Stimulation of reparative dentin formation by ex vivo gene therapy using dental pulp stem cells electrotransfected with growth/differentiation factor 11 (Gdf11).
Dental pulp progenitor/stem cells have the capacity to differentiate into odontoblasts and they provide a potential for dentin repair and regeneration by gene therapy. To develop a successful ex vivo gene therapy to induce reparative dentin formation rapidly and effectively after treatment of caries, we developed a three-dimensional pellet culture system of pulp cells electrotransfected with growth/differentiation factor 11 (Gdf11). The viability after electrotransfection was more than 85%, and the efficiency was about 70% as determined by flow cytometry. After 10 days of culture, the total amount of type I and type III collagen was 3-fold higher in the pEGFP-Gdf11-transfected pellet than in the control. Real-time RT-PCR analysis demonstrated that the expression of markers of odontoblast differentiation (alkaline phosphatase, dentin matrix protein 1 [Dmp1], dentin sialophosphoprotein [Dspp], enamelysin, and phosphate-regulating gene with homologies to endopeptidases on X-chromosome [Phex]) was increased in the pEGFP-Gdf11-transfected pellet compared with the control on day 14. On the basis of this in vitro evaluation, an in vivo investigation in the dog was performed. Autogenous transplantation of Gdf11-transfected cells cultured as a pellet on amputated pulp stimulated reparative dentin formation. Thus, Gdf11 gene therapy may be potentially used in endodontic treatment in dentistry.
Nakashima
Misako
M
Division of Oral Rehabilitation, Department of Clinical Oral Molecular Biology, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan. misako@dent.kyushu-u.ac.jp
Iohara
Koichiro
K
Ishikawa
Masaki
M
Ito
Masataka
M
Tomokiyo
Atsushi
A
Tanaka
Takamasa
T
Akamine
Akifumi
A
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000235:genetics*; D002417:Cattle; D002454:Cell Differentiation; D049109:Cell Proliferation; D002470:Cell Survival; D024042:Collagen Type I / Q000378:metabolism; D024061:Collagen Type III / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000378:metabolism; D003782:Dental Pulp / Q000166:cytology*; D003804:Dentin / Q000737:chemistry* / Q000502:physiology*; D004285:Dogs; D018274:Electroporation; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D055412:Growth Differentiation Factors; D018613:Microscopy, Confocal; D009804:Odontoblasts / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D012038:Regeneration; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology*; D013552:Swine; D013997:Time Factors; D014070:Tooth / Q000166:cytology*; D014162:Transfection; D019076:Transgenes / Q000235:genetics
D019485:Bone Morphogenetic Proteins; D024042:Collagen Type I; D024061:Collagen Type III; D017931:DNA Primers; D018076:DNA, Complementary; C492266:GDF11 protein, human; D055412:Growth Differentiation Factors; D000469:Alkaline Phosphatase