The role of matrix viscoelasticity as a biophysical cue in macrophage phenotype regulation in the early fracture hematoma

Bone fracture healing begins with the formation of a hematoma, a dynamic injury niche characterized by complex, intertwined inflammatory processes that set the trajectory for subsequent tissue repair. Prior studies have shown that the phenotypic composition of immune cells within the fracture hematoma critically influences the course of bone repair. Effective healing requires a coordinated transition of the immune niche from an early pro-inflammatory state to a subsequent anti-inflammatory, reparative state. During this transition, macrophage phenotype regulation is an important regulatory process that helps shape the pro- and anti-inflammatory environment and thereby supports the reconstruction of the disrupted extracellular matrix (ECM). The ECM of the fracture hematoma is generally considered viscoelastic and undergoes compositional changes over time as matrix reconstruction progresses. However, how changes in hematoma viscoelasticity during the early phase of bone healing influence macrophage phenotypes has not been addressed. In-depth mechanical characterization of ex vivo human fracture tissue samples using viscoelastic modeling revealed that the stress-relaxation behavior of fracture hematoma can be decomposed into an early component, τ1, and a late component, τ2. Within this modeling framework, the late relaxation time constant τ2 correlated positively with days post-injury, likely reflecting changes in matrix composition as matrix repair progressed. Based on this observation, this thesis tests the hypothesis that τ2 represents a mechanical property of the ECM that regulates macrophage phenotype. To address this question, ionically crosslinked alginate hydrogels with defined molecular weights were used as artificial ECM, enabling controlled tuning of viscoelastic stress-relaxation behavior and cell–matrix adhesion properties in 3D culture. ECM stress-relaxation properties were tuned by varying alginate molecular weight and crosslinking, while maintaining comparable ECM elasticity, to generate hydrogels that mimic the mechanical properties of the fracture hematoma during the early healing phase. To study the impact of ECM with low- or high-τ2 on macrophage phenotype, human monocyte-derived macrophages were cultured in hydrogels for up to 48 hours. Macrophage phenotypes were assessed by surface marker profiling, cytokine secretion analysis, and single-cell transcriptomic profiling. The multimodal characterization revealed that ECM with low τ2, resembling early fracture hematoma, induced pro-inflammatory macrophage phenotypes and activated glycolytic and hypoxia-responsive transcriptional programs. In contrast, ECM with high τ2 values, resembling a maturer fracture hematoma, induced less inflammatory phenotypes and enhanced the expression of oxidative metabolic genes. Notably, these effects were reversible, dependent on continued mechanical stimulation, and occurred independently of integrin-mediated adhesion or physical confinement, underscoring the specific contribution of stress-relaxation dynamics. To assess whether the engineered viscoelastic in vitro model mimics biologically relevant inflammatory states, transcriptional programs induced by low- and high-τ2 ECM were compared with macrophage gene-expression signatures observed in vivo. The low τ2-associated gene programs aligned with pro-inflammatory macrophage signatures at early post-injury time points and diminished as healing progressed, mirroring the transition from the pro-inflammatory to the anti-inflammatory phase during fracture repair. Together, these findings reveal that the viscoelastic properties of the ECM in fracture hematoma provide instructive mechanical cues that guide macrophage phenotype during early fracture healing. This work advances mechanobiological understanding of immune regulation in fracture repair and identifies stress-relaxation as a critical material parameter with potential relevance for the design of immunomodulatory biomaterials in regenerative medicine.