title: Osteogenic differentiation of human periodontal ligament cells after transfection with recombinant lentiviral vector containing follicular dendritic cell secreted protein.
abstract: OBJECTIVE
  Follicular dendritic cell secreted protein (FDC-SP), has been identified in human periodontal ligament (PDL) in a recent study. It is suggested that the expression of FDC-SP might be associated with the osteogenic differentiation and mineralization of human periodontal ligament cells (hPDLCs). However, the intrinsic mechanism regarding this is still unclear. The aim of this study was to establish hPDLCs with safe and efficient overexpression of FDC-SP and to elucidate the influence of FDC-SP transfection on hPDLC osteogenesis in periodontal regeneration.
  
  
  METHODS
  We first applied a recombinant lentiviral vector containing FDC-SP to transfect hPDLCs via different multiplicity of infection (MOI) levels (1, 10, 20, 50 and 100). Western blot was performed to confirm the expression of FDC-SP. MTT assay was employed to evaluate the proliferation status of transfected cells. Then, the extent of osteogenic differentiation was investigated by simultaneous monitoring of alkaline phosphatase (ALP) activity assessment, immunofluorescent staining, the expression patterns of osteoblastic markers and mineralization staining.
  
  
  RESULTS
  We found that hPDLCs transfected via MOI 20, 50 and 100 exhibited expression of FDC-SP protein compared with MOI 1 and 10. There was no significant effect of FDC-SP transfection (at different MOI levels of 1, 10 and 20) on the proliferation of hPDLCs, whereas higher MOI levels (50 and 100) inhibited cell proliferation ability. In addition, ALP activity decreased significantly in FDC-SP-transfected hPDLCs at day 7. When stained with alizarin red, cells overexpressing FDC-SP formed less mineralized nodules at 21 d post-induction of differentiation, compared with the control cultures. Osteogenic inhibition was also confirmed by ALP immunostaining. Moreover, mRNA expression levels of osteoblastic markers decreased after FDC-SP transfection, which were in accordance with western blot results.
  
  
  CONCLUSIONS
  Our data suggest that MOI 20 is optimal to transfect hPDLCs, which achieves safe and efficient overexpression of FDC-SP in transfected cells. Moreover, FDC-SP overexpression inhibits osteogenic differentiation of hPDLCs. The present study contributes to a better understanding of the biological functions governing FDC-SP-induced hPDLC differentiation.
authors.affiliation: State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China; Dental Implant Center, West China Hospital of Stomatology, Sichuan University, Chengdu, China.
authors.forename: L
authors.initials: L
authors.lastname: Xiang
authors.forename: L
authors.initials: L
authors.lastname: Ma
authors.forename: Y
authors.initials: Y
authors.lastname: He
authors.forename: N
authors.initials: N
authors.lastname: Wei
authors.forename: P
authors.initials: P
authors.lastname: Gong
mesh_terms: D000293:Adolescent; D000469:Alkaline Phosphatase / Q000032:analysis; D000880:Anthraquinones; D015153:Blotting, Western; D002113:Calcification, Physiologic / Q000235:genetics / Q000502:physiology; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D002648:Child; D024042:Collagen Type I / Q000032:analysis; D004396:Coloring Agents; D050659:Core Binding Factor Alpha 1 Subunit / Q000032:analysis; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D010006:Osteoblasts / Q000166:cytology / Q000502:physiology; D010012:Osteogenesis / Q000235:genetics / Q000502:physiology*; D010513:Periodontal Ligament / Q000166:cytology*; D011506:Proteins / Q000235:genetics / Q000502:physiology*; D012038:Regeneration / Q000235:genetics / Q000502:physiology; D000076182:Sp7 Transcription Factor; D014157:Transcription Factors / Q000032:analysis; D014162:Transfection
chemical_list: D000880:Anthraquinones; D024042:Collagen Type I; D004396:Coloring Agents; D050659:Core Binding Factor Alpha 1 Subunit; C465580:FDCSP protein, human; D011506:Proteins; C493709:RUNX2 protein, human; D000076182:Sp7 Transcription Factor; C478163:SP7 protein, human; D014157:Transcription Factors; C010078:alizarin; D000469:Alkaline Phosphatase
keywords: follicular dendritic cell secreted protein; human periodontal ligament cells; lentiviral vector; osteogenic differentiation