Endothelial Cell Migration on Fibers Mimicking The Early Fracture Environment

Abstract

Bone is a tissue with many important functions, such as structural support, organ protection and mineral homeostasis. Additionally, after trauma, it can naturally regenerate into fresh, fully functional bone tissue. However, several factors can significantly impede the healing process and cause the formation of non-union tissue. Costs for hospitalization and treatments that prevent such non-unions can become a significant personal and socio-economic burden. To develop more efficient treatments
that can tackle this, a thorough understanding of the bone fracture healing process is needed. It is currently known that bone heals via a multistage sequential process of inflammation, soft-callus formation, hard-callus formation and then bone remodelling. Moreover, during this process there is a need for restoration of a vascular network for nutrient delivery and waste removal. Whereas the regulation of vascular network during later stages is understood, knowledge of the mechanisms and
driving forces of vascularization during earlier phases in bone healing is currently lacking. Nevertheless, during this process, cell migration towards the site of fracture is of great importance. This process is affected through a range of extracellular signals, which are dependent on the properties of the extracellular matrix. It has been shown that the structural properties and the composition of the fracture tissue are two of the key regulators in guiding cellular migration. This study has integrated the use of a novel electrospinning technique to mimic the effects of the early fracture composition on (endothelial) cell migration. Fibrinogen and gelatin fibers, which are reflective of the early fracture environment, have been spun using an acidic solvent system. Characterization of the fibers showed that the electrospinning process caused minor changes in the protein structure of fibrinogen, and none in that of gelatin. Furthermore, endothelial cells were cultured with a cell free zone on top of
fibrinogen and gelatin patterned substrates to mimic cell migration during fracture revascularization. Cellular migration increased with higher fiber density, and gelatin fibers generally showed higher rates of cell migration. Furthermore, gelatin fibers showed higher amounts of cell polarization than fibrinogen fibers, likely driven by the higher stiffness of gelatin fibers. Moreover, the addition of fibers showed to
reflect different modes of in vivo vascularization of cell migration on the substrates. To further analyze the effects of fracture composition on endothelial cell migration, fiber membranes were electrospun on a transwell-like device, and cell migration from a fibrin gel was assessed. The physical constraints posed by the fiber membranes were shown to impede cellular migration from the fibrin gel, and no significant differences were found between cell migration on the membranes. Overall, it became evident
that the structural properties and the composition of the fibrous micro-environment regulate cellular migration, and can in turn affect the bone regeneration as a whole. Ultimately, further optimization and characterization of the used models are needed, such that they can more closely reflect the fracture healing environment.