Bone sialoprotein gene transfer to periodontal ligament cells may not be sufficient to promote mineralization in vitro or in vivo.
BACKGROUND
To improve regenerative therapies, it is important to understand the cells and factors modulating periodontal tissues. Our group has focused on bone sialoprotein (BSP), a mineralized tissue-selective protein considered to be involved in the initiation of cementogenesis and osteogenesis. In this study, we examined whether gene transfer of BSP into periodontal ligament (PDL) cells would result in an increased ability of PDL cells to promote mineralization in vitro and in vivo.


METHODS
PDL cells obtained from CD-1 mice were immortalized using simian virus (SV) 40 large T antigen (TAg) and designated SV-PDL cells. SV-PDL cells were infected in vitro with LacZ gene-expressing control adenovirus vector. A 1,000 plaque-forming unit (pfu) titer was selected (based on X-gal staining) and cells were infected with mouse BSP-expressing replication-deficient adenoviral vector to determine the mRNA expression and protein level of BSP. Total RNA was isolated from cells on days 2, 4, and 6. Media were obtained on days 3, 5, and 7 for protein determination. Northern blot analysis was performed for mRNA expression and Western blot analysis for protein expression. To test the effect of BSP gene transfer on the mineralization of PDL cells, in vitro (von Kossa) and in vivo (severe combined immunodeficiency [SCID] mice) experiments were performed.


RESULTS
Under normal conditions, PDL cells do not express BSP transcripts and do not promote significant mineralization. SV-PDL cells infected with a BSP viral vector expressed and secreted substantial levels of BSP as confirmed by Northern and Western blot analysis. BSP mRNA and protein levels were strong on day 2 and still apparent on day 6, although not as great. However, no mineral nodule formation was noted either in vitro or in vivo.


CONCLUSIONS
Although BSP is an important and necessary protein for mineralization, it may not be sufficient for promoting mineralization without the addition or removal of other factors. Further studies will help to clarify the specific factors required for promoting mineralization, a required step for designing predictable periodontal regenerative therapies.
Hakki
Sema S
SS
Department of Periodontology, Faculty of Dentistry, Selcuk University, Konya, Turkey. sshakki@selcuk.edu.tr
Wang
Dian
D
Franceschi
Renny T
RT
Somerman
Martha J
MJ
D016475:3T3 Cells; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000957:Antigens, Viral, Tumor / Q000235:genetics; D015152:Blotting, Northern; D015153:Blotting, Western; D002113:Calcification, Physiologic / Q000235:genetics*; D025021:Cementogenesis / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D058574:Integrin-Binding Sialoprotein; D051379:Mice; D008815:Mice, Inbred Strains; D016513:Mice, SCID; D010012:Osteogenesis / Q000235:genetics; D010513:Periodontal Ligament / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D012038:Regeneration / Q000235:genetics; D012795:Sialoglycoproteins / Q000235:genetics* / Q000502:physiology; D013539:Simian virus 40 / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
D000957:Antigens, Viral, Tumor; C546872:Ibsp protein, mouse; D058574:Integrin-Binding Sialoprotein; D012333:RNA, Messenger; D012795:Sialoglycoproteins