Developing a microfluidic device incorporating submicron topographies for studying bone regeneration

Abstract

A major challenge facing the study of bone regeneration today is the inability to mimic the tissue microenvironment and the factors comprising it. Organ-on-chip based microfluidic systems today aim at doing so by dynamically culturing living cells in them. These are preferred owing to their small feature sizes, laminar flows, high throughput, small reagent quantities, and reduced dependency on animal studies. Substrate topography even at the submicron and nanoscale is known to induce osteogenesis by infleuncing cells at ligand/receptor levels but poses a major challenge in terms of fabrication and upscaling. The two photon polymerisation (2PP) process allows producing features in a spatially and dimensionally controlled fashion. The method is typically known for fabricating micro-sized structures but remains largely unexplored for producing features in the submicron/nano scale. This study aims at creating reproducible submicron pillar based topographies using 2PP and integrating them into a microfluidic device for studying their effect on bone regeneration. Uniform submicron patterns were produced using optimal process parameters and writing strategy. Further, the patterns were expanded to areas up to 2 mm $\times$ 1.7 mm and their uniformity was assessed. To demonstrate multiscale fabrication, these were also integrated onto a 2PP fabricated micro-scaffold in a single step process. The topography enhanced the surface hydrophilicity and could withstand flow rates of up to 8 ml/min. The human mesenchymal stem cells (hMSCs) dynamically cultured in these integrated devices showed a healthy morphology on the pattern with no visible signs of cytotoxicity even after a period of 5 days. This study marks a step towards printing controlled submicron topographies using 2PP that can be upscaled (both spatially and dimensionally), incorporated into biocompatible microfluidic chips, and thus help in achieving the optimal microenvironment for bone regeneration studies in the future.