Macrophage-Driven Regulation of Collagen Formation in a 3D Human Tendon Mod

Abstract

Tendon injuries affect millions of people annually and tendons rarely regain full mechanical function after healing. Macrophages are key regulators of tendon healing, shifting between pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes across the phases of healing, yet their specific effects on tenocyte behavior and extracellular matrix formation remain unclear. This project aimed to investigate how M1 and M2 macrophage phenotypes affect the biomechanical properties, tenocyte phenotype and activity, and extracellular matrix formation in 3D human tendon constructs. Primary human tenocytes isolated from the semitendinosus tendon were embedded in a fibrin-based hydrogel, where cell-mediated contraction led to formation of a 3D in vitro tendon construct. Macrophage–tenocyte crosstalk was established using a Transwell-based paracrine co-culture system. The effects of macrophage–tenocyte crosstalk on tendon constructs were evaluated using uniaxial tensile testing, reverse transcription quantitative PCR, and hematoxylin and eosin staining. M1-stimulated constructs exhibited significantly increased expression of MMP-1, MMP-13, TIMP-1, and IL-6, together with decreased scleraxis expression, indicating enhanced matrix remodeling, increased inflammatory signaling, and an altered tenocyte phenotype. M2-stimulated constructs exhibited no significant changes in the evaluated mechanical properties and displayed tissue organization comparable to controls. However, significant increases in TIMP-1 and IL-6 expression were observed, indicating altered matrix remodeling and inflammatory signaling despite the absence of detectable mechanical effects. Histological assessment demonstrated no significant differences in histological scores between both groups and the control. These findings suggest that pro-inflammatory M1 macrophage signaling negatively affects tendon construct mechanical properties and extracellular matrix integrity, while M2 macrophage signaling exerts a more moderate influence. These results highlights the importance of macrophage phenotype balance during tendon healing and may contribute to future development of improved regenerative strategies for tendon repair.