Impregnation of plasmid DNA into three-dimensional scaffolds and medium perfusion enhance in vitro DNA expression of mesenchymal stem cells.
This article describes the development of an in vitro culture system to enhance the expression of a plasmid DNA for mesenchymal stem cells (MSCs) by a combination of plasmid DNA impregnation into three-dimensional cell scaffolds and culture methods. Gelatin was cationized by introducing spermine to the carboxyl groups for complexation with the plasmid DNA. As the MSC scaffold, poly(glycolic acid) (PGA) fiber fabrics, collagen sponges, and collagen sponges reinforced by incorporation of PGA fibers were used. A complex of cationized gelatin and plasmid DNA encoding bone morphogenetic protein 2 (BMP-2) was impregnated into the scaffolds. Plasmid DNA was released from PGA-reinforced collagen sponge for longer than from the other scaffolds. MCS were seeded into each type of scaffold and cultured by static, stirring, and perfusion methods. When MSCs were cultured in PGA-reinforced sponge, the level of BMP-2 expression was significantly enhanced by perfusion culture compared with the other culture methods, and the time of expression was prolonged. Irrespective of the culture method, the expression level was significantly higher from plasmid DNA impregnated in scaffold than by plasmid DNA in medium. The alkaline phosphatase activity and osteocalcin content of MSCs cultured in PGA-reinforced sponge by the perfusion method were significantly higher compared with those of other methods, and a significantly higher amount of plasmid DNA internalized into MSCs was observed. We conclude that a combination of plasmid DNA-impregnated PGA-reinforced sponge and the perfusion method was promising to promote in vitro gene expression for MSCs.
Hosseinkhani
Hossein
H
Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
Inatsugu
Yasuyuki
Y
Hiraoka
Yosuke
Y
Inoue
Sachiko
S
Shimokawa
Hitoyata
H
Tabata
Yasuhiko
Y
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D001854:Bone Marrow Cells / Q000166:cytology; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D003432:Cross-Linking Reagents / Q000737:chemistry; D003470:Culture Media / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D003890:Desiccation; D005269:Femur / Q000166:cytology; D015870:Gene Expression; D005976:Glutaral / Q000737:chemistry; D006358:Hot Temperature; D007700:Kinetics; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D015675:Osteocalcin / Q000032:analysis; D010012:Osteogenesis; D010477:Perfusion; D010957:Plasmids; D011100:Polyglycolic Acid / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D051381:Rats; D011916:Rats, Inbred F344; D023822:Tissue Engineering / Q000379:methods*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism; D014466:Ultraviolet Rays
D001672:Biocompatible Materials; C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D003432:Cross-Linking Reagents; D003470:Culture Media; D016212:Transforming Growth Factor beta; D015675:Osteocalcin; D011100:Polyglycolic Acid; D003094:Collagen; D004247:DNA; D000469:Alkaline Phosphatase; D005976:Glutaral