Chapter 21 Evaluation of Precise Optimal Cyclic Strain for Tenogenic Differentiation of MSCs Yasuyuki Morita, Toshihiro Sato, Sachi Watanabe, and Yang Ju Abstract Although there are a number of papers relating to tenogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) using uniaxial cyclic stretching stimulation with homogeneous strain field, it has been pretty hard to figure out the optimal normal strain in the stretch direction for the differentiation. In the present study, our group has developed a non-uniform strain field system to elucidate the optimal normal strain in one-time experiment in principle. A relationship between the normal strain of membrane and expression levels of the differentiation marker proteins, type I collagen (Col I) and tenascin-C (Tnc), derived from stretched cells was obtained. Finally, the rigorous optimal normal strains were clarified 7.9 and 8.5 % for Col I and Tnc, respectively. Additionally, we found that a dependence of protein expression levels with the normal strain of membrane was different in each protein, which would be crucial in the field of embryology and regenerative medicine. Keywords Digital image correlation (DIC) • Optimal strain • Mechanical stimulation • Tenogenic differentiation • Mesenchymal stem cell (MSC) 21.1 Introduction Tendon tissue engineering in vitro could be the best medical treatment since tendon tissues have poor self-curing ability. The main problems are, however, the low number of cells obtained from explanted tendon tissue since tendons are relatively acellular and low cell density of tenocytes [1]. Hence, bone marrow mesenchymal stem cells (BMSCs) are commonly used in this field of study due to their high proliferative capacity and pluripotency [2, 3]. Promoting differentiation of BMSCs into tenocytes effectively in vitro is one of the vital issues. Mechanical stimulation [4] would have advantages from cost and simplicity perspectives although there are some techniques, e.g. biochemical stimulation [5], micro-/nano-structure [6], co-culture [7], etc. for BMSC-to-tenocyte differentiation. Thus, a number of research groups have investigated the differentiation by using cyclic uniaxial stretch stimulation [8]. Our group has been also involved in this research field [9, 10]. In general, this kind of experiment has been carried out by means of PDMS elastic chamber like shown in Fig. 21.1. Cells are seeded onto the bottom of the chamber which is coated with cell-adhesion factor, and then mechanical loading is imposed to the cells by stretching the chamber uniaxially. Song et al. indicated that 1.0 Hz of cyclic frequency works effectively for proliferation of human BMSCs (hBMSCs) in vitro [11]. Although it has been found that the optimal strain rate of cyclic uniaxial stretch for enhancing tenogenic differentiation of hBMSCs was roughly 8–10 % in the stretch direction [9, 10, 12], the rigorous strain rate of the uniaxial stretching which is crucial for tendon tissue engineering has not been elucidated yet. The main reason must be that the numerous experimental conditions of the strain rate need to be performed though those experiments are impractical from perspectives of cost and efficiency. Recently, our group has developed a new 2D culture system using a non-uniform strain field [13]. Then by using the system, the axial strain threshold of cells was determined with high reliability, and the preferential axial strain of cells was suggested firstly as a new characteristic of cells [13]. Exploring cellular functions and activities using non-uniform deformation fields has been getting more important and paid attention nowadays [13–15]. In the present report, we determined the optimal strain rate of cyclic stretching for hBMSC-to-tenocyte differentiation described above by means of the 2D culture system of a non-uniform strain field. Digital image correlation (DIC) method [16] was applied to quantify the non-uniform strain distribution of the membrane. Expression levels of marker proteins for tenogenic differentiation, type Y. Morita (*) • T. Sato • S. Watanabe • Y. Ju Department of Mechanical Science and Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan e-mail: morita@mech.nagoya-u.ac.jp #The Society for Experimental Mechanics, Inc. 2017 C.S. Korach et al. (eds.), Mechanics of Biological Systems and Materials, Volume 6, Conference Proceedings of the Society for Experimental Mechanics Series, DOI 10.1007/978-3-319-41351-8_21 149
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