Mechano-immunomodulation of macrophages influences the regenerative environment of fracture healing through the regulation of angiogenesis and osteogenesis

Successful completion of the initial inflammatory phase is critical for the establishment of a regenerative environment conducive to long-term fracture healing. Mechanical signals are among the most potent regulators of bone repair, yet whether local mechanics can modulate inflammation and associated immune response remains poorly understood. In this study, we develop a 3D in vitro model comprising of a purpose-built bioreactor that can replicate distinct loading conditions experienced during ambulation of fixated or unfixed large bone defects, and a haematoma mimetic fibrin hydrogel mirroring the local tissue composition, mechanical properties, and immune environment. Harnessing this system, we demonstrated that macrophages, key regulators of the early immune response, are mechanoresponsive and sensitive to the loading magnitude of local compressive forces. Specifically, moderate loading (5 % strain) as experienced within semi-rigid fixation, was capable of driving a hybrid phenotype with a higher regenerative secretome in M0 macrophages, while inhibiting inflammation in pro-inflammatory M1-like macrophages which supported capillary-size vascular formation. Conversely, higher loading (35 % strain), representative of mechanically unstable defects, was shown to elicit a poor regenerative immune response detrimental to vascular growth and long-term mineralisation. Collectively, our findings highlight mechanical cues as potent stimuli to modulate early immune responses, thus informing the development of novel materials and mechanotherapies to enhance bone repair. STATEMENT OF SIGNIFICANCE: Mechano-immunology is an emerging field that aims at interrogating how mechanical cues shape immune cell phenotype and function. This study presents for the first time, the design and validation of a purpose-built 3D in vitro platform of a dynamically loaded bone fracture haematoma. Utilising this model, we demonstrate that macrophages are mechanoresponsive and sensitive to compressive loading magnitude, with moderate loading (5 % strain) producing a hybrid regenerative macrophage phenotype and secretome, while excessive loading (35 % strain) produced a secretome detrimental to angiogenesis and osteogenesis. Moreover, moderate strain can also dampen inflammation in a model of an inflamed compromised fracture. This knowledge may inform the development of novel mechano-immunomodulatory materials and therapeutics that target the early inflammation phase for bone repair.