Journal of Prevention and Treatment for Stomatological Diseases ›› 2021, Vol. 29 ›› Issue (10): 656-662.doi: 10.12016/j.issn.2096-1456.2021.10.002

• Basic Study • Previous Articles     Next Articles

Effects of graphene on the proliferation, migration and morphology of dental pulp stem cells

SUN Jingxuan(),LI Yanping,PAN Shuang,HE Lina,SUN Xiangyu,ZHANG Shuang,NIU Yumei()   

  1. The First Affiliated Hospital of Harbin Medical University, School of Stomatology, Harbin Medical University, Harbin 150001, China
  • Received:2021-02-10 Revised:2021-04-06 Online:2021-10-20 Published:2021-06-23
  • Contact: Yumei NIU E-mail:2018021032@hrbmu.edu.cn;yumeiniu@163.com
  • Supported by:
    National Natural Science Foundation of China(81970924);Postdoc-toral Fund Project of China(2018M641871)

Abstract:

Objective To investigate the effects of graphene on the proliferation, migration and cell morphology of dental pulp stem cells (DPSCs). Methods Graphene powder was prepared by the oxidation-reduction method, and a 0.5 mg/mL graphene dispersion was prepared. Raman spectroscopy and atomic force microscopy were used to characterize the structure and surface morphology of graphene. DPSCs were isolated and cultured in vitro. MTT assay was used to detect the effects of different concentrations of graphene dispersions (0, 1, 5, 10, 20, 50, 100 μg/mL) on the proliferation and wound healing assay was used to detected the migration abilities of DPSCs. The effects of graphene on the morphology of DPSCs were observed by immunofluorescence staining. Results In the present study, compared with the control group (0 μg/mL), the proliferation of DPSCs in the 100 μg/mL group was inhibited at 72 h (P < 0.05), and the proliferation of DPSCs in the other groups was not significantly affected (P > 0.05). Graphene dispersions at 10 and 20 μg/mL promoted the migration of DPSCs (P < 0.05). After being cultured in 20 μg/mL graphene dispersions for 3 days, the DPSCs showed a large and orderly cytoskeletal structure, and the spread area of cells was not significantly different from that of the control group (0 μg/mL) (P > 0.05), while some cells had the morphological characteristics of nerve cells. Conclusion Graphene has good biocompatibility and is expected to be a suitable material for tissue engineering within fitting concentration.

Key words: graphene, graphene oxide, structure characterization, Raman spectrum, atomic force microscopy, dental pulp stem cells, cell proliferation, cell migration, tissue engineering

CLC Number: 

  • R78

Figure 1

Isolation, culture and identification of dental pulp stem cells a: morphology of primary DPSCs on day 7 (× 100), and long spindle-shaped cells were observed to crawl out around the tissue; b: on the 20th day of primary DPSC culture, the cells were gradually fused (× 100); c: after 14 days of induction of mineralization, a large number of DPSCs were postive with ALP staining (× 200); d: DPSCs were positive for STRO-1 expression. STRO-1: stromal cell antigen; DPSCs: dental pulp stem cells"

Figure 2

Raman spectra of GO and graphene The characteristic D-peak (1 350 cm-1) and G-peak (1 580 cm-1) of graphene and GO can be clearly seen. The G peak represents the characteristic C=C bond on the surface of graphene and GO, while the D peak represents the transformation of the sp2 carbon atom in the aromatic ring of graphene into the sp3 structure after oxidation. After reduction, the ID/IG value of graphene decreases (GO: ID/IG=1.06; Graphene: ID/IG=0.88), which indicates that after reduction, part of the sp3 structure is reduced to the sp2 structure. GO: graphene oxide"

Figure 3

The surface morphologies of GO and graphene were observed by atomic force microscopy a:GO; b: graphene; GO and graphene mainly exist in the form of monolayers; compared with GO, after reduction, the graphene presents a smoother thin sheet with a more uniform size distribution and a reduced thickness. GO: graphene oxide"

Figure 4

Effects of graphene on the proliferation ability of dental pulp stem cells *: P < 0.05 vs. the control group (0 μg/mL)"

Figure 5

Influence of graphene on the migration ability of DPSCs After 6 h of culture, DPSCs crawled out of the edges of cell scratches in each group. After 12 h, the areas of cell scratches decreased in all groups. After 24 h, the areas of cell scratches decreased significantly, and some areas in the 10 and 20 μg/mL groups had fused. **P < 0.01, ***P < 0.001, compared with the control group (0 μg/mL); DPSCs: dental pulp stem cells"

Figure 6

Effect of graphene on the morphology of DPSCs (× 100) DPSCs were cultured in a 20 μg/mL graphene dispersion for 3 days, and the spindle cell body of the experimental group contracted and transformed into a round cell body. The protrusion became longer and had a nerve cell-like morphology, while the morphology of cells in the 0 μg/mL group did not change significantly. DPSCs: dental pulp stem cells"

Figure 7

Effect of graphene on the morphology of DPSCs a: actin and nuclear staining(× 200), after 3 days of culture, the cells in the 20 μg/mL graphene dispersion group had a large spreading area and high luciferin density, with a thick and orderly cytoskeleton structure, and some cells showed long synapses and polygonal morphology; b: fluorescence areas were compared between the experimental group and the control group (0 μg/mL); DPSCs: dental pulp stem cells"

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