Sandwich culture of mesenchymal stem cells between bio-functional hydrogels as a novel 3-D culture method for regulating their osteoblastic differentiation

摘要

Introduction: The objective of this study is to develop a novel 3-dimensional (3-D) culture technique that can maintain mesenchymal stem cells (MSC) in sandwich culture between bio-functional hydrogels, thereby regulating osteoblastic differentiation of MSC. As the bio-functional hydrogels, poly(acrylamide) (PAAm) hydrogels with the immobilization of a cell signaling protein were fabricated. Ephrin B2 of an Eph signal ligand was utilized as the cell signaling protein for regulating the osteoblastic differentiation of MSC. Since a direct binding between ephrinB2 and EphB4 via the cell-cell contact is required to activate the ephrinB2-EphB4 signaling, the immobilization of ephrinB2 in an orientation-regulated manner was designed to achieve the efficient ligand-receptor binding. Materials and Methods: Acrylamide and N,N'-methylenebisacrylamide (BIS) were co-polymerized to prepare polyacrylamide hydrogels with varied elasticity. The elasticity was assessed in terms of the storage modulus of hydrogels measured by a rheometer (Rheostress Ⅰ, Thermo Haake, Inc.). To allow MSC to attach on the surface of the hydrogel, rat tail collagen type Ⅰ was immobilized by use of Sulfosucctnimidyl ester. A recombinant chimeric protein of ephrinB2 and Fc domain (ephrinB2-Fc) was immobilized via protein A in an orientation-regulated manner on the surface of hydrogels. Briefly, protein A was immobilized on N-hydroxysuccinimide ester-conjugated hydrogels. The protein A-immobilized hydrogel was exposed to the solution of ephrinB2-Fc to fabricate ephrinB2-immobilized substrates. As a control, ephrinB2-Fc was chemically conjugated on the collagen type l-immobilized hydrogel without the protein A immobilization. Human MSC were sandwiched between the resulting bio-functional hydrogels and maintained in culture for 5 days. RUNX2 expression of MSC was measured by quantitative polymerase chain reaction to evaluate their osteoblastic differentiation. Results and Discussion: In sandwich culture between the ephrinB2-immobilized hydrogels, MSC showed enhanced RUNX-2 expression, which is similar to those on the corresponding hydrogel in the two-dimensional (2-D) culture condition (Figure 1). The RUNX-2 expression could be modulated by not only the bottom hydrogels in the 2-D culture but also the top hydrogels in the sandwich culture. Figure 1. Runx2 expression of MSC cultured for 5 days in different culture conditions. *p＜0.05; significance against culture conditions without ephrinB2. Conclusions: The present study suggests that cell signaling pathways for osteoblastic differentiation in MSC could be activated by immobilized ephrinB2 even in the sandwiched condition.