Introduction: Fibrinogen undergoes significant developmental changes during the transition from neonatal to adult life. Neonates possess a unique molecular variant of fibrinogen, fetal fibrinogen that is characterized by increased sialic acid and phosphorus content, increased negative charge, slower thrombin clotting time, and decreased activity compared to adult fibrinogen. Previous studies have also shown that there are differences in the fibrin and fibrinogen network properties of adults and neonates, specifically, greater fibrin alignment in neonatal networks. Improved wound healing has been associated with fiber alignment which affects the binding and migration of cells. The specific aim of our study was to further understand the differences in wound healing between adults and neonates. This was done by creating microfluidic devices to investigate the migratory preference of fibroblasts in neonatal and adult fibrinogen. Microfluidics were used to evaluate the assembly of endothelial cells in adult and cord plasma and allow for control over cell culture conditions to mimic the microenvironment experienced by cells in vivo. Furthermore, a microfluidic model allowed for the assessment of cellular migration independent of other phenomena that might influence migration in vivo as well as high-resolution images to be taken and the ability to monitor cell behavior in real time within the fibrinogen and plasma matrices.
Materials and
Methods: Given the aim of the study, a microfluidic “mini-well” model was designed to study the migration of human dermal fibroblast neonate (HDFn) cells in adult and neonatal fibrin(ogen). The mini- well design consisted of a glass slide with a bonded section of PDMS that had been punctured with a 3.0 mm biopsy punch and an overlapping 1.5 mm biopsy punch. Although we were able to get results using this technique, it was hard to replicate the exact configuration every time since the wells were being manually punched. To ensure consistency between trials, we designed a 3D microdevice mold using SolidWorks and printed it using a CADworks3D microfluidics printer. The design of the new devices was two side channels joining a central channel via a half PDMS wall. A Fibrin-GelMA mix was put in the central channel and HDFn-GelMA mix was added to the right channel and exposed to UV light to allow for cross-linking of the GelMA molecules forming a solid hydrogel matrix, which encapsulated the HDFn cells. Growth media was then added to the left channel and extra growth media was added to the top of the device, and the device was incubated at 37 degrees Celsius for 7 days before being fixed and imaged.
Results, Conclusions, and Discussions: Migration analyses were done via confocal imaging which revealed the cord fibrinogen + Factor XII condition showed increased migration of fibroblasts and decreased overall clot density. However, additional trials must be done in order to get conclusive results. Our study has established a starting point for the better understanding of how fibrin matrices influence fibroblast cell behavior, migration, and network formation. Such investigations hold the potential to advance our comprehension of hemostasis in both adult and neonatal contexts. Thus enhancing the understanding of vascular development and pathological conditions associated with fibrin matrices.
