THE SYNERGISTIC EFFECTS OF STARLING FLOWS AND A DISTRIBUTED AND DELOCALIZED NUTRIENT SOURCE ON BONE MARROW STROMAL CELL CULTURE IN HOLLOW FIBRE MEMBRANE BIOREACTORS

Objectives: The development of large dimensions bone tissue engineered constructs is limited by the difficult delivery of nutrients to cells situated in the inner part of them. In this study, hollow fibre membrane bioreactors (HFMB) were used to allow a distributed and delocalized nutrients supply in 3D cm-scale constructs. Low to high spontaneus convective flows effect on cell distribution, proliferation and collagen deposition were investigated. Methods: HFMB where seeded with 4.5x10 super(8) BMSC cells/mL. Cells were fed with culture medium from the membranes lumen in a recirculation modality for 12 days. Bioreactors were operated to establish low to high convective flows towards the ECS. At the end of the culture cells were processed for Scanning Electron Microscopy (SEM). Histological sections were stained with H&E to evidence cell radial organization and with DAPI to analyze cell nuclei distribution along the bioreactor length. Osteoblast phenotype maintenance was confirmed by pro-collagen immuno-staining, collagen deposition detected with Masson-Goldner stain. Results: HFMB operated under low convective flows presented a uniform axial cell distribution and poor cell proliferation. Cells were found at the membrane surface forming a thin layer around them. SEM analysis confirmed the low proliferation rate under this operating condition. Histological analysis showed how in the presence of high spontaneous convective flows cells were dragged at the bioreactor outlet. Despite this evidence, this operating condition seems to be the most promising in obtaining large bone tissue engineered substitutes as demonstrated by the rearrangement of ostegenic cells in aggregates 2.5cm thick. SEM analysis showed how cell number grew significantly respect to the seeding density forming a continuous thick multilayer covering the surface of several adjacent membranes. Conclusions: A delocalized and distributed nutrients supply coupled to bioreactor design promoting the occurrence of high Starling flows in the cell compartment may contribute to obtain clinical-scale 3D tissue engineered constructs.