title: Enhanced ectopic bone formation using a combination of plasmid DNA impregnation into 3-D scaffold and bioreactor perfusion culture.
abstract: The objective of this study is to enhance in vivo ectopic bone formation by combination of plasmid DNA impregnation into three-dimensional (3-D) cell scaffolds and a developed in vitro culture method. Gelatin was cationized by introducing spermine (Sm) to the carboxyl groups for complexation with the plasmid DNA. As the MSC scaffold, collagen sponge reinforced by incorporation of poly(glycolic acid) (PGA) fibers was used. A complex of the cationized gelatin and plasmid DNA of BMP-2 was impregnated into the scaffold. MCS were seeded into each scaffold and cultured by a static and perfusion methods. When MSC were cultured in the PGA-reinforced collagen sponge, the level of BMP-2 expression was significantly enhanced by the perfusion culture compared with static method. When the osteoinduction activity of the PGA-reinforced collagen sponges seeded with PBS, MSC, naked plasmid DNA-BMP-2, cationized gelatin-plasmid DNA-BMP-2 complex, and transfected MSC by static and perfusion method, were studied following the implantation into the back subcutis of rats in terms of histological and biochemical examinations, homogeneous bone formation was histologically observed throughout the sponges seeded with cationized gelatin-plasmid DNA of BMP-2 complex and transfected MSC by perfusion method, although the extent of bone formation was higher for the later one. The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of sponges seeded with transfected MSC by perfusion method were significantly high compared with those seeded with other agents. We conclude that combination of plasmid DNA-impregnated PGA-reinforced collagen sponge and the perfusion method was promising to promote the in vitro gene expression for MSC and in vivo ectopic bone formation.
authors.affiliation: National Institute for Materials Science (NIMS), Nano and Biomaterial Research Building, International Center for Young Scientists (ICYS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan.
authors.forename: Hossein
authors.initials: H
authors.lastname: Hosseinkhani
authors.forename: Masaya
authors.initials: M
authors.lastname: Yamamoto
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Inatsugu
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Hiraoka
authors.forename: Sachiko
authors.initials: S
authors.lastname: Inoue
authors.forename: Hitoyata
authors.initials: H
authors.lastname: Shimokawa
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Tabata
mesh_terms: D000818:Animals; D001672:Biocompatible Materials; D019149:Bioreactors; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D003094:Collagen; D004247:DNA / Q000008:administration & dosage / Q000378:metabolism*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000502:physiology; D009999:Ossification, Heterotopic / Q000235:genetics* / Q000378:metabolism*; D010477:Perfusion; D010957:Plasmids / Q000008:administration & dosage / Q000378:metabolism*; D011100:Polyglycolic Acid; D051381:Rats; D011916:Rats, Inbred F344; D046509:Tissue Culture Techniques; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology
chemical_list: D001672:Biocompatible Materials; C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D016212:Transforming Growth Factor beta; D011100:Polyglycolic Acid; D003094:Collagen; D004247:DNA