Osteoclastogenic Potential of Tissue-Engineered Periosteal Sheet: Effects of Culture Media on the Ability to Recruit Osteoclast Precursors

Abstract

Cell culture media influence the characteristics of human osteogenic periosteal sheets. We have previously found that a stem cell medium facilitates growth and collagen matrix formation in vitro and osteogenesis in vivo. However, it has not yet been demonstrated which culture medium is superior for osteoclastogenesis, a prerequisite for reconstruction of normal bone metabolic basis. To address this question, we compared chemotaxis and osteoclastogenesis in tissue-engineered periosteal sheets (TPSs) prepared with two types of culture media. Periosteal tissues obtained from adult volunteers were expanded with the conventional Medium 199 or with the stem cell medium, MesenPRO. Hematopoietic enhanced-green-fluorescent-protein (EGFP)-nude mice were prepared by γ-irradiation of Balb/c nu/nu mice and subsequent transplantation of bone marrow cells from CAG-EGFP C57BL/6 mice. TPSs were implanted subcutaneously into the chimeric mice and retrieved after intervals for immunohistopathological examination. EGFP⁺ cells were similarly recruited to the implantation site in both the TPSs prepared, whereas the distribution of CD11b⁺ cells was significantly lower in the TPS prepared with the stem cell medium. Instead, osteoclastogenesis was higher in the TPS prepared with the stem cell medium than in the one prepared with the conventional medium. These findings suggest that the stem cell medium is preferable for the preparation of more functional TPSs.

Keywords: CD11b; bone marrow transplantation; green fluorescent protein; osteoclastogenesis; periosteal sheets; tartrate-resistant acid phosphatase.

Figure 1

An illustration depicting possible mechanisms of tissue-engineered periosteal sheet (TPS)-induced osteoclastogenesis.

Figure 2

Effects of culture media on the growth and osteoblast differentiation of tissue-engineered periosteal sheets (TPSs) in vitro. (a) Growth rates, (b) alkaline phosphatase activity staining, and (c) Alizarin Red S staining for biomineralization. The broken lines represent the edges of TPSs. * p < 0.05 vs. Medium 199.

Figure 3

Effects of culture media on mRNA expression levels of chemotactic factors, (a) stromal cell derived factor-1 (SDF-1), (b) chemokine C-C motif ligand (CCL1), (c) CCL2, (d) CCL5, (e) CCL3, and (f) CCL4, in tissue-engineered periosteal sheets (TPSs) in vitro. N.S.: not significant.

Figure 4

Effects of culture media on new bonelike tissue formation and tartrate-resistant acid phosphatase+ (TRAP+) cell induction in the implanted TPSs in vivo. (a,b) Hematoxylin and eosin (HE) staining and (c,d) TRAP staining. Panels (b,d) represent higher magnification of the corresponding regions shown in Panels (a,c). Yellow arrows represent bonelike tissues. TRAP+ cells were stained red. The black arrow indicates representative TRAP+ multinucleated cells (osteoclasts).

Figure 5

Effects of culture media on the infiltration of enhanced green fluorescent protein⁺ (EGFP⁺) and CD11b⁺ cells. (a) EGFP immunostaining (inset: higher magnification of the selected region), (b) CD11b immunostaining. Individual positive cells were stained dark brown. (c) Quantification of the immunohistochemical data. N.S.: not significant.