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● 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