Healthy tendons are mainly composed of aligned collagen hierarchically organized from collagen fibrils to fiber bundles with a scarce cellular population mainly composed of tenocytes and tendon stem/progenitor cells. However, injured tendon acquires a fibrotic state characterized by a loss of ECM alignment and increased cellularization. The lack of reliable 3D models that recreate the organization and microenvironment of healthy and diseased tendons is one of the main obstacles faced by the scientific community. To recreate the architecture of healthy and diseased tendons, electrospun nanofiber scaffolds with anisotropic and isotropic nanotopography were developed. These scaffolds were coated with a shell consisting of cell-laden hydrogels encapsulating human adipose-derived stem cells (hASCs) to include the living component. To show the versatility of the system, extracellular vesicles (EVs) were encapsulated in the hydrogel as biological cues. The living fibers were characterized by microscopy and morphological analysis. The morphology and phenotype of cells was evaluated using microscopy, gene expression analysis and immunostainings for tendon markers.Introduction
Method
Tendinopathies represent a significant health burden, causing inflammation, pain, and reducing quality of life. The pivotal role of macrophages (Mφ) characterized by their ability to differentiate into proinflammatory (M1) or anti-inflammatory (M2) phenotypes depending on the microenvironment, has gained significant interest in tissue inflammation research. Additionally, existing literature states that the interplay between tenocytes and immune cells during inflammation involves unidentified soluble factors (SF). This study aimed to investigate the effect of extracellular vesicles (EVs) and SF derived from polarized Mφ on tendon cells to provide deeper insights of their potential therapeutic applications in the context of inflammation. Human monocytes were isolated from blood donor buffy coats and differentiated into M1, M2, and hybrid M1/M2 phenotypes. Subsequently, EVs were isolated from the conditioned media from polarized Mφ and comprehensively characterized. In parallel, the elution media containing SF was collected. Furthermore, the EVs and SF were released independently onto tenocytes from human donors, previously induced with IL-1β to simulate an inflammatory environment. Finally, mRNA levels of tendon-related markers were evaluated by qPCR after the exposure to these EVs and SF.Introduction
Method
