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Effects of Bone Morphogenetic Protein Distribution and Shear Stress on 3-D Human Mesenchymal Stem Cell Construct Development In a 3-D Perfusion Bioreactor System

Teng Ma, Feng Zhao, Katelyn Sellgren, and Ravindran Chella. Chemical and Biomedical Engineering, Florida State University, 2525 Pottsdamer Street, Tallahassee, FL 32310

The process of creating functional 3-D tissues constructs in the bioreactor system using stem cells and scaffolds is influenced by the dynamic culture environment characterized by dynamic stresses and concentration gradients of essential nutrients and regulatory molecules. Among these microenvironmental parameters, media flow and molecule distributions are crucial regulators of 3-D hMSC tissue development. Our previous study has found that hMSCs actively respond to shear stresses at a range of 110-5 to 110-4 Pa, orders of magnitude lower than those observed on planar surfaces (Zhao et al., 2007). However, media flow also influences macromolecule transport and these two effects are strongly coupled in the 3-D constructs. In this study, we have combined the experimental and modeling approaches to delineate the effects of shear stress and distribution of bone morphogenetic proteins (BMP) by operating the modular perfusion bioreactor system developed in our lab (Zhao et al., 2005) in either transverse or parallel perfusion modes. BMPs are members of the TGF-β superfamily and are autologously secreted by hMSC, which have been demonstrated to be potent inducers of its osteogeneic differentiation. First, the effects of media flow on the developmental characteristics of hMSCs grown in 3-D poly (ethylene terephthalate) (PET) matrices were determined. The two perfusion modes have different hydrodynamic characteristics but identical volumetric flowrate of 0.2 mL/min, residence time, and initial cell seeding population. A 1.6 times higher proliferation rate, higher CFU-F formation, and stem cell gene expression at day 20 were observed in parallel perfusion in comparison with those under transverse perfusion. The transverse flow also upregulated the osteogenic differentiation potential at day 20 as measured by the osteonectin gene expression and calcium deposition in the matrices. In addition, ECM of hMSCs was patterned differently under the two perfusion modes, in which transverse perfusion directed a graded ECM distribution along the flow direction, as opposed to a denser and more uniform spatial patterning of ECM on both sides of 3-D constructs dictated by parallel perfusion flow mode. Hydrodynamic modeling results showed cells in transverse flow mode were exposed to media flow at ~ 3 μm/s and shear stress in the range of 110-3 Pa. Concentration gradients of BMPs and the fraction of bound receptors in the constructs under various flow conditions are modeled to determine the spatial distribution of BMPs on hMSC proliferation and osteogenic differentiation. The spatial distribution of alkaline phosphatase in 3-D constructs will be determined to experimentally analyze the cellular response to long-term accumulation of BMP under various flow conditions. Combining modeling and experimental results will not only help to delineate the effects of the biomechanical microenvironment and macromolecule transport but also improve tissue engineering strategy using perfusion bioreactor systems.

Zhao F, Chella R and Ma T. Effects of shear stress on 3-D human mesenchymal stem cell construct development in a versatile perfusion bioreactor system: experiments and hydrodynamic modeling. Biotechnology and Bioengineering, 2007;96:584-595.

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