The role of lipase and alpha-amylase in the degradation of starch/poly(epsilon-caprolactone) fiber meshes and the osteogenic differentiation of cultured marrow stromal cells.
The present work studies the influence of hydrolytic enzymes (alpha-amylase or lipase) on the degradation of fiber mesh scaffolds based on a blend of starch and poly(epsilon-caprolactone) (SPCL) and the osteogenic differentiation of osteogenic medium-expanded rat bone marrow stromal cells (MSCs) and subsequent formation of extracellular matrix on these scaffolds under static culture conditions. The biodegradation profile of SPCL fiber meshes was investigated using enzymes that are specifically responsible for the enzymatic hydrolysis of SPCL using concentrations similar to those found in human serum. These degradation studies were performed under static and dynamic conditions. After several degradation periods (3, 7, 14, 21, and 30 days), weight loss measurements and micro-computed tomography analysis (specifically porosity, interconnectivity, mean pore size, and fiber thickness) were performed. The SPCL scaffolds were seeded with rat MSCs and cultured for 8 and 16 days using complete osteogenic media with and without enzymes (alpha-amylase or lipase). Results indicate that culture medium supplemented with enzymes enhanced cell proliferation after 16 days of culture, whereas culture medium without enzymes did not. No calcium was detected in groups cultured with alpha-amylase or without enzymes after each time period, although groups cultured with lipase presented calcium deposition after the eighth day, showing a significant increase at the sixteenth day. Lipase appears to positively influence osteoblastic differentiation of rat MSCs and to enhance matrix mineralization. Furthermore, scanning electron microscopy images showed that the enzymes did not have a deleterious effect on the three-dimensional structure of SPCL fiber meshes, meaning that the scaffolds did not lose their structural integrity after 16 days. Confocal micrographs have shown cells to be evenly distributed and infiltrated within the SPCL fiber meshes up to 410 microm from the surface. This study demonstrates that supplementation of culture media with lipase holds great potential for the generation of bone tissue engineering constructs from MSCs seeded onto SPCL fiber meshes, because lipase enhances the osteoblastic differentiation of the seeded MSCs and promotes matrix mineralization without harming the structural integrity of the meshes over 16 days of culture.
Martins
Ana M
AM
3Bs Research Group-Biomaterials, Biodegradables and Biomimetics, Department of Polymer Engineering, University of Minho, Campus de Gualtar, Braga, Portugal.
Pham
Quynh P
QP
Malafaya
Patrícia B
PB
Sousa
Rui A
RA
Gomes
Manuela E
ME
Raphael
Robert M
RM
Kasper
F Kurtis
FK
Reis
Rui L
RL
Mikos
Antonios G
AG
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000378:metabolism*; D018613:Microscopy, Confocal; D010012:Osteogenesis; D011091:Polyesters / Q000378:metabolism*; D016062:Porosity; D051381:Rats; D017550:Spectroscopy, Fourier Transform Infrared; D013213:Starch / Q000378:metabolism*; D017154:Stromal Cells / Q000166:cytology* / Q000201:enzymology / Q000648:ultrastructure; D054457:Tissue Scaffolds / Q000737:chemistry*; D015431:Weight Loss; D055114:X-Ray Microtomography; D000516:alpha-Amylases / Q000378:metabolism*
D011091:Polyesters; C016240:polycaprolactone; D013213:Starch; D008049:Lipase; D000469:Alkaline Phosphatase; D000516:alpha-Amylases; D002118:Calcium