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INTRODUCTION: Healthy tendon is characterized by its highly organized extracellular matrix composed of aligned collagen fibers. Nonetheless, in tendinopathic situations this characteristic architecture is lost and causes morphological adaptations of the resident tendon fibroblasts (TFs) together with dramatic changes in load distribution . The impact of these biomechanical alterations have started to emerge and our group has recently reported that tenocytes adhering to surfaces that mimic the disorganized tendinopathic tissue exhibit a more pronounced immunoresponse compared to those on aligned topographies . In the present study we expand our knowledge on this topic and determine the impact of topographical cues and mechanical load on macrophage polarization and on the immune response of tendon fibroblasts to the macrophage-derived factors. METHODS: Naïve (M0) macrophages (THP-1 cell line) and primary human TFs were cultured on aligned or random oriented nanofiber substrates (mimicking characteristic matrix of ‘healthy’ and ‘diseased’ tissue, respectively) and clamped within a custom-made bioreactor for conditioning by either static load (1% strain) or cyclic overloading (7% strain) for 24h (Fig. 1-I). In addition, after 24h of mechanical stimulation macrophage polarization was assessed by analyzing the surface markers CCR7 (M1-like) and MRC1 (M2-like) on the gene expression level and was further validated by flow cytometry (Fig.1-II). The macrophage conditioned medium (CM) was further used to stimulate TFs in static culture on aligned substrates for 30 min and the translocation of NFκB-p65 was quantified from fluorescent stained cells (Fig.1-III-IV). RESULTS: Disorganized topographies alone promoted the polarization of M0 macrophages towards a pro-inflammatory phenotype (M1-like) (Figure 1-II). Expression of CCR7, a marker of M1-like macrophages, was significantly enhanced at both the RNA and protein levels upon cyclic mechanical overloading (Fig. 1-II). Furthermore, factors secreted by mechanically loaded macrophages cause a significantly elevated translocation of the NFκB-p65 subunit in TFs than those exposed to the supernatant of statically loaded macrophages (Fig. 1-IV). On the other side, expression of tenogenic markers in TFs exposed to the loading conditions was not significantly affected (not shown). DISCUSSION: In this work we present a novel approach to study the impact of mechanical and topographical cues on tendinopathic disease. Our results reveal that those features characteristic to tendinopathy favor the polarization of M0 macrophages towards the M1-like phenotype and as result promote the secretion of larger amounts of inflammatory factors. Consequently, nuclear translocation of the p65 subunit of the NFκB complex in TFs was dramatically increased when stimulated with medium conditioned by mechanically overloaded macrophages adhering to disorganized nanofiber matrices. Because these surfaces have also been shown to predispose the immunoresponse of TFs2, it is likely that the combined effect of these features on immune and tendon cells strongly influence the healing phase of this disease. In fact, the response of macrophages to substrate topography and mechanical stimulus appears to be more dramatic than that of the stromal fibroblasts, suggesting a central role for macrophages as mechano-regulatory cells in tendon repair or disease. SIGNIFICANCE: A better understanding of the role of immune cells in tendinopathic situations and of how the structural and mechanical properties of the diseased tissue impact their function is essential for the development of improved therapeutic approaches. Using a minimal in vitro model of tendon matrix organization we have uncovered the impact of two major features of tendinopathic tissue, chaotic architecture and mechanical overloading, and demonstrate the potentially central role that these factors may play in the crosstalk of immune cells and tendon fibroblasts. REFERENCES: 1) Fearon, A. et al., J. Sci. Med. Sport 17, 346–350 (2014). 2) Schoenenberger, A.D. et al., Acta Biomater. 71, 306–317 (2018).
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