In Vitro Localization of Bone Growth Factors in Constructs of Biodegradable Scaffolds Seeded with Marrow Stromal Cells and Cultured in a Flow Perfusion Bioreactor

Abstract

Tissue engineering strategies aim at controlling the behavior of individual cells to stimulate tissue formation. This control is achieved by mimicking signals that manage natural tissue development or repair. Flow perfusion bioreactors that create culture environments with minimal diffusion constraints and provide cells with mechanical stimulation may closely resemble in vivo conditions for bone formation. Therefore, these culturing systems, in conjunction with an appropriate scaffold and cell type, may provide significant insight towards the development of in vitro tissue engineering models leading to improved strategies for the construction of bone tissue substitutes. The objective of this study was to investigate the in vitro localization of several bone growth factors that are usually associated with bone formation in vivo by culturing rat bone marrow stromal cells seeded onto starch-based biodegradable fiber meshes in a flow perfusion bioreactor. The localization of several bone-related growth factors-namely, transforming growth factor-beta1, platelet-derived growth factor- A, fibroblast growth factor-2, vascular endothelial growth factor, and bone morphogenetic protein- 2-was determined at two different time points in scaffolds cultured under perfusion conditions at two different flow rates using an immunohistochemistry technique. The results show the presence of regions positively stained for all the growth factors considered, except platelet-derived growth factor-A. Furthermore, the images obtained from the positively stained sections suggest an increase in the immunohistochemically stained area at the higher flow rate and culture time. These observations demonstrate that flow perfusion augments the functionality of scaffold/cell constructs grown in vitro as it combines both biological and mechanical factors to enhance cell differentiation and cell organization within the construct. This study also shows that flow perfusion bioreactor culture of marrow stromal cells, combined with the use of appropriate biodegradable fiber meshes, may constitute a useful model to study bone formation and assess bone tissue engineering strategies in vitro.

Effect of Low-Frequency Pulsatile Flow on Expression of Osteoblastic Genes by Bone Marrow Stromal Cells

“…Indeed, BMP-2, -4, and -7 are known to stimulate expression of bone ECM proteins BSP and OC, 47 while perfusion culture has been shown qualitatively to induce synthesis of growth and differentiation factors, including BMP-2 and TGF-b 1 . 12 However, this study-which may be the first to quantitatively characterize the induction of growth factors under flow-showed that different flow regimens resulted in different levels of expression of BMP-2, -7 (Fig. 5a), and TGF-b 1 (Fig.…”

“…Separately, perfusion culture of BMSCs has been shown to stimulate synthesis and accumulation of growth and differentiation factors, including bone morphogenetic protein (BMP)-2, fibroblast growth factor (FGF)-2, VEGF-A, and TGF-b 1 . 12 Together these data suggest that growth and differentiation factors-which accumulate in the ECM in response to hydrodynamic shear stress-may guide osteoblastic differentiation and contribute to enhanced expression of bone ECM proteins.…”

Flow Perfusion Improves Seeding of Tissue Engineering Scaffolds with Different Architectures

“…6,16,[23][24][25]30,34,48 However, static seeding is not an ideal method due to low efficiencies and poor cell distribution throughout the scaffold's porous network. 36,62,64 Consequently, it was important to evaluate the effects of flow perfusion in the seeding of polymeric scaffolds for tissue engineering.…”

In vivo short-term and long-term host reaction to starch-based scaffolds

“…Natural-origin biomaterials have been considered for many years as a way to improve, in comparison to synthetic polymers, in vivo biofunctionality and to modulate/avoid a harmful host response due to their similarities with biological molecules. Starch-based scaffolds, processed using several methodologies aiming at a range of TE applications [19][20][21][22][23][24][25][26], have demonstrated great potential in this field. Very promising results for bone tissue regeneration have in particular been obtained with a blend of starch and polycaprolactone (SPCL) [20][21][22][23]25,[27][28][29][30].…”

“…Starch-based scaffolds, processed using several methodologies aiming at a range of TE applications [19][20][21][22][23][24][25][26], have demonstrated great potential in this field. Very promising results for bone tissue regeneration have in particular been obtained with a blend of starch and polycaprolactone (SPCL) [20][21][22][23]25,[27][28][29][30]. SPCL scaffolds, with adequate physicochemical and mechanical properties for bone TE [19,22] and adequate degradability rate [24,25,31], have been shown to support mesenchymal stem cell growth and differentiation [19,20], and to be excellent supporting structures for endothelial cells [22,23,32,33].…”