Journal of Prevention and Treatment for Stomatological Diseases ›› 2021, Vol. 29 ›› Issue (2): 81-87.doi: 10.12016/j.issn.2096-1456.2021.02.002

• Basic Study • Previous Articles     Next Articles

Exosomes derived from lipopolysaccharide-preconditioned dental folic cells regulate osteogenic differentiation of periodontal ligament cell in periodontitis

SHI Weiwei1,2,3(),DING Yi3,TIAN Weidong1,2,4,GUO Shujuan1,2,3()   

  1. 1. Engineering Research Center of Oral Translational Medicine, Ministry of Education, Sichuan University, Chengdu 610041, China
    2. National Engineering Laboratory for Oral Regenerative Medicine, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
    3. State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases, Department of Periodontics, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
    4. State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases, Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
  • Received:2020-02-19 Revised:2020-04-28 Online:2021-02-20 Published:2020-12-21
  • Contact: Shujuan GUO E-mail:494808794@qq.com;guo.shujuan@yahoo.com
  • Supported by:
    grants from National Key Research and Development Program of China(2017YFA0104800)

Abstract:

Objective The purpose of this study was to investigate the effect of different concentrations of exosomes (Exos) secreted from dental folic cells (DFCs) preconditioned with lipopolysaccharide (LPS) on the osteogenic differentiation ability of periodontal cells in periodontitis (p-PDLCs) in patients to provide a basis for the prevention and treatment of periodontal disease. Method Tissue block and enzyme digestion methods were used to culture DFCs and p-PDLCs. Exosomes were isolated from 250 ng/mL LPS-preconditioned DFCs 24 h later. The characteristics of exosomes were detected by transmission electron microscopy, particle size analysis and Western blotting. The effects of 10 μg/mL and 100 μg/mL exosomes on the osteogenic differentiation of p-PDLCs were detected by RT-PCR and Alizarin red staining. Results LPS-pretreated DFC-derived exosomes (L-Exos) are vesicle-like structures with a size between 30-100 nm that positively express CD63 and Alix. Compared with the control group, exosomes significantly upregulated Periostin, Col Ⅰ, and Col Ⅲ expression at 100 μg/mL (P < 0.05), while TGF- β1 was significantly upregulated at 10 μg/mL (P < 0.01). At 7 days after osteogenic induction, mineralized nodules were significantly more abundant in the exosome group than in the control group (P < 0.01), and the results were better at a concentration of 100 μg/mL (P < 0.01). Conclusion 100 μg/mL L-Exos are better than 10 μg/mL L-Exos in enhancing the osteogenic differentiation ability of p-PDLCs.

Key words: lipopolysaccharide, dental folic cells, periodontal ligament cells, exosomes, osteogenic differentiation, collagen fiber, mineralization, periodontal regeneration

CLC Number: 

  • R781

Table 1

Primer sequence"

Gene Forward Reverse
Periostin 5′-CACTCTTTGCTCCCACCAATA-3′ 5′-ATTTCCTTCCAGCGTCTCAA-3′
Col Ⅰ 5′-AACATGGAGACTGGTGAGACCT-3′ 5′-CGCCATACTCGAACTGGAATC-3′
Col Ⅲ 5′-TGGAGGATGGTTGCACGAAA-3′ 5′-ACAGCCTTGCGTGTTCGATA-3′
TGF-β1 5′-CACGTGGAGCTGTACCAGAA-3′ 5′-GAACCCGTTGATGTCCACTT-3′
GAPDH 5′-GTTTGGTATCGTGGAAGGACTC-3′ 5′-GTAGAGGCAGGGATGATGTTCT-3′

Figure 1

The cell culture of DFCs and p-PDLCs a:dental folic cells(DFCs);b: periodontal ligament cells of periodontitis(p-PDLCs) ; bar=500 μm"

Figure 2

Flow cytometry analysis for DFCs DFCs positively expressed CD73, CD90 and CD146, while negatively expressed CD31, CD106 and CD34; DFCs: dental folic cells"

Figure 3

Identification of exosomes a: transmission electron microscopy detected that L-Exos were vesicle-like structures; b: nanoparticle tracking analysis determined that the size distribution of L-Exos was mainly approximately 100 nm; c: surface markers of exosomes (CD63, Alix) and cell markers (Actin) were detected by Western blotting; L-Exos: LPS-pretreated DFC-derived exosomes"

Figure 4

The expression of L-Exos-induced p-PDLC osteogenic and adhesion-related genes was detected by RT-PCR ColⅠ: CollagenⅠ; Col Ⅲ: Collagen Ⅲ; TGF-β1: transforming growth factor-β1; L-Exos: LPS pretreated DFCs derived exosome; p-PDLCs: periodontal ligament cells of periodontitis; **: P < 0.01 vs. Control; ##: P < 0.01 vs. 10 μg/mL"

Figure 5

Alizarin red staining and quantitative analysis of L-exos-induced p-PDLC osteogenesis induction after 7 d a: general and microscopic view of Alizarin red staining; b: semiquantitative analysis of Alizarin red staining; L-Exos: LPS-pretreated DFC-derived exosomes; p-PDLCs: periodontal ligament cells of periodontitis; **: P < 0.01 vs. Control; ##: P < 0.01 vs. 10 μg/mL"

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