Type III Collagen Promotes a Pro‐Fibrotic Microenvironment for In Vitro Tendon Healing Microphysiological Systems

Microphysiological systems of tendon healing, or Tendon-on-Chips, are promising platforms for studying scarring and fibrotic repair and can be relevant for the development of novel anti-fibrotic therapeutics. Effective fibrosis models must accurately recreate the myofibroblast microenvironment of signals that modulate their activation and drive peritendinous adhesions. These include not only the cellular interactions between resident tendon cells and proliferating immune and endothelial cells, but importantly, the dynamic extracellular matrix changes. In this study, we explore the extracellular matrix of healing tendon by investigating the role of type III collagen in inflammatory fibrosis. To address this, we evaluated the structural and biological effects of increased type III collagen ratio in an in vitro hydrogel model. Collagen hydrogels with 20% type III and 80% type I collagen had smaller fibrils and pores compared to those with 5% type III and 95% type I collagen. While these structural differences did not significantly alter viscoelastic or diffusive transport properties, higher type III collagen led to significant cellular effects that correlate with known in vivo features. Activated myofibroblasts were less contractile in high type III collagen hydrogels. Monocytes exhibited significantly hindered migration in a chemotaxis experiment. Furthermore, endothelial cells demonstrated increased angiogenic sprouting and microvascular network formation when suspended in high type III collagen hydrogels. These findings demonstrate that type III collagen precipitates a vascular fibrotic phenotype in injured tendons. Clinical Significance: Use of type III collagen to simulate fibrotic extracellular matrix will increase the accuracy of human-relevant tendon microphysiological systems for improved antifibrotic drug development.