Abstract
Introduction: How to control inflammation, especially induction of macrophage polarization that are regulated by Extra-cellular matrix (ECM) is a prerequisite for tissue regeneration in damaged organs. In this study, we administered clustered myoblast cells with ECM framework into hind limb ischemia (HLI) model, and evaluated the change in macrophages and effect on tissue regeneration. Methods: Myoblast cells isolated from C57BL/6 mice were expanded to form cell patch with ECM framework (10 6 cells/sheet) and then fragmented and administered as cluster cells (CC group, n=6). As control groups, non-clustered single myoblast cells (SC, n=6) or saline (SA, n=6) was administered. Blood perfusion was evaluated with Laser Doppler Perfusion imaging (LDPI). Leg muscles were obtained and analyzed regarding angiogenesis, skeletal muscle regeneration, and inflammation including macrophage status. Results: LDPI showed significant improvement in CC compared with SC (P=0.001) and SA (P<0.0001) at day 28. Also, markedly augmented angiogenesis and muscle regeneration were detected in CC. Regarding inflammation, the number of CD68+ macrophages is significantly larger in CC throughout within 7 days after administration, but the character of the macrophages was quite different during time courses. Although in SC and SA macrophage polarization was rarely detected within 7 days, marked polarization was detected at day 5 only in CC. At day 5, CD11c+/CD206- (inflammatory, M1) and CD11c-/CD206+ (anti-inflammatory, M2) macrophages are discerned conspicuously (M1: 148.7±104.4/mm 2 , M2: 185.2±46.3/mm 2 , non-polarized CD11c+/CD206+: 140.9±99.4/mm 2 ), while at day 3 non-polarized macrophages were almost exclusive (M1: 2.0±2.3/mm 2 , M2: 3.1±4.0/mm 2 , CD11c+/CD206+: 410.2±90.6/mm 2 ). Consistently significant increase in IL-10 and TGF-β, that are cytokines secreted by M2 macrophages, was detected at day 5 and 7 in CC. Conclusions: Myoblast cells administration with ECM framework, which was formed during cell patch formation, augmented macrophage polarization to show anti-inflammatory effect, creating appropriate microenvironment for angiogenesis and muscle regeneration in HLI.
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