title: 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.
authors.affiliation: 3 Bs Research Group-Biomaterials, Biodegradables and Biomimetics, University of Minho, Campus de Gualtar, Braga, Portugal.
authors.forename: Manuela E
authors.initials: ME
authors.lastname: Gomes
authors.forename: Carla M
authors.initials: CM
authors.lastname: Bossano
authors.forename: Carol M
authors.initials: CM
authors.lastname: Johnston
authors.forename: Rui L
authors.initials: RL
authors.lastname: Reis
authors.forename: Antonios G
authors.initials: AG
authors.lastname: Mikos
mesh_terms: D000818:Animals; D001672:Biocompatible Materials / Q000138:chemical synthesis*; D019149:Bioreactors; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism*; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D005346:Fibroblast Growth Factors / Q000378:metabolism; D007335:Insulin-Like Growth Factor II; D008297:Male; D010477:Perfusion; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D011091:Polyesters / Q000138:chemical synthesis; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013213:Starch / Q000138:chemical synthesis; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism; D023822:Tissue Engineering; D016212:Transforming Growth Factor beta / Q000378:metabolism; D053773:Transforming Growth Factor beta1; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D001672:Biocompatible Materials; C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; C035170:IGF2 protein, human; D010982:Platelet-Derived Growth Factor; D011091:Polyesters; D011506:Proteins; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D042461:Vascular Endothelial Growth Factor A; C075278:platelet-derived growth factor A; C492325:starch polycaprolactone; D005346:Fibroblast Growth Factors; D007335:Insulin-Like Growth Factor II; D013213:Starch