The Role of Lipase and α-Amylase in the Degradation of Starch/Poly(ɛ-Caprolactone) Fiber Meshes and the Osteogenic Differentiation of Cultured Marrow Stromal Cells

Abstract

The present work studies the influence of hydrolytic enzymes (a-amylase or lipase) on the degradation of fiber mesh scaffolds based on a blend of starch and poly(e-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 (a-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 a-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 mm 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.

Dose effect of tumor necrosis factor-α on in vitro osteogenic differentiation of mesenchymal stem cells on biodegradable polymeric microfiber scaffolds

“…By modulating key processing variables, including the PCL concentration, flow rate, collector distance, and applied voltage, PCL microfiber scaffolds with a specific pore size, porosity, and fiber diameter can be reproducibly generated [22]. Scaffold pore size and porosity are key determinants of cell attachment and migration, and also of nutrient transport, all of which affect cell function, proliferation, and differentiation [23–25]. Electrospun PCL fibers with sub-micron diameters have been shown to enhance MSC adhesion and spreading [22].…”

Effect of temporally patterned TNF-α delivery on in vitro osteogenic differentiation of mesenchymal stem cells cultured on biodegradable polymer scaffolds

“…PCL was selected for the generation of electrospun microfiber mesh scaffolds because it is biocompatible and biodegradeable, with a slow degradation rate that would be beneficial in future in vivo implantation studies by providing structural support of a bone defect during tissue ingrowth, then degrading to allow for bone remodelling (16, 23, 32, 33). As in previous studies of PCL microfiber meshes (16, 21, 23), successful cell attachment and infiltration to a depth of 200-500 μm was observed (Figure 6).…”

Level and distribution of secreted and cell-associated N-acetyl, N-glycolylneuraminic, and deaminoneuraminic acids in osteosarcoma cells isolated from patients

“…Alkaline phosphatase (ALP) activity assay ALP activity was determined kinetically by monitoring the conversion of p-nitrophenyl phosphate (p-NPP) to p-nitrophenol 17 . Osteoblasts and osteosarcoma cells were seeded at 10 4 cells per well in 96-well-plates in four replications for each case.…”