Porous Polymer-Ceramic Systems for Tissue Engineering Support the Formation of Mineralized Bone Matrix
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Mat. Res. Soc. Symp. Proc. Vol. 414 01996 Materials Research Society
EXPERIMENT Matrix Fabrication A 50:50 poly(lactide/glycolide) copolymer (PLAGA), [DuPont Medisorb, lot S2127 S144] and particulate hydroxyapatite (HA), [Howmedica, lot HAG 89272B], were used to fabricate 3dimensional macroporous matrices. Macroporous 3-dimensional polymer matrices were fabricated using techniques described elsewhere(7). Briefly, a 40% w/v solution of PLAGA in chloroform was created to suspend HA and NaCl crystals (100-250 gm diameter). One gram each of PLAGA, HA and salt were vortex mixed with 2.5 ml chloroform. After dissolution of the polymer, 1.0 ml of a 1% aqueous solution of poly(vinyl alcohol), [Polysciences, lot 413322] was added to stabilize the mixture. The resulting emulsion was cast into molds, allowed to solidify 24h, and dried under vacuum 48h. NaCI was leached from the polymer matrix by immersion of the specimens in deionized water at 37'C for 48h. The resulting pore size averaged 1504m in diameter. The polymer was again vacuum dried prior to testing.
Cell culture Primary osteoblast-like cultures were established using the method described by Schwartz(15). Ten neonatal Sprague-Dawley rat calvaria were surgically excised and cleaned in phosphate buffered saline (PBS) containing 2x penicillin-streptomycin (Sigma Chemical Co., St. Louis, MO). The calvaria were subjected to sequential collagenase/trypsin digestions. Digestion solution was prepared by dissolving 137 mg collagenase (type I, Worthington Biochemicals) and 50 mg trypsin (Sigma, type III) in 10 ml PBS solution. Calvaria were added to the digestion solution, incubated for 20 minutes, after which the digestion solution was removed and transferred to centrifuge tubes. Each digestion population was centrifuged, resuspended and plated onto 75 cm 2 polystyrene tissue culture flasks. Cells were grown in Ham's F-12 medium (GIBCO, Grand Island, NY), supplemented with 12% fetal bovine serum (Sigma) and maintained at 370 C with 5% C02. Medium was changed after the first 24 hours and then every subsequent 48 hours. Cells were grown to confluency and then seeded onto 3-dimensional matrices at a plating density of Ix 104 cells per well. Cell populations were cultured on the polymer matrices up to 21 days. Cell Proliferation Tissue culture plates (24 well, Falcon) were coated with 12% poly(hydroxyethylmethacrylate) (Polysciences, Warrington, PA), to ensure that the cells would grow only on the polymer discs contained in the wells and not on the tissue culture polystyrene surface (TCPS). Polymers were held to the bottom of the well using sterilized inert, silicon-based, high temperature vacuum grease (VWR Scientific, San Francisco, CA). The cells were plated on the polymer discs at a plating density of 1 x 104 per well. Cell proliferation was determined at 24 hours, 3, 7, 14 and 21 days. At these times, the discs were gently washed with phosphate buffered saline (PBS) to remove any unattached cells. The adherent cells were removed from the substrate by incubation in 1 ml o
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