(761c) Effects of Macroscopic Flow On hMSC Microenvironment and Osteogenic Differentiation In Perfusion Bioreactor

Human mesenchymal stem cells (hMSCs) have multi-lineage differentiation potentials and combining hMSCs with 3D scaffolds is an important strategy to enhance functional outcomes in bone tissue regeneration.  In this approach, perfusion bioreactor plays a critical role in regulating 3D construct microenvironment and in providing controlled physiochemical and biomechanical environments.  To date, media flow in the bioreactors is either parallel (e.g., spinner flask and rotating wall vessel) or transverse to the 3D scaffolds (e.g., direct perfusion).  Although bioreactors of both flow conditions have been extensively used in bone tissue engineering, the impact of macroscopic flow on the ECM microenvironment and hMSC fate is unknown.  Using the in-house modular perfusion bioreactor system, we investigated the effects of the macroscopic flows (i.e., parallel or transverse) on the formation of construct microenvironment and its subsequent influence on hMSC proliferation and osteogenic differentiation in the porous poly(ethylene terephthalate) scaffolds.  The parallel flow effectively retained ECM proteins and growth factors (e.g., FGF-2) within the scaffolds, preserving hMSC proliferation potential with higher CFU-F numbers and upregulation of OCT-4 expressions.  In contrast, the transverse flow enhanced hMSC osteogenic differentiation with higher ALP activity and calcium deposition.  The analysis of the transcriptional profiles of the osteogenic gene (e.g., ALP, Runx-2, and OSX) and the up-regulation of osteogenic proteins (e.g., BMP-2, ALP, and OC) suggest the enhanced hMSC osteogenic differentiation as a combined result of shear stress stimulation and the depletion of pro-mitotic growth factors.  Utilizing the media flow to influence the formation of ECM microenvironment before and after chemical induction, we are currently investigating the optimal bioreactor strategy for enhancing bone construct development in 3D scaffolds.  The results will provide important insight into the role of macroscopic flow on construct microenvironment formation and bone construct function.