plates as
received without coating. Five samples were tested at each time
point for each group (n=5). Two-way ANOVA was used for
comparison. In addition, a pilot assessment for cell differentiation
on the material was performed using the assay for alkaline
phosphatase protein activity and alizarin red stain for mineralization.
Results
Figure 1 shows that the optimal HAP-Gel to enTMOS ratio was 4:1
(20 wt% of enTMOS), which produced cement with the highest
strength (96.9 ± 27.7MPa) comparable to that (95-110 MPa) of the
Maximum
Compressive
Stress (Mpa)
commercial PMMA bone cement. The low (10%) and high (27%)
enTMOS contents weakened the cement due to insufficient crosslink and too thick of the silica matrix, respectively.
150
120
90
60
30
0
0%
10%
20%
27% enTMOS
Fig 1. Box plot showing that compressive strength varies with the
amount of enTMOS used.
According to the weight loss and shrinkage data, the setting time of
the cement occurred at 13 hours after mixing. The TGA data further
confirmed that the initial setting was attributed to the dehydration
that aggregates HAP-Gel particles into a dense matter. The TGA
data also suggested that the dried cement contains 65-70%
inorganic, 20-27% organic, and 7% water.
Osteoblasts (MC3T3-E1) adhered and grew normally on the
surfaces of the gelatinous hydroxyapatite silica matrix. No
difference (p>0.05) in cell growth was found between the matrixcoated and the normal Petri dish (control) (Fig. 2). Results also
showed that there were no differences in alkaline phosphatase
activity (synthesis function) between cells on the material and the
control. Alizarin red stain further confirmed mineralization for both
the cement and the control.
1.5
Absorbance
Introduction
Polymethyl methacrylate (PMMA) bone cement has been used
successfully to fill in the space of fractured vertebrae and between
the prosthesis and bone. However, PMMA cement is not used for
craniofacial skeletal reconstruction, partially, due to its nonabsorbable nature and exothermal reaction. Commercially available
calcium phosphate based cements may be used for craniofacial
applications in non-load bearing areas such as small, periodontal
defects. Nevertheless, the critical size defects in craniofacial
skeleton that may require multiple-phase surgery to achieve
adequate reparation and function cannot be restored by any
cementeous bone replacements. Developing a bioabsorbable
material to mimic natural bone will provide great potential not only
in craniofacial reconstruction, but also to enhance remedy for spinal,
hip and forearm fractures. The purpose of this research is to develop
cementeous formula of a nanostructural, gelatinous hydroxyapatite
composite that imitates the actual bony polymer-ceramic (collagenhydroxyapatite) nano-bonding structures. Biocompatibility, setting
characteristics and mechanical strength of the new cement are
reported.
Control
Cement
1
0.5
0
7
10
13 Days
1
4
Fig. 2. Comparison of MC3T3-E1 cell proliferation profiles on the
materials (cement) and the control (Petri dish).
Discussion
Our previous studies1,2 have largely characterized the
nanocomposite system including (1) thermal analyses show the
similar decomposition pattern between our nanocomposite and
natural bone; (2) FT-IR spectra reveal similar organic-inorganic
binding mechanisms between our material and hydroxyapatitecollagen system,Error! Bookmark not defined. and natural bone; (3)
amide and carboxyl chains of the gelatins are subjected to
modification by cross-linking agents. The gelatinous hydroxyapatite
in organosilica polymeric matrix shown in the present study is a
unique material. It has formable properties allowing laboratorial
molding of the porous scaffolds and surgical injection of
cementitous bone replacement. Compressive strength is comparable
to that of natural cortical bone. Pilot data also suggest that the
material is subjected to remodeling in vivo within 4-8 weeks (data
not shown). This new cement is expected to have many dental and
orthopedic applications.
Acknowledgement
This study is supported by NC Biotech Center, NIH/NIDCR
K08DE018695, R41DE020971, UNC Research Council and AAOF.
References
1. Chang MC, Ko CC, Douglas WH. Biomaterials, 24(17):2853-2862,
2003.
2. Luo T-J M., Ko C.C., Chiu C-K, Llyod J., Huh H. J. Sol-Gel Sci &
Tech. 53:459–465, 2010.
Poster No. 1910 • ORS 2011 Annual Meeting