Growth differentiation factor-5 modified by bisphosphonate promotes osteogenic differentiation of MC3T3-E1 cells

doi:10.12307/2023.981

Chinese Journal of Tissue Engineering Research ›› 2024, Vol. 28 ›› Issue (3): 373-379.doi: 10.12307/2023.981

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Growth differentiation factor-5 modified by bisphosphonate promotes osteogenic differentiation of MC3T3-E1 cells

Li Lisi1, 2, 3, Zhang Chengdong2, Li Xiaolong2, Ye Ziyu3, Pu Chao2, Yang Zaijun1, 3, Shi Feng1, 3, Xiao Dongqin2

1Organization and Repair Materials Engineering Technology Collaborative Innovation Center, West China Normal University, Nanchong 637000, Sichuan Province, China; 2Second Clinical College of North Sichuan Medical College·Institute of Tissue Engineering and Stem Cells of Nanchong Central Hospital, Nanchong 637000, Sichuan Province, China; 3College of Life Sciences, West China Normal University, Nanchong 637000, Sichuan Province, China

Received:2022-12-19 Accepted:2023-02-11 Online:2024-01-28 Published:2023-07-10
Contact: Yang Zaijun, PhD, Professor, Organization and Repair Materials Engineering Technology Collaborative Innovation Center, West China Normal University, Nanchong 637000, Sichuan Province, China; College of Life Sciences, West China Normal University, Nanchong 637000, Sichuan Province, China Shi Feng, PhD, Associate professor, Organization and Repair Materials Engineering Technology Collaborative Innovation Center, West China Normal University, Nanchong 637000, Sichuan Province, China; College of Life Sciences, West China Normal University, Nanchong 637000, Sichuan Province, China Xiao Dongqin, PhD, Associate researcher, Second Clinical College of North Sichuan Medical College·Institute of Tissue Engineering and Stem Cells of Nanchong Central Hospital, Nanchong 637000, Sichuan Province, China
About author:Li Lisi, Master candidate, Organization and Repair Materials Engineering Technology Collaborative Innovation Center, West China Normal University, Nanchong 637000, Sichuan Province, China; Second Clinical College of North Sichuan Medical College·Institute of Tissue Engineering and Stem Cells of Nanchong Central Hospital, Nanchong 637000, Sichuan Province, China; College of Life Sciences, West China Normal University, Nanchong 637000, Sichuan Province, China
Supported by:
National Natural Science Foundation of China, No. 82002289 (to XDQ); Natural Science Foundation of Sichuan Province, No. 2022NSFSC0685 (to XDQ); Natural Science Foundation of Sichuan Province, No. 23NSFSC3371 (to ZCD); Nanchong City-School Cooperative Scientific Research Project. No. 22SXJCQN0002 (to XDQ); Nanchong City-School Cooperative Scientific Research Project. No. 22SXQT0385 (to PC)

Abstract

Abstract: BACKGROUND: As a member of bone morphogenetic proteins, growth differentiation factor-5 shows promising potential in the application of cartilage and bone repair. The affinity of growth differentiation factor-5 onto bone tissue determines protein use efficiency, so it is of great significance to prepare growth differentiation factor-5 with bone targeting capability.
OBJECTIVE: To modify growth differentiation factor-5 using bisphosphonates and investigate the effects of modified protein on the growth of preosteoblasts in mice.
METHODS: Pamidronate disodium/growth differentiation factor-5 complex was prepared using chemical crosslinking to couple growth differentiation factor-5 with pamidronate disodium. The functional groups and structures of the complex were characterized using Fourier transform infrared spectroscopy and circular dichromatography. To determine the bone targeting in vitro, the binding of the modified growth differentiation factor-5 with calcium phosphate and in vitro release amount of growth differentiation factor-5 were measured with an ELISA kit. Growth differentiation factor-5 (control group) and the pamidronate disodium/growth differentiation factor-5 complex (experimental group) were co-cultured with preosteoblasts MC3T3-E1. Individually cultured cells were blank controls. The effect of the complex on cell proliferation and differentiation was evaluated.
RESULTS AND CONCLUSION: (1) The infrared spectroscopy and circular dichromatography results indicated that the bisphosphonate/growth differentiation factor-5 complex was successfully prepared without significant changes in the protein secondary structure. In vitro protein adsorption results showed that growth differentiation factor-5 adsorption on calcium phosphate was increased by about one time after coupling with a bisphosphonate. In the presence of cysteine, growth differentiation factor-5 could be released from the bisphosphonate/growth differentiation factor-5 complex. (2) CCK-8 assay results showed that the absorbance value of the experimental group cultured for 4 and 7 days was higher than that of the control group and blank control group (P < 0.000 1).
After 7 days of culture, the expression of alkaline phosphatase in the experimental group was significantly higher than that in the control group and blank control group (P < 0.000 1). After 13 days of culture, the content of calcium nodules in the experimental group was significantly higher than that in the control group and the blank control group (P < 0.000 1). The results of qRT-PCR showed that the mRNA expression of alkaline phosphatase, osteocalcin and Runx2 in the experimental group was higher than that in the control group and the blank control group after 7 days of culture (P < 0.01, P < 0.001, P < 0.000 1). (3) These findings exhibit that bisphosphonate modification can enhance the binding capacity of growth differentiation factor-5 to calcium phosphate as well as improve its biological activity.

Key words: growth differentiation factor-5, bisphosphonate, release, MC3T3-E1 cell, proliferation, differentiation

CLC Number:

Li Lisi, Zhang Chengdong, Li Xiaolong, Ye Ziyu, Pu Chao, Yang Zaijun, Shi Feng, Xiao Dongqin. Growth differentiation factor-5 modified by bisphosphonate promotes osteogenic differentiation of MC3T3-E1 cells[J]. Chinese Journal of Tissue Engineering Research, 2024, 28(3): 373-379.

Figures/Tables 10

References

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Growth differentiation factor-5 modified by bisphosphonate promotes osteogenic differentiation of MC3T3-E1 cells

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