A quantitative analysis of cell bridging kinetics on a scaffold using computer vision algorithms.

Tissue engineering involves the seeding of cells into a structural scaffolding to regenerate the architecture of damaged or diseased tissue. To effectively design a scaffold, an understanding of how cells collectively sense and react to the geometry of their local environment is needed. Advances in the development of melt electro-writing have allowed micron and submicron polymeric fibres to be accurately printed into porous, complex and three-dimensional structures. By using melt electrowriting, we created a geometrically relevant in vitro scaffold model to study cellular spatial-temporal kinetics. These scaffolds were paired with custom computer vision algorithms to investigate cell nuclei, cell membrane actin and scaffold fibres over different pore sizes (200-600 µm) and time points (28 days). We find that cells proliferated much faster in the smaller (200 µm) pores which halved the time until confluence versus larger (500 and 600 µm) pores. Our analysis of stained actin fibres revealed that cells were highly aligned to the fibres and the leading edge of the pore filling front, and we found that cells behind the leading edge were not aligned in any particular direction. This study provides a systematic understanding of cellular spatial temporal kinetics within a 3D in vitro model to inform the design of more effective synthetic tissue engineering scaffolds for tissue regeneration. STATEMENT OF SIGNIFICANCE: Advances in the development of melt electro-writing have allowed micron and submicron polymeric fibres to be accurately printed into porous, complex and three-dimensional structures. By using melt electrowriting, we created a geometrically relevant in vitro model to study cellular spatial-temporal kinetics to provide a systematic understanding of cellular spatial temporal kinetics within a 3D in vitro model. The insights presented in this work help to inform the design of more effective synthetic tissue engineering scaffolds by reducing cell culture time; which is valuable information for the implant or lab-grown-meat industries.