title: Enhancement of periodontal tissue regeneration by transplantation of osteoprotegerin-engineered periodontal ligament stem cells.
abstract: BACKGROUND
  The objective of the present study was to evaluate the capacity of a tissue-engineered complex of human osteoprotegerin (hOPG)-transfected periodontal ligament stem cells (PDLSCs) seeding on beta-tricalcium phosphate (β-TCP) to regenerate alveolar bone defects in New Zealand rabbits.
  
  
  METHODS
  PDLSCs were isolated from rabbit periodontal ligament tissues and expanded in vitro to enrich PDLSC numbers, and their proliferative activities and differentiation capability were evaluated under specific induction conditions. Lentiviral vector containing hOPG and enhanced green fluorescent protein (EGFP) was constructed by using Gateway technology and transfected into rabbit PDLSCs. The expression of hOPG was determined with quantitative real-time reverse transcription-polymerase chain reaction and Western blot. The PDLSCs with or without engineered hOPG were seeded on β-TCP scaffolds prior to transplantation. Morphological characterization of cells and materials was done by scanning electron microscope. Twenty rabbits with alveolar bone defects were randomly allocated into four groups and transplanted with β-TCP, PDLSCs/β-TCP, and hOPG-transfected PDLSCs/β-TCP or were left untreated as a control. Animals were sacrificed 12 weeks after operation for histological observation and histomorphometric analysis.
  
  
  RESULTS
  PDLSCs expressed STRO-1 and vementin and favored osteogenesis and adipogenesis in conditioned media. Expressions of hOPG were significantly upregulated after transfection of the lentiviral vector into PDLSCs. PDLSCs attached and spread well on β-TCP, and there was no significant difference in growth of PDLSCs on β-TCP between the hOPG transfection group and the non-transfection group. The histological observation and histomorphometric analysis showed that the hOPG-transfected PDLSCs/β-TCP complex exhibited an earlier mineralization and more bone formation inside the scaffold than control, β-TCP, and PDLSCs/β-TCP complexes. Implantation of hOPG-transfected PDLSCs contributed to new bone formation as determined by EGFP gene expression under circularly polarized light microscopy.
  
  
  CONCLUSIONS
  The present study demonstrated the feasibility of β-TCP scaffolds for primary PDLSC culture and expression of hOPG gene in vitro and in vivo, and hOPG-transfected PDLSCs could serve as a potential cell source for periodontal bone regeneration, which may shed light on the potential of systemic hOPG gene therapy in combination with PDLSC tissue engineering as a good candidate in periodontal tissue engineering for alveolar bone regeneration.
authors.affiliation: Institute of Stomatology, General Hospital of Chinese PLA, No. 28, Fuxing Road, Haidian District, Beijing, 100853, China. fangsu6513@163.com.|Department of Stomatology, The 306th Hospital of Chinese PLA, No.9 Anxiang Beili, Chaoyang District, Beijing, 100101, China. fangsu6513@163.com.
authors.forename: Fang
authors.initials: F
authors.lastname: Su
authors.affiliation: Institute of Stomatology, General Hospital of Chinese PLA, No. 28, Fuxing Road, Haidian District, Beijing, 100853, China. shisenliu00@163.com.|Department of Stomatology, Navy General Hospital of Chinese PLA, No. 6 Fucheng Road, Haidian District, Beijing, 100048, China. shisenliu00@163.com.
authors.forename: Shi-Sen
authors.initials: SS
authors.lastname: Liu
authors.affiliation: Institute of Stomatology, General Hospital of Chinese PLA, No. 28, Fuxing Road, Haidian District, Beijing, 100853, China. junlima91@163.com.
authors.forename: Jun-Li
authors.initials: JL
authors.lastname: Ma
authors.affiliation: Institute of Stomatology, General Hospital of Chinese PLA, No. 28, Fuxing Road, Haidian District, Beijing, 100853, China. dongshengwang65@163.com.
authors.forename: Dong-Sheng
authors.initials: DS
authors.lastname: Wang
authors.affiliation: Institute of Stomatology, General Hospital of Chinese PLA, No. 28, Fuxing Road, Haidian District, Beijing, 100853, China. linglinge87@163.com.
authors.forename: Ling-Ling
authors.initials: LL
authors.lastname: E
authors.affiliation: Institute of Stomatology, General Hospital of Chinese PLA, No. 28, Fuxing Road, Haidian District, Beijing, 100853, China. hongchenliu19@163.com.
authors.forename: Hong-Chen
authors.initials: HC
authors.lastname: Liu
mesh_terms: D016301:Alveolar Bone Loss / Q000628:therapy*; D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism; D001861:Bone Regeneration / Q000502:physiology*; D002130:Calcium Phosphates / Q000627:therapeutic use; D002454:Cell Differentiation; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins / Q000235:genetics; D016557:Guided Tissue Regeneration, Periodontal / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D010012:Osteogenesis / Q000187:drug effects / Q000502:physiology; D053244:Osteoprotegerin / Q000378:metabolism; D010510:Periodontal Diseases / Q000473:pathology / Q000628:therapy; D010513:Periodontal Ligament / Q000166:cytology*; D010519:Periodontium / Q000473:pathology; D011817:Rabbits; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology; D023822:Tissue Engineering / Q000379:methods; D054457:Tissue Scaffolds
chemical_list: D000954:Antigens, Surface; D002130:Calcium Phosphates; D053244:Osteoprotegerin; C485817:beta-tricalcium phosphate; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins