The influence of pore size on colonization of poly(l-lactide-glycolide) scaffolds with human osteoblast-like MG 63 cells in vitro
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A degradable copolymer of l-lactide and glycolide (PLG) was synthesized by ring opening polymerization using zirconium acetylacetonate [Zr(acac)4] as a biocompatible initiator. The structure of the copolymer was studied by nuclear magnetic resonance spectroscopy (NMR) and gel permeation chromatography (GPC). Porous scaffolds of defined microstructure were prepared by solvent casting/salt particulate leaching, which resulted in the creation of three types of scaffolds with the same porosity (87% ± 1%) but with different diameters of the pores (600, 200 and 40 μm) and degree of interconnectivity. The potential of the scaffolds for cell colonization was tested in a conventional static cell culture system using human osteoblast-like MG 63 cells. As revealed by conventional fluorescence and confocal microscopy on days 5 and 7 after seeding, the cells on the scaffolds of large or medium pore size infiltrated the inside part of the material, whereas on the scaffolds of small pore size, the cells were retained on the material surface. On day 7 after seeding, the highest number of cells was found on the scaffolds of the largest pore size (more than 120,000 cells per sample of the diameter 15 mm and thickness 2 mm), whereas on the scaffolds with medium and smallest pore diameter, the number of cells was almost three times lower and similar for both pore sizes. These results corresponded well with the incorporation of bromodeoxyuridine into newly synthesized DNA, which was significantly higher in cells on scaffolds of the largest pore size than on the material with medium and smallest pore diameter. As indicated by the MTT test, the mitochondrial activity in cells on scaffolds with medium pore size was similar to that on the material with the highest pore size, and significantly higher than on scaffolds of the smallest pore diameter. These results suggest that PLG scaffolds with the largest pore diameter (600 μm) and better pore interconnectivity are the most suitable for colonization with osteogenic cells.
This study was supported by the Polish Budget Fund for Scientific Research (project No. 3 T08D 019 28), by the Ministry of Education, Youth and Sports of the Czech Republic (COST project, Action 527.130, grant No. 1P05OC012), and by the Grant Agency of the Czech Republic (grant No. 106/06/1576). We also thank Ms. Katarina Mitrova (Inst. Physiol., Acad. Sci CR) for her excellent technical assistance with confocal microscopy and Mr. Robin Healey (Czech Technical University, Prague) for language revision of the manuscript.
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