title: Dickkopf-related protein 3 negatively regulates the osteogenic differentiation of rat dental follicle cells.
abstract: The present study aimed to investigate the effect of Dickkopf-related protein 3 (DKK3) on osteogenic differentiation of rat dental follicle cells (DFCs). A PCR array analysis of Wnt pathway activation in DFCs identified genes dysregulated by mineral induction. Among them, DKK3expression levels were decreased, and further experiments were conducted to investigate its role in DFC osteogenesis. By comparing DFCs grown in normal growth and mineral‑induction media for 4 weeks, the present study confirmed that DKK3 was a potential target gene of osteogenesis through reverse transcription-quantitative polymerase chain reaction (RT‑qPCR) and western blotting (WB). A short hairpin RNA (shRNA) was introduced into DFCs using a lentiviral vector to inhibit DKK3 expression. An alkaline phosphatase (ALP) activity assay and Alizarin Red staining were performed to observe the DKK3‑shRNA DFCs. In addition, the osteogenic differentiation of DKK3‑shRNA DFCs was analyzed by RT‑qPCR and WB. In vivo, DKK3‑shRNA DFCs seeded on hydroxyapatite/β-tricalcium phosphate (HA/TCP) scaffolds were transplanted into the subcutaneous tissue of mice with severe combined immunodeficiency, followed by hematoxylin‑eosin and Masson staining. The results confirmed that DKK3 expression was downregulated during mineral induction in rat DFCs. Lentivirus‑mediated expression of DKK3 shRNA in DFCs promoted calcified‑nodule formation, ALP activity and the expression of β‑catenin, runt‑related transcription factor 2 and osteocalcin, compared with control cells. In vivo, the implanted section presented the majority of newly formed osteoid matrices and collagen, with limited space between the HA/TCP scaffolds and matrices. In conclusion, DKK3 expression negatively regulates the osteogenic differentiation of DFCs and, conversely, downregulation of DKK3 may enhance DFC osteogenesis.
authors.affiliation: Department of Prosthodontics, Guangdong Provincial Key Laboratory of Stomatology, Guanghua School of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China.
authors.forename: Xinchun
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Operative Dentistry and Endodontics, Guangdong Provincial Key Laboratory of Stomatology, Guanghua School of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Du
authors.affiliation: Department of Operative Dentistry and Endodontics, Guangdong Provincial Key Laboratory of Stomatology, Guanghua School of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China.
authors.forename: Junqi
authors.initials: J
authors.lastname: Ling
authors.affiliation: Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Sun Yat‑sen University, Guangzhou, Guangdong 510080, P.R. China.
authors.forename: Weiqiang
authors.initials: W
authors.lastname: Li
authors.affiliation: Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Sun Yat‑sen University, Guangzhou, Guangdong 510080, P.R. China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liao
authors.affiliation: Department of Operative Dentistry and Endodontics, Guangdong Provincial Key Laboratory of Stomatology, Guanghua School of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China.
authors.forename: Xi
authors.initials: X
authors.lastname: Wei
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D003795:Dental Sac / Q000166:cytology* / Q000378:metabolism*; D005260:Female; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D008297:Male; D008903:Minerals / Q000494:pharmacology; D010012:Osteogenesis / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D060449:Wnt Signaling Pathway / Q000187:drug effects
chemical_list: D015415:Biomarkers; C498626:Dkk3 protein, rat; D036341:Intercellular Signaling Peptides and Proteins; D008903:Minerals; D034741:RNA, Small Interfering