The cytocompatibility and osteoinduction of water-soluble phosphorylated chitosan (P-chitosan), that was modified by PO23 groups, with neonatal rat osteoblasts in vitro was studied. To identify the biocompatibility of P-chitosan with osteoblasts, the biofunctions of osteoblast cells, in the presence of three different concentrations of P-chitosan solutions as culture media and DMEM with 10% FBS as controls, were determined. The osteoblast differences in the P-chitosan solutions and on chitosan films were also investigated with immunocytochemistry. It was found, based on the ALP activity and mineralization assay that water-soluble P-chitosan has excellent cytocompatibility compared to the chitosan without phosphorylated modification. P-chitosan concentrations as high as 2% had a significant influence on cytocompatibility and osteoinduction; one tissue (or derived product) caused a second undifferentiated tissue to differentiate into bone. Water-soluble P-chitosan could be a promising osteoinductive biomaterial for tissue engineering and orthopedic uses.
2. Xu, J., McCarthy, S.P., Gross, R.A. and Kaplan, D.L. (1996). Chitosan Film Acylation and Effects on Biodegradability , Macromolecules, 29: 3436–3440 .
3.
3. Sashiwa, H., Saimoto, H., Shigemasa, Y., Ogawa, R. and Tokura, S. (1990). Lysozyme Susceptibility of Partially Deacetylated Chitin , Int. J. Biol. Macromol., 12: 295–296 .
4.
4. Chatelet, C., Damour, O. and Domard, A. (2001). Influence of the Degree of Acetylation on Some Biological Properties of Chitosan Films , Biomaterials, 22: 261–268 .
5.
5. Sakaguchi, T., Hirokoshi, T. and Nakajima, A. (1981). Adsorption of Uranium by Chitin Phosphate and Chitosan Phosphate , Agric. Biol. Chem., 45(10): 2191–2195 .
6.
6. Nishi, N., Maekita, Y., Nishimura, S., Hasegawa, O. and Tokura, S. (1987). Highly Phosphorylated Derivatives of Chitin, Partially Deacetylated Chitin and Chitosan as New Functional Polymers: Metal Binding Property of the Insolubilized Materials , Int. J. Biol. Macromol., 9: 109–114 .
7.
7. Wang, X., Ma, J., Wang, Y. and He, B. (2001). Structural Characterization of Phosphorylated Chitosan and their Applications as Effective Additives of Calcium Phosphate Cements , Biomaterials, 22: 2247–2255 .
8.
8. Wang, X., Ma, J., Wang, Y. and He, B. (2002). Bone Repair in Radii and Tibias of Rabbits with Phosphorylated Chitosan Reinforced Calcium Phosphate Cements , Biomaterials, 23: 4167–4176 .
9.
9. Nishi, N., Ebina, A., Nishimura, S., Tsutsumi, A., Hasegawa, O. and Tokura, S. (1986). Highly Phosphorylated Derivatives of Chitin, Partially Deacetylated Chitin and Chitosan as New Functional Polymers: Preparation and Characterization , Int. J. Biol. Macromol., 8: 311–317 .
10.
10. Ripamonti, U. and Duneas, N. (1996). Tissue Engineering of Bone by Osteoinductive Biomaterials , MRS Bull., 21: 36–39 .
11.
11. Puleo, D.A., Holleran, L.A., Doremus, R.H. and Bizios, R. (1991). Osteoblast’s Responses to Orthopaedic Implant Materials in Vitro , J. Biomed. Mat. Res., 25: 711–723 .
12.
12. Sringa, E., Filanti, C., Giunciuglio, D., Albini, A. and Manduca, P. (1996). Osteoblastic Cells from Rat Long Bone, Characterization of their Differentiation in Culture , Bone, 16: 663–670 .
13.
13. Bronwyn, M.K., Fiona, J.M. and John, H.M. (2000). Serum-Free Cultures of Rat Post-natal and Fetal Brainstem Neurons , Development Brain Research, 120: 199–210 .
14.
14. Cai, K., Yao, K., Lin, S., Yang, Z., Li, X., Xie, H., Qing, T. and Gao, L. (2002). Poly(D, L-Lactic Acid) Surfaces Modified by Silk Fibroin: Effects on the Culture of Osteoblast In Vitro , Biomaterials, 23: 1153–1160 .
15.
15. Thomas, J.W., Celaletdin, E., Robert, H.D., Richard, W.S. and Rena, B. (2000). Enhanced Functions of Osteoblasts on Nanophase Ceramics , Biomaterials, 21: 1803–1810 .
16.
16. Zund, G., Ye, Q., Hoerstrup, S.P., Shoeberlein, A., Schmid, A.C., Grunenfelder, J., Vogt, P. and Turina, M. (1999). Tissue Engineering in Cardiovascular Surgery: MTT, a Rapid and Reliable Quantitative Method to Assess the Optimal Human Cell Seeding on Polymeric Meshes , Eur. J. Cardio-thoracic. Surg., 15: 519–524 .
17.
17. Ciapetti, G., Cenni, E., Pratelli, L. and Pizzoferrato, A. (1993). In Vitroevaluation of Cell/Biomaterial Interaction by MTT Assay , Biomaterials, 14: 359–364 .
18.
18. Lowry, O.H., Roberts, N.R., Wu, M., Hixton, W.S. and Crawford, E.J. (1954). The Quantitive Histochemistry of Brain III, Enzyme Measurements , J. Bio. Chem., 207: 19–37 .
19.
19. Lieberherr, M., Vreven, J. and Vaes, G. (1973). The Acid and Alkaline Phosphatases, and Phosphoprotein Assay , Biochem. Biophys. Acta, 293: 160–169 .
20.
20. Siggelkow, Heide, Hilmes, Dirk, Rebenstorff, Katja, Kurre, Wiebke, Engel, Iris, Hüfner, Michael (1998). Analysis of Human Primary Bone Cells by Florescence Activated Cell Scanning: Methodological Problems and Preliminary Results , Clinica Chimica Acta, 272: 111–125 .
21.
21. Auf’m-Kolk, B., Hauschka, P.V. and Schwarta, E.R. (1985). Characterization of Human Bone Cells in Culture , Calcif. Tissue Int., 37: 228–235 .
22.
22. Wergedal, J.E. and Baylink, D.J. (1984). Characterization of Cells Isolated and Cultured from Human Bone , In: Proc. Soc. Exp. Bio. Med., 176: 60–69 .
23.
23. Du, C., Cui, F.Z., Zhu, X.D. and De Groot, K. (1999). Three-Dimensional Nano-HAp/Collagen Matrix Loading with Osteogenic Cells in Organ Culture , J. Biomed. Mat. Res., 44(4): 407–415 .
24.
24. Becker, J., Schuppan, D., Benzian, H., Bals, T., Cantaluppi, C. and Reichard, P. (1986). Immunohistochemical Distribution of Collagen Types IV, V, and Procollagen Type Iand III in Human Alveolar Bone and Dentine , J. Histochem. Cytochem., 34: 1417–1429 .
25.
25. Walsh, S., Dodds, R.A., James, I.E., Bradbeer, J.N. and Gowen, M. (1994). Monoclonal Antibodies with Selectivity Reactivity Against Osteoblasts and Osteocytes in Human Bone , J. Bone Miner. Res., 11: 1687–1696 .
26.
26. Belows, C.G., Aubin, J.E., Heersche, N.M. and Antosz, M.E. (1982). Mineralized Bone Nodules Formed In Vitro from Enzymatically Released Tat Calvaria Cell Population , J. Cell. Biol., 92: 452–461 .
27.
27. Collin, P., Nefussi, J.R., Wetterwald, A., Nicolas, V., Boy-Le fever, M-L. and Fleisch, H. et al. (1992). Expression of Collagen, Osteocalcin, and Bone Alkaline Phosphatase in a Mineralizing Rat Osteoblastic Cell Culture , Calcif. Tissue. Int., 50: 175–183 .
28.
28. Gerstenfld, L.C., Chipman, S.D., Kelly, C.M., Hodgnes, K.J., Lee, D.D. and Landis, W.J. (1998). Collagen Expression, Ultrastructure Assembly and Mineralization in Cultures of Chicken Embryo Osteoblasts , J. Cell. Biol., 106: 979–989 .
29.
29. Wiestner, M., Fische, S., Dessau, W. and Muller, P.K. (1981). Collagen Type Synthesis by Isolated Calvaria Cells , Exp. Cell. Res., 133: 115–125 .
30.
30. Ahmad, Mansur, McCarthy, MaryBeth, Gronowicz, Gloria (1999). An In Vitro Model for Mineralization of Human Osteoblast-like Cells on Implant Materials , Biomaterials, 20: 211–220 .
