ABSTRACT In this study, we use a pluripotent mesenchymal stem cell (MSC) model, C3H/10T1/2, to evaluate three calcium phos-phate (CaP) materials, namely the hydroxyapatite (HA), α-tricalcium phosphate (α-TCP) and β-tricalcium phosphate (β-TCP). 10T1/2 cell was chosen as it has advantages over its counterparts in terms of ease of maintenance, free of ethical concerns and also more reproducible results. ALP enzymatic assay, RT-qPCR, DAPI staining and SEM were employed to assess the osteoinductivity of these materials. A good reference material which also acts as a scientific control is necessary for comparisons of results from different experimental batches and hence other materials such as titanium, Nunclon plastic surface, BD Falcon plastic surface and gold coated porous HA were also tested. The results show that ceramics induce a more sustained osteo-differentiation state as compared with plastics. Inductivity was found to be acting in descending order of strength with HA > β-TCP > α-TCP, which is reversed in terms of their impact on proliferation rate (HA < TCP). This is also consistent with the results observed in SBF study in terms of calcium phosphate precipitate area coverage (HA > TCP) and in vivo osteoinductivity in terms of incidence and quality of bone described previously (HA > β-TCP > α-TCP). These confirm the suitability of using 10T1/2 cells in cell culture assay of osteoinductivity.
Cite this paper
nullJ. Lee, K. Wang, W. TSANG and K. Chow, "Comparative In Vitro Osteoinductivity Study of CaP Ceramics (HA, α-TCP, β-TCP) using 10T1/2 Cells with Different Controls and Possible Correlations with Other Systems," Journal of Biomaterials and Nanobiotechnology, Vol. 2 No. 2, 2011, pp. 162-171. doi: 10.4236/jbnb.2011.22021.
  K. A. Hing, “Bioceramic Bone Graft Substitutes: In-flence of Porosity and Chemistry,” International Journal of Applied Ceramic Technology, Vol. 2, No. 3, 2005, pp. 184-199. doi:10.1111/j.1744-7402.2005.02020.x
 P. Habibovic and K. de Groot, “Osteoinductive Biomate-rials-Properties and Relevance in Bone Repair.” Journal of Tissue Engineering and Regenerative Medicine, Vol. 1, No. 1, 2007, pp. 25-32. doi:10.1002/term.5
 R. Z. Legeros, S. Lin, R. Rohanizadeh, D. Mijares and J. P. Legeros, “Biphasic Calcium Phosphate Bioceramics: Preparation, Properties and Applications,” Journal of Materials Science: Materials in Medicine, Vol. 14, No. 3, 2003, pp. 201-209. doi:10.1023/A:1022872421333
 R. Z. LeGeros, “Calcium Phosphate-Based Osteoinduc-tive Materials,” Chemical Reviews, Vol. 108, No. 11, 2008, pp. 4742-4753. doi:10.1021/cr800427g
 F. Bronner, M. C. Farach-Carson, A. G. Mikos and SpringerLink, “Engineering of Functional Skeletal Tis-sues,” Vol. 3, 2007, pp. 178
 M. F. Pittenger, A. M. Mackay, S. C. Beck, R. K. Jaiswal, R. Douglas, J. D. Mosca, M. A. Moorman, D. W. Simon-etti, S. Craig and D. R. Marshak, “Multilineage Potential of Adult Human Mesenchymal Stem Cells,” Science, Vol. 284, No. 5411, 1999, pp. 143-147.
 W. A. Silva Jr., D. T. Covas, R. A. Panepucci, R. Proto-Siqueira, J. L. C. Siufi, D. L. Zanette, A. R. D. Santos and M. A. Zago, “The Profile of Gene Expression of Human Marrow Mesenchymal Stem Cells,” Stem Cells, Vol. 21, No. 6, 2003, pp. 661-669.
 N. Jaiswal, S. E. Haynesworth, A. I. Caplan and S. P. Bruder, “Osteogenic Differentiation of Purified, Cul-ture-Expanded Human Mesenchymal Stem Cells in Vi-tro,” Journal of Cellular Biochemistry, Vol. 64, No. 2, 1997, pp. 295-312.
 H. Yuan, Z. Yang, J. D. De Bruijn, K. De Groot and X. Zhang, “Material-Dependent Bone Induction by Calcium Phosphate Ceramics: A 2.5-Year Study in Dog,” Bioma-terials, Vol. 22, No. 19, 2001, pp. 2617-2623.
 J. T. Y. Lee, W. H. Tsang and K. L. Chow, “Simple Modifications to Standard TRIzol? Protocol Allow High- Yield RNA Extraction from Cells on Resorbable Materi-als,” Journal of Biomaterials and Nanobiotechnology, Vol. 2, No. 1, 2011, pp. 41-48.
 Y. Dohi, M. Akahane and H. Ohgushi, “Molecular Structure of Osteocalcin and the Role as An Essential Marker in the Osteogenic Differentiation Cascade,” Journal of Nara Medical Association, Vol. 59, No. 3-4, 2008, pp. 83-96.
 J. B. Lian, G. S. Stein, A. Javed, A. J. Van Wijnen, J. L. Stein, M. Montecino, M. Q. Hassan, T. Gaur, C. J. Lengner and D. W. Young, “Networks and Hubs for the Transcriptional Control of Osteoblastogenesis,” Reviews in Endocrine and Metabolic Disorders, Vol. 7, No. 1-2, 2006, pp. 1-16. doi:10.1007/s11154-006-9001-5
 J. B. Lian and G. S. Stein, “Runx2/Cbfa1: A Multifunc-tional Regulator of Bone Formation,” Current Pharma-ceutical Design, Vol. 9, No. 32, 2003, pp. 2677-2685. doi:10.2174/1381612033453659
 S. Maeno, Y. Niki, H. Matsumoto, H. Morioka, T. Yatabe, A. Funayama, Y. Toyama, T. Taguchi and J. Tanaka, “The Effect of Calcium Ion Concentration on Osteoblast Viabil-ity, Proliferation and Differentiation in Monolayer and 3D Culture,” Biomaterials, Vol. 26, No. 23, 2005, pp. 4847- 4855. doi:10.1016/j.biomaterials.2005.01.006
 M. M. Dvorak, A. Siddiqua, D. T. Ward, D. H. Carter, S. L. Dallas, E. F. Nemeth and D. Riccardi, “Physiological Changes in Extracellular Calcium Concentration Directly Control Osteoblast Function in the Absence of Cal-ciotropic Hormones,” Proceedings of the National Aca- demy of Sciences of the United States of America, Vol. 101, No. 14, 2004, pp. 5140-5145.
 M. M. Dvorak and D. Riccardi, “Ca2+ as an Extracellular Signal in Bone,” Cell calcium, Vol. 35, No. 3, 2004, pp. 249-255. doi:10.1016/j.ceca.2003.10.014
 M. Ahlstrom, M. Pekkinen, U. Riehle and C. Lamberg- Allardt, “Extracellular Calcium Regulates Parathyroid Hormone-Related Peptide Expression in Osteoblasts and Osteoblast Progenitor Cells,” Bone, Vol. 42, No. 3, 2008, pp. 483-490. doi:10.1016/j.bone.2007.10.025
 T. L. Arinzeh, T. Tran, J. Mcalary and G. Daculsi, “A Comparative Study of Biphasic Calcium Phosphate Ce-ramics for Human Mesenchymal Stem-Cell-Induced Bone Formation,” Biomaterials, Vol. 26, No. 17, 2005, pp. 3631-3638. doi:10.1016/j.biomaterials.2004.09.035
 C. G. Bellows, J. N. M. Heersche and J. E. Aubin, “Inor-ganic Phosphate Added Exogenously or Released from β-Glycerophosphate Initiates Mineralization of Osteoid nodules in Vitro,” Bone and Mineral, Vol. 17, No. 1, 1992, pp. 15-29. doi:10.1016/0169-6009(92)90707-K
 C. S. Adams, K. Mansfield, R. L. Perlot and I. M. Shapiro, “Matrix Regulation of Skeletal Cell Apoptosis. Role of Calcium and Phosphate Ions,” Journal of Bio-logical Chemistry, Vol. 276, No. 23, 2001, pp. 20316- 20322. doi:10.1074/jbc.M006492200
 Z. Meleti, I. M. Shapiro and C. S. Adams, “Inorganic phosphate Induces Apoptosis of Osteoblast-Like Cells in Culture,” Bone, Vol. 27, No. 3, 2000, pp. 359-366.
 J. Chen, J. Weng, Q. Zhang, J. Feng, Y. Cao and X. Zhang, “Effect of Post-Treatment on Dissolution and Biomineralization on Surface of HA Coatings in Simu-lated Body Fluid (SBF),” Materials Research Society Symposium - Proceedings, Vol. 599, 2000, pp. 55-60.
 R. Detsch, H. Mayr and G. Ziegler, “Formation of Os-teoclast-Like Cells on HA and TCP Ceramics,” Acta Biomaterialia, Vol. 4, No. 1, 2008, pp. 139-148.
 S. Langstaff, M. Sayer, T. J. N. Smith and S. M. Pugh, “Resorbable Bioceramics Based on Stabilized Calcium Phosphates. Part II: Evaluation of Biological Response,” Biomaterials, Vol. 22, No. 2, 2001, pp. 135-150.
 R. Tang, M. Hass, W. Wu, S. Gulde and G. H. Nancollas, “Constant Composition Dissolution of Mixed Phases II. Selective Dissolution of Calcium Phosphates,” Journal of colloid and Interface Science, Vol. 260, No. 2, 2003, pp. 379-384. doi:10.1016/S0021-9797(03)00048-1
 T. Suzuki, T. Yamamoto, M. Toriyama, K. Nishizawa, Y. Yokogawa, M. R. Mucalo, Y. Kawamoto, F. Nagata and T. Kameyama, “Surface Instability of Calcium Phosphate Ceramics in Tissue Culture Medium and the Effect on Adhesion and Growth of Anchorage-Dependent Animal Cells,” Journal of Biomedical Materials Research, Vol. 34, No. 4, 1997, pp. 507-517.
 X. Lu and Y. Leng, “Theoretical Analysis of Calcium Phosphate Precipitation in Simulated Body Fluid,” Bio-materials, Vol. 26, No. 10, 2005, pp. 1097-1108.
 S. V. Dorozhkin and M. Epple, “Biological and Medical Significance of Calcium Phosphates,” Angewandte Che-mie-International Edition, Vol. 41, No. 17, 2002, pp. 3130-3146. doi:10.1002/1521-3773(20020902)41:17<3130::AID-ANIE3130>3.0.CO;2-1
 M. Vallet-Regí and J. M. González-Calbet, “Calcium Phosphates as Substitution of Bone Tissues,” Progress in Solid State Chemistry, Vol. 32, No. 1-2, 2004, pp. 1-31. doi:10.1016/j.progsolidstchem.2004.07.001
 Ferńndez E., F. J. Gil, M. P. Ginebra, F. C. M. Driessens, J. A. Planell and S. M. Best, “Calcium Phosphate Bone Cements for Clinical Applications. Part I: Solution Che- mistry,” Journal of Materials Science: Materials in Medicine, Vol. 10, No. 3, 1999, pp. 169-176.
 R. Garimella, J. B. Sipe and H. C. Anderson, “A Simple and Non-Radioactive Technique to Study the Effect of Monophosphoesters on Matrix Vesicle-Mediated Calci-fication,” Biological Procedures Online, Vol. 6, No. 1, 2004, pp. 263-267. doi:10.1251/bpo97
 K. Hamada, M. Kon, T. Hanawa, K. Yokoyama, Y. Mi-yamoto and K. Asaoka, “Hydrothermal Modification of Titanium Surface in Calcium Solutions,” Biomaterials, Vol. 23, No. 10, 2002, pp. 2265-2272.
 E. A. dos Santos, M. Farina, G. A. Soares and K. Anselme, “Chemical and Topographical Influence of Hydroxyapatite and Beta-Tricalcium Phosphate Surfaces on Human Osteoblastic Cell Behavior.” Journal of bio-medical materials research. Part A, Vol. 89, No. 2, 2009, pp. 510-520. doi:10.1002/jbm.a.31991
 G. C. Reilly, S. Radin, A. T. Chen and P. Ducheyne, “Differential Alkaline Phosphatase Responses of Rat and Human Bone Marrow Derived Mesenchymal Stem Cells to 45S5 Bioactive Glass,” Biomaterials, Vol. 28, No. 28, 2007, pp. 4091-4097.
 E. A. Abou Neel, T. Mizoguchi, M. Ito, M. Bitar, V. Salih and J. C. Knowles, “In Vitro Bioactivity and Gene Expres-sion by Cells Cultured on Titanium Dioxide Doped Phos-phate-Based Glasses,” Biomaterials, Vol. 28, No. 19, 2007, pp. 2967-2977. doi:10.1016/j.biomaterials.2007.03.018
 M. A. Aronow, L. C. Gerstenfeld, T. A. Owen, M. S. Tassinari, G. S. Stein and J. B. Lian, “Factors that Pro-mote Progressive Development of the Osteoblast Pheno-type in Cultured Fetal Rats Calvaria Cells,” Journal of Cellular Physiology, Vol. 143, No. 2, 1990, pp. 213-221.
 A. J. Engler, S. Sen, H. L. Sweeney and D. E. Discher, “Matrix Elasticity Directs Stem Cell Lineage Specifica-tion,” Cell, Vol. 126, No. 4, 2006, pp. 677-689.
 C. Desbois, D. A. Hogue and G. Karsenty, “The Mouse Osteocalcin Gene Cluster Contains Three Genes with Two Separate Spatial and Temporal Patterns of Expres-sion,” Journal of Biological Chemistry, Vol. 269, No. 2, 1994, pp. 1183-1190.
 S. Rahman, A. Oberdorf, M. Montecino, S. M. Tanhauser, J. B. Lian, G. S. Stein, P. J. Laipis and J. L. Stein, “Mul-tiple Copies of the Bone-Specific Osteocalcin Gene in Mouse and Rat,” Endocrinology, Vol. 133, No. 6, 1993, pp. 3050-3053. doi:10.1210/en.133.6.3050.