(572bg) Effects of Perfusion Flow on Hmsc Construct Development In Chitosan-Based Biomimetic 3-D Scaffolds

Biomimetic scaffolds mimic the in vivo extracellular matrix environments of the target tissues and have been used to augment the therapeutic potential of human mesenchymal stem cells (hMSC) for a broad range of diseases. Previous studies from our lab have used hydroxyapatite, chitosan, and gelatin (HCG) to fabricate a three dimensional scaffold that mimics the biological composition of bone tissue and have demonstrated the potency of the HCG composite scaffold on hMSC growth, retention, and osteogenic differentiation.¹ However, the successful regeneration of functional 3-D constructs, for substantial bone defects, from HCG scaffolds and hMSCs requires uniform spatial cell distribution and growth throughout the entire 3-D scaffold in a controlled environment. To meet the demand, we incorporated the 3-D HCG scaffolds into the perfusion bioreactor system developed in our lab and investigated the effects of cell seeding, flow rate, and flow modes on hMSC development.^2,3 The in-house perfusion bioreactor system has multiple, individually controlled flow chambers that share the same media source and inoculum facilitating the comparison of various operation conditions. The perfusion system also has the ability for dynamic cell seeding by depth filtration that enhances seeding efficiency and initial cell distribution. During the cell growth phase, the perfusion flow in each chamber can be controlled as either parallel or perpendicular to the construct, meeting the varying nutrient demands over the course of construct development. In the current study we fabricated an HCG scaffold with an average pore size of 93 ± 27 ìm to fit within our current bioreactor system. Dynamically seeded human mesenchymal stem cells showed a notable 67 % seeding efficiency, significantly higher than the static seeding efficiency. A higher cell number increase compared to static culture was observed after 10 days of transverse perfusion growth at a flow rate of 0.1 mL/min. SEM has confirmed the presence of cells throughout the 1 mm thickness of the scaffold. Cell metabolic activities were monitored by measuring glucose consumption and lactate production. A steady increase in metabolic activity beyond day 15 was found to accompany the cell number increase. Currently, we are investigating the effects of flow rate under parallel and transverse conditions on CFU-F, alkaline phosphatase (ALP), and calcium levels of cells retained from perfusion samples for up to 30 days of perfusion culture. Our overall objective is to develop a functional bone construct from hMSC and the chitosan-based biomimetic scaffold capable of large bone defect repair.

1. F. Zhao, W. Grayson, T. Ma*, B. Bunnell, and W. Lu. Effects of Hydroxyapatite on 3-D Human Mesenchymal Stem Cell Tissue Development. Biomaterials, 2006, 27:1859-1867.

2. F. Zhao and T. Ma* Perfusion bioreactor system for human mesenchymal stem cell tissue engineering: Dynamic cell seeding and construct development, F Biotechnology and Bioengineering, Vol. 91, 482-493, 2005.

3. F. Zhao, R. Chella, and T. Ma* Effects of Shear Stress on 3-D Human Mesenchymal Stem Cell Construct Development in a Perfusion Bioreactor System: Experiments and Hydrodynamic Modeling. . Biotechnology and Bioengineering, 96:584-595, 2007