Manufacturing high throughput in vitro models resembling the tissue microenvironment is highly demanded for studying bone regeneration. Tissues such as bone have complex multiscale architectures inside which cells reside. To this end, engineering a microfluidic platform incorporated with three-dimensional (3D) microscaffolds and submicron/nanoscale topographies can provide a promising model for 3D cell cultures. There are, however, certain challenges associated with this goal, such as the need to decorate large surfaces area with high-fidelity 3D submicron structures. Here, we succeeded in fabricating a microfluidic platform embedded with a large area (mm range) of reproducible submicron pillar-based topographies. Using the two-photon polymerization (2PP) as a 3D printing technique based on direct laser writing, uniform submicron patterns were created through optimization of the process parameters and writing strategy. To demonstrate the multiscale fabrication capabilities of this approach, submicron pillars of various heights were integrated onto the surfaces of a 3D microscaffold in a single-step 2PP process. The created submicron topography was also found to improve the hydrophilicity of the surface while being able to withstand flow rates of up to 8 mL/min. The material (IP-Dip resin) used for patterning did not have cytotoxic effects against human mesenchymal stromal cells after 3 days of dynamic culture in the microfluidic device. This proof-of-principle study, therefore, marks a significant step forward in manufacturing submicron structure-on-a-chip models for bone regeneration studies.@en