title: Combination of enzymes and flow perfusion conditions improves osteogenic differentiation of bone marrow stromal cells cultured upon starch/poly(epsilon-caprolactone) fiber meshes.
abstract: Previous studies have shown that alpha-amylase and lipase are capable of enhancing the degradation of fiber meshes blends of starch and poly(epsilon-caprolactone) (SPCL) under dynamic conditions, and consequently to promote the proliferation and osteogenic differentiation of bone marrow stromal cells (MSCs). This study investigated the effect of flow perfusion bioreactor culture in combination with enzymes on the osteogenic differentiation of MSCs. SPCL fiber meshes were seeded with MSCs and cultured with osteogenic medium supplemented with alpha-amylase, lipase, or a combination of the two for 8 or 16 days using static or flow conditions. Lipase and its combination with alpha-amylase enhanced cell proliferation after 16 days. In addition, the flow perfusion culture enhanced the infiltration of cells and facilitated greater distribution of extracellular matrix (ECM) throughout the scaffolds in the presence/absence of enzymes. A significant amount of calcium was detected after 16 days in all groups cultured in flow conditions compared with static cultures. Nevertheless, when alpha-amylase and lipase were included in the flow perfusion cultures, the calcium content was 379 +/- 30 microg/scaffold after as few as 8 days. The highest calcium content (1271 +/- 32 microg/scaffold) was obtained for SPCL/cell constructs cultured for 16 days in the presence of lipase and flow. Furthermore, von Kossa staining and tetracycline fluorescence of histological sections demonstrated mineral deposition within the scaffolds for all groups cultured for 16 days under flow. However, all the data corroborate that lipase coupled with flow perfusion conditions improve the osteogenic differentiation of MSCs and enhance ECM mineralization.
authors.affiliation: University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Taipas, Guimarães, Portugal.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Martins
authors.forename: Anita
authors.initials: A
authors.lastname: Saraf
authors.forename: Rui A
authors.initials: RA
authors.lastname: Sousa
authors.forename: Catarina M
authors.initials: CM
authors.lastname: Alves
authors.forename: Antonios G
authors.initials: AG
authors.lastname: Mikos
authors.forename: F Kurtis
authors.initials: FK
authors.lastname: Kasper
authors.forename: Rui L
authors.initials: RL
authors.lastname: Reis
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001681:Biological Assay; D001854:Bone Marrow Cells / Q000166:cytology*; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004798:Enzymes / Q000494:pharmacology*; D005453:Fluorescence; D008049:Lipase / Q000494:pharmacology; D008297:Male; D008855:Microscopy, Electron, Scanning; D010012:Osteogenesis / Q000187:drug effects*; D010477:Perfusion / Q000379:methods*; D011091:Polyesters / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D012212:Rheology / Q000187:drug effects; D013194:Staining and Labeling; D013213:Starch / Q000494:pharmacology*; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D013752:Tetracycline; D054457:Tissue Scaffolds / Q000737:chemistry*; D000516:alpha-Amylases / Q000494:pharmacology
chemical_list: D004798:Enzymes; D011091:Polyesters; C016240:polycaprolactone; D013213:Starch; D004247:DNA; D008049:Lipase; D000469:Alkaline Phosphatase; D000516:alpha-Amylases; D013752:Tetracycline; D002118:Calcium