Journal of Jilin University(Medicine Edition) ›› 2022, Vol. 48 ›› Issue (4): 954-961.doi: 10.13481/j.1671-587X.20220415

• Research in basic medicine • Previous Articles     Next Articles

Effect of TGF-β3-loaded methacrylated heparin on osteogenic differentiation of dental pulp stem cells and its mechanism

Xinying ZOU,Shuang GAO,Hong ZHAO,Xin LIU,Yuanhang ZHAO,Jiazhuo SONG,Linlin YAN,Zhimin ZHANG()   

  1. Department of Endodontics,Stomatology Hospital,Jilin University,Changchun 130021,China
  • Received:2021-10-26 Online:2022-07-28 Published:2022-07-26
  • Contact: Zhimin ZHANG E-mail:zhangzhim@jlu.edu.cn

Abstract:

Objective: To investigate the promotion effect of transforming growth factor β3(TGF-β3)-loaded methacrylamide heparin(HepMA) on the osteogenic differentiation of dental pulp stem cells(DPSCs), and to elucidate its possible mechanism.

Methods

The hDPSCs were extracted and cultured in vitro and identified by flow cytometry. A light-cured HepMA hydrogel was synthesized, and the surface morphology of the material was observed by scanning electron microscope.The HepMA loaded with TGF-β3 (TGF-β-HepMA) at different concentrations (20, 40, 60, 80 and 100 μg?L-1) was synthesized and its extract was prepared to culture the hDPSCs for 1, 3 and 5 d; meanwhile, control group was established, and the optimal concentration of TGF-β3 loaded was screened out by CCK-8 method. Enzyme-linked immunosorbent assay (ELISA) was used to detect the cumulative release amounts of TGF-β3 in TGF-β-HepMA at different time points and the drug release curve was drawn.The HDPSCs were divided into control group, HepMA group and HE PMA group loaded with the optimal concentration of TGF-β3 (TGF-β3-HepMA). After being cultured for 24 h in the medium containing the corresponding extracts, the osteogenic induction solution was prepared to induce hHDPSCs for 7, 14 and 21 d, respectively. ALP staining and Alizarin red staining were used to detect the cell ALP staining and calcium nodule formation in each group. Real-time fluorescentce quantitative PCR(RT-qPCR) was used to detect the mRNA expression levels of osteogenic related factors in the cells in each group.

Results

The hDPSCs isolated and cultured were spindle-shaped under light microscope.The flow cytometry results showed positive expressions of CD105 and CD90 and negative expressions of CD34 and CD45 in the cultured hDPSCs, confirming that the cells isolated and cultured in this experiment were the hDPSCs. The scanning electron microscope (SEM) observation showed that the average pore size of HepMA was 50-70 μm. The ELISA results showed that TGF-β3 released slowly after a sudden release phase within 7 d and reached the equilibrium around 21 d.The CCK8 method results showed that compared with 0 μg?L-1 TGF-β3-HepMA group, the proliferation rate of hDPSCs in 60 μg?L-1 TGF-β 3-HEPMA group was increased(P<0.05), so the optimal concentration of TGF-β3 loaded was 60 μg?L-1. Compared with control group and HepMA group, the ALP staining area in tTGF-β3-HepMA group was the largest and the most stained in color when osteogenesis induction for 7 and 14 d, and the ALP staining in cells on the 14th day was more significant than that on the 7th day. Compared with control group and the HepMA group, the mineralized calcium nodule area stained with Alizarin red was the largest in TGF-β3-HepMA group at 21 d of osteogenic induction. The RT-qPCR results showed that compared with control group, the mRNA expression levels of Runx2, ALP, OCN and COL-Ⅰ in the cells in HepMA and TGF-β3-HepMA groups were significantly increased after 7 and 14 d of culture(P<0.05 or P<0.01); compared with HepMA group, the mRNA expression levels of Runx2, ALP, OCN and COL-Ⅰ in the cells in TGF-β3-HepMA group were significantly increased (P<0.05).

Conclusion

HepMA loading TGF-β3 can improve the osteogenic differentiation of hDPSCs, and its mechanism may be related to the up-regulation of the expressions of osteogenic related factors.

Key words: Transforming growth factor-beta 3, Dental pulp stem cells, Methyl acryloyl heparin, Osteogenic differentiation

CLC Number: 

  • R782