Proliferation and Mineralization of Human Osteosarcoma Cells on

Proliferation and Mineralization of Human Osteosarcoma Cells on Different Polyarylates and Polycarbonate Substrates Milca I. 1 Aponte-Román , Dr. Robert 2 Dubin , Dr. Joachim 2 Kohn , Dr. Adrian 1 Mann , Dr. David 3 Denhardt , Departments of 1 Materials Science Engineering, and 3 Cell Biology & Neuroscience, 2 Center for Biomaterials. Rutgers University, The Sate University of New Jersey Introduction The goal of this research was to characterize the response of human osteosarcoma cells to polymer substrate variation in vitro and to establish correlation between cellular response and polymer chemistry/structure. In this particularly project mineralization by human osteosarcoma cell line, Saos-2, when grown on various polymers was examined. The biodegradable and biocompatible polymers examined are members of the "polyarylate" and “polycarbonates” combinatorial library developed in the Dr. J. Kohn’s research group at Rutgers University. Alizarin Red-S assay was used to compare the mineralization on each of the substrates. Substrates Polyarylate- and polycarbonate-coated glass coverslips were prepared in Kohn’s lab. Chemico-physical properties affecting polymer flexibility, protein adhesion and degradation were altered by the inclusion of DT, poly(ethylene glycol) (PEG), and Iodine (I 2) into the backbone of the poly(DTE carbonate), and by alterations in the pendent chain and diacid componet of the polyarylate family of polymers. Cell Culture Saos-2 cells were initially cultured on top of polyarylate- and polycarbonate-coated coverslips in HAM’s F-12 medium supplemented with 10% fetal bulvine serum, 10 m. M Hepes p. H 7. 5, + penicillin and streptomycin, and + glutamine. At day 7, 10 m. M -glycerolphosphate, 10 n. M dexamethasone (DEX), and 50 g/m. L ascorbic acid were added to induce mineralization, and the addition repeated at every medium change until the end of experiment. Cultures were grown at 37 o. C in a humidified 5% CO 2 atmosphere. Alizarin Red-S Assay Alizarin red is a dye, which binds selectively to calcium. Every week the medium of one plate was removed and the culture wells were briefly washed with PBS and stained for 10 min with 1% alizarin red-S p. H 4. 14. Cultures were then rinsed with water and PBS, to remove the stain not associated with calcium mineral deposit. A destaining procedure was followed using 10% cetylpyridinium chloride in 10 nm sodium phospahete p. H 7. 0 for 15 min in order to measure the optical absorbance. Absorbance measurements were done with a microplate reader with 570 nm. Results Alizarin, is the main colorant found in the madder plant Rubia tinctorum A. Polyarylates 1. Poly(DTE suberate) 2. Poly(DTB suberate) 3. Poly(DTH suberate) 4. Poly(DTO suberate) 5. Poly(DTD suberate) 6. Poly(DTE succinate) 7. Poly(DTH succinate) 8. Poly(DTD succinate) 9. Poly(DTE dodecandioate) 10. Poly(DTH dodecandioate) 11. Poly(DTD dodecandioate) 12. Poly(DTH adipate) 13. Poly(DTH sebacate) PLGA poly(DL-lactate co-glycolate) PLA poly(DL-lactate) TCP tissue culture plastic B. Polycarbonates 1. 2. 3. 5. 7. 8. 9. 1. 5. 7. 8. 9. Poly(DTE Carbonate) Poly(DTE-co-4% PEG l. K Carbonate) Poly(DTE-co-8% PEG l. K Carbonate) Poly(DTE-co-10% DT carbonate) Poly(DTE-co-10% DT-co-4% PEG l. K Carbonate) Poly(DTE-co-10% DT-co-8% PEG l. K Carbonate) Poly(I 2 DTE-co-4% PEG l. K Carbonate) Poly(I 2 DTE-co-8% PEG l. K Carbonate) Poly(I 2 DTE-co-10% I 2 DT-co-4% PEG l. K Carbonate) Poly(I 2 DTE-co-10% I 2 DT-c 0 -8% PEG l. K Carbonate) 17. PLGA-resomer 506 (Boehringer Ingelheim) PLLA-resomer L 206 (B. I Chemicals) TCP tissue culture plastic Plate with polycarbonate-coated coverslips after stained with alizarin red-S. Future Work This work was done in the Cell Biology & Neuroscience Department as a part of my Integrative Graduate Education and Research Traineeship (IGERT). The main idea was to rotate through the laboratory of my interdisciplinary secondary advisor, Dr. Denhardt, and learn about cell culture. For now on I will be working using a Near Field Scanning Optical Microscope (NSOM) to nanopattern surfaces coated with Self Assembled Monolayers (SAMs). The idea is to functionalize the SAM with bioactive chemicals by using the laser from the NSOM to locally initiate a chemical reaction. Discussion and Preliminary Conclusions • All Saos-2 cultures on polyarylate-coated coverslips formed calcium phospahate mineral with minimal disparity between each other. • All Saos-2 cultures on polycarbonate-coated coveslips formed calcium phosphate mineral. However, low light absorbance was obtained in polycarbonate substrates with %4 and 8% PEG (code #2 and #3 from polycarbonate series). PEG in %8 resulted in poor cell attachment and little spreading. • In contrast, no significant variations were obtained on the polycarbonate cultures where PEG is also present in 4% and 8% (code #10, #11, #14, and #15 from polycarbonate series). When Iodine (I 2) is incorporated it seems to suppress the effect of PEG. It is possible that the Iodine may work to suppress the PEG by steric hindrance rather than making the polymer more hydrophobic. • These experiments need to be repeated before firm conclusions can be drawn. Acknowledgments The author would like to express her gratitude to, • Dr. Denhardt from the Department of Cell Biology & Neuroscience, for letting me work in his lab and teaching me about cell culture. • Dr. Robert Dubin, for providing us with the culture plates coated with polycarbonate and polyarylates, and valuable discussion. • Christian Kazanecki, Melissa Weidner and Jennifer Luo for their help in cell culture and for sharing their lab with me. • IGERT for supporting this work.