SCD  Vol.2 No.4 , October 2012
Increased osteogenesis with hydroxyapatite constructs combined with serially-passaged bone marrow-derived mesenchymal stem cells
ABSTRACT
We have previously reported on both the osteogenic potential of hydroxyapatite (HA) combined with bone marrow-derived mesenchymal stem cells (BMSCs) and a method involving osteogenic matrix cell sheet transplantation of BMSCs. In the present study, we assessed the osteogenic potential of serially-passaged BMSCs, both in vitro and in vivo. We also assessed whether an additional cell-loading technique can regain the osteogenic potential of the constructs combined with serially-passaged BMSCs. The present study revealed that passage (P) 1 cells cultured in osteogenic-induced medium showed strong positive staining for alkaline phosphatase (ALP) and Alizarin Red S, whereas P3 cells showed faint staining for ALP, with no Alizarin Red S staining. Staining of P1, P2 and P3 cells were progressively weaker, indicating that the osteogenic potential of the serially-passaged rat BMSCs is lost after P3 in vitro. The in vivo study showed that little bone formation was observed in the HA constructs seeded with P3 cells, 4 weeks after subcutaneous implantation. However, P3 cell/HA constructs which had increased cell-loading showed abundant bone formation within the pores of the HA construct. ALP and osteocalcin mRNA expression in these constructs was significantly higher than that of constructs with regular cell-seeding. The present study indicates that the osteogenic potential of the constructs with serially-passaged BMSCs is increased by additional cell-loading. This method can be applied to cases requiring hard tissue reconstruction, where BMSCs require serial expansion of cells.

Cite this paper
Akahane, M. , Ueha, T. , Shimizu, T. , Inagaki, Y. , Kido, A. , Imamura, T. , Kawate, K. and Tanaka, Y. (2012) Increased osteogenesis with hydroxyapatite constructs combined with serially-passaged bone marrow-derived mesenchymal stem cells. Stem Cell Discovery, 2, 133-140. doi: 10.4236/scd.2012.24018.
References
[1]   Owen, M. (1988) Marrow stromal stem cells. Journal of Cell Science, 10, 63-76.

[2]   Ohgushi, H. and Caplan, A.I. (1999) Stem cell technology and bioceramics: From cell to gene engineering. Journal of Biomedical Materials Research, 48,913-927. doi:10.1002/(SICI)1097-4636(1999)48:6<913::AID-JBM22>3.0.CO;2-0

[3]   Ohgushi, H., Yoshikawa, T., Nakajima, H., Tamai, S., Dohi, Y. and Okunaga, K. (1999) Al2O3 doped apatitewollastonite containing glass ceramic provokes osteogenic differentiation of marrow stromal stem cells. Journal of Biomedical Materials Research, 44, 381-388. doi:10.1002/(SICI)1097-4636(19990315)44:4<381::AID-JBM3>3.0.CO;2-E

[4]   Sonal, R., Jackson, J.D., Brusnahan, S.K., O’Kane, B. J. and Sharp, J.G. (2012) Characterization of a mesenchymal stem cell line that differentiates to bone and provides niches supporting mouse and human hematopoietic stem cells. Stem Cell Discovery, 2, 5-14. doi:10.4236/scd.2012.21002

[5]   Brazelton, T.R., Rossi, F.M., Keshet, G.I. and Blau, H.M. (2000) From marrow to brain: Expression of neuronal phenotypes in adult mice. Science, 290, 1775-1779. doi:10.1126/science.290.5497.1775

[6]   Jiang, Y., Jahagirdar, B.N., Reinhardt, R.L., Schwartz, R.E., Keene, C.D., Ortiz-Gonzalez, X.R., Reyes, M., Lenvik, T., Lund, T., Blackstad, M., Du, J., Aldrich, S., Lisberg, A., Low, W.C., Largaespada, D.A. and Verfaillie, C.M. (2002) Pluripotency of mesenchymal stem cells derived from adult marrow. Nature, 418, 41-49. doi:10.1038/nature00870

[7]   Krause, D.S. (2002) Plasticity of marrow-derived stem cells. Gene Therapy, 9, 754-758. doi:10.1038/sj.gt.3301760

[8]   Ter Brugge, P.J. and Jansen, J.A. (2002) In vitro osteogenic differentiation of rat bone marrow cells subcultured with and without dexamethasone. Tissue Engineering, 8, 321-331. doi:10.1089/107632702753725076

[9]   Matsushima, A., Kotobuki, N., Tadokoro, M., Kawate, K., Yajima, H., Takakura, Y. and Ohgushi, H. (2009) In vivo osteogenic capability of human mesenchymal cells cultured on hydroxyapatite and on beta-tricalcium phosphate. Artificial Organs, 33,474-481. doi:10.1111/j.1525-1594.2009.00749.x

[10]   Akahane, M., Shigematsu, H., Tadokoro, M., Ueha, T., Matsumoto, T., Tohma, Y., Kido, A., Imamura, T. and Tanaka, Y. (2010) Scaffold-free cell sheet injection results in bone formation. Journal of Tissue Engineering and Regenerative Medicine, 4, 404-411. doi:10.1002/term.259

[11]   Nakamura, A., Akahane, M., Shigematsu, H., Tadokoro, M., Morita, Y., Ohgushi, H., Dohi, Y., Imamura, T. and Tanaka, Y. (2010) Cell sheet transplantation of cultured mesenchymal stem cells enhances bone formation in a rat nonunion model. Bone, 46, 418-424. doi:10.1016/j.bone.2009.08.048

[12]   Nakamura, A., Dohi, Y., Akahane, M., Ohgushi, H., Nakajima, H., Funaoka, H. and Takakura, Y. (2009) Osteocalcin secretion as an early marker of in vitro osteogenic differentiation of rat mesenchymal stem cells. Tissue Engineering Part C: Methods, 15, 169-180. doi:10.1089/ten.tec.2007.0334

[13]   Akahane, M., Ohgushi, H., Yoshikawa, T., Sempuku, T., Tamai, S., Tabata, S. and Dohi, Y. (1999) Osteogenic phenotype expression of allogeneic rat marrow cells in porous hydroxyapatite ceramics. Journal of Bone and Mineral Research, 14, 561-568. doi:10.1359/jbmr.1999.14.4.561

[14]   Bianco, P. and Robey, P.G. (2001) Stem cells in tissue engineering. Nature, 414, 118-121. doi:10.1038/35102181

[15]   Dong, J., Kojima, H., Uemura, T., Kikuchi, M., Tateishi, T. and Tanaka, J. (2001) In vivo evaluation of a novel porous hydroxyapatite to sustain osteogenesis of transplanted bone marrow-derived osteoblastic cells. Journal of Biomedical Materials Research, 57,208-216. doi:10.1002/1097-4636(200111)57:2<208::AID-JBM1160>3.0.CO;2-N

[16]   Petite, H., Viateau, V., Bensaid, W., Meunier, A., de Pollak, C., Bourguignon, M., Oudina, K., Sedel, L. and Guillemin, G. (2000) Tissue-engineered bone regeneration. Nature Biotechnology, 18, 959-963. doi:10.1038/79449

[17]   Shigematsu, H., Akahane, M., Dohi, Y., Nakamura, A., Ohgushi, H., Imamura, T. and Tanaka, Y. (2010) Osteogenic potential and histological characteristics of mesenchymal stem cell sheet/hydroxyapatite constructs. The Open Tissue Engineering and Regenerative Medicine Journal, 2, 63-70. doi:10.2174/1875043500902010063

[18]   Akahane, M., Nakamura, A., Ohgushi, H., Shigematsu, H., Dohi, Y. and Takakura, Y. (2008) Osteogenic matrix sheet-cell transplantation using osteoblastic cell sheet resulted in bone formation without scaffold at an ectopic site. Journal of Tissue Engineering and Regenerative Medicine, 2, 196-201. doi:10.1002/term.81

[19]   Wakitani, S., Imoto, K., Yamamoto, T., Saito, M., Murata, N. and Yoneda, M. (2002) Human autologous culture expanded bone marrow mesenchymal cell transplantation for repair of cartilage defects in osteoarthritic knees. Osteoarthritis and Cartilage, 10, 199-206. doi:10.1053/joca.2001.0504

[20]   Ohgushi, H., Kotobuki, N., Funaoka, H., Machida, H., Hirose, M., Tanaka, Y. and Takakura, Y. (2005) Tissue engineered ceramic artificial joint—Ex vivo osteogenic differentiation of patient mesenchymal cells on total ankle joints for treatment of osteoarthritis. Biomaterials, 26, 4654-4661. doi:10.1016/j.biomaterials.2004.11.055

[21]   Kawate, K., Yajima, H., Ohgushi, H., Kotobuki, N., Sugimoto, K., Ohmura, T., Kobata, Y., Shigematsu, K., Kawamura, K., Tamai, K. and Takakura, Y. (2006) Tissue-engineered approach for the treatment of steroid-induced osteonecrosis of the femoral head: transplantation of autologous mesenchymal stem cells cultured with beta-tricalcium phosphate ceramics and free vascularized fibula. Artifical Organs, 30, 960-962. doi:10.1111/j.1525-1594.2006.00333.x

[22]   M. Akahane, T.U., Shimizu, T., Shigematsu, H., Kido A., Omokawa, S., Kawate, K., Imamura, T. and Y. Tanaka. (2010) Cell Sheet Injection as a technique of osteogenic supply. International Journal of Stem Cells, 3, 138-143.

[23]   McCulloch, C.A., Strugurescu, M., Hughes, F., Melcher, A.H. and Aubin, J.E. (1991) Osteogenic progenitor cells in rat bone marrow stromal populations exhibit self-renewal in culture. Blood, 77, 1906-1911.

[24]   Aubin, J.E. (1998) Advances in the osteoblast lineage. Biochemistry and Cell Biology, 76, 899-910. doi:10.1139/o99-005

[25]   Kadiyala, S., Young, R.G., Thiede, M.A. and Bruder, S.P. (1997) Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro. Cell Transplantation, 6, 125-134. doi:10.1016/S0963-6897(96)00279-5

[26]   Anil Kumar, P.R., Varma, H.K. and Kumary, T.V. (2005) Rapid and complete cellularization of hydroxyapatite for bone tissue engineering. Acta Biomaterialia, 1, 545-552. doi:10.1016/j.actbio.2005.05.002

[27]   Ogose, A., Hotta, T., Hatano, H., Kawashima, H., Tokunaga, K., Endo, N. and Umezu, H. (2002) Histological examination of beta-tricalcium phosphate graft in human femur. Journal of Biomedical Materials Research, 63, 601-604. doi:10.1002/jbm.10380

[28]   Yamamoto, T., Onga, T., Marui, T. and Mizuno, K. (2000) Use of hydroxyapatite to fill cavities after excision of benign bone tumours. Clinical results. Journal of Bone & Joint Surgery, British Volume, 82, 1117-1120. doi:10.1302/0301-620X.82B8.11194

[29]   Schindler, O.S., Cannon, S.R., Briggs, T.W. and Blunn, G.W. (2008) Composite ceramic bone graft substitute in the treatment of locally aggressive benign bone tumours. Journal of Orthopaedic Surgery (Hong Kong), 16, 66-74.

 
 
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