浓缩生长因子联合骨髓间充质干细胞膜片的生物学性能及其在骨缺损修复中的作用

doi:10.13481/j.1671-587X.20240607

Abstract

Abstract:

Objective To discuss the effect of concentrated growth factor （CGF） on the performance of bone marrow mesenchymal stem cells （BMSCs） sheets, and to clarify the role of CGF-containing composite cell sheets（CS） in the bone defect repairment. Methods In in vitro experiments, the BMSCs were isolated and cultured from two 3-week-old SD rats； Alizarin Red S and Oil Red O staining were used to identify the osteogenic and adipogenic capabilities of BMSCs； CGF liquid extracts （CGFe） was prepared from three 3-week-old SD rats. The cells were divided into control group, traditional CS （BMSC-CS） group, and CGF-containing composite CS （CGF/BMSC-CS） group. The morphology of the CS in two groups was observed by HE staining. Alizarin Red and alkaline phosphatase （ALP） staining were used to detect the osteogenic differentiation of the CS in various groups； cell scratch assay was used to detect the migration abilities of the cells in various groups； real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the mRNA expression levels of ALP, collagen are type 1 (COL-1), Runt-related transcription factor 2 （RUNX2）, and osteocalcin (OCN) in the cells in various groups. In in vivo experiments, 15 SD rats were randomly divided into control group, BMSC-CS group, and CGF/BMSC-CS group; micro computed tomography （Micro-CT） was used to detect the bone formation parameters in skull defects of the rats in various groups; HE staining and Masson staining were used to observe the morphology of skull defect tissue of the rats in various groups. Results The third-generation BMSCs were spindle-shaped, closely arranged, and grew in a vortex cluster. The Alizarin red staining results showed obvious calcium nodules, and the Oil red O staining showed red lipid droplets, confirming the cells’ ability to undergo osteogenic and adipogenic differentiation. The CS were white and semi-transparent, with slightly curled edges. The peeled CS were irregularly curled and wrinkled. Compared with BMSC-CS group, the CS in CGF/BMSC-CS group were whiter, less transparent, significantly increased in thickness and extensibility, less prone to breakage, and had a certain degree of stickiness and plasticity. The HE staining results showed that compared with BMSC-CS group, the number of the cells of CS in CGF/BMSC-CS group was increased, with denser arrangement and more abundant extracellular matrix (ECM), which wrapped and connected the cells to form an integral sheet-like structure. The Alizarin red and ALP staining results showed that compared with control group, the ALP activity and mineralization uplift value of CS in BMSC-CS group were significantly increased （P<0.05）； compared with control group and BMSC-CS group, the number of osteoblasts and red mineralized nodules in the CS in CGF/BMSC-CS group was significantly increased, with obvious deepening of the staining, increased positive area, and the ALP activity and mineralization uplift value were significantly increased （P<0.05）. Compared with BMSC-CS group， the ALP activity and mineralization uplif value of the CS in CGF/DMSC-CS group were increased （P<0.05）. The cell scratch assay results showed that after 24 of culture, compared with control group, the migration rates of the cells in BMSC-CS group and CGF/BMSC-CS group were significantly increased (P<0.05). Compared with BMSC-CS group, the migration rate of the cells in CGF/BMSC-CS group was significantly increased (P<0.01). After 48 h of culture, compared with control group, the migration rate of the cells in CGF/BMSC-CS group was significantly increased （P<0.05). The RT-qPCR results showed that compared with control group, the expression levels of COL-1 and OCN mRNA in the cells in BMSC-CS group were significantly increased (P<0.01), and the expression levels of ALP, COL-1, OCN, and RUNX2 mRNA in the cells in CGF/BMSC-CS group were significantly increased (P<0.01). Compared with BMSC-CS group, the expression levels of ALP, COL-1, and OCN mRNA in the cells in CGF/BMSC-CS group were significantly increased(P<0.01). The Micro-CT detection results showed that in control group, the boundary of the rat skull defect area was clear, with almost no new bone formation. In BMSC-CS group, a small amount of new bone formed only at the edge of the bone defect in skull of the rats, with a significant gap in the central area of the defect. In CGF/BMSC-CS group, new bone formed along the edge of the bone defect towards the central area in skull of the rats, repairing most of the bone defect. Compared with control group, the bone volume (BV) and trabecular number (Tb.N) of the rats in BMSC-CS group were significantly increased (P<0.05)； the bone volume (BV), bone volume fraction [BV/tissue volume (TV)], trabecular thickness (Tb.Th), and trabecular number (Tb.N) in skull of the rats in CGF/BMSC-CS group,were significantly increased (P<0.05). Compared with BMSC-CS group, the BV, BV/TV, Tb.Th, and Tb.N in skull of the rats in CGF/BMSC-CS group were significantly increased (P<0.01). The HE and Masson staining observation showed that in control group, almost no new bone formed in the skull defect tissue of the rats, with only a large amount of collagen fibers connecting the two sides of the bone ends. In BMSC-CS group, a small amount of new bone formed only at the edge of the bone defect in skull tissue of the rats, with the central area of the defect containing dense collagen fibers connected to the newly formed bone at the defect edge. In CGF/BMSC-CS group, new bone tissue could be seen at the edge of the bone defect, and bone islands formed in the central area of the defect, surrounded by osteocytes and a large amount of collagen fibers. The Masson staining observation results showed that the cytoplasm and osteoid were red, and the collagen was blue. In CGF/BMSC-CS group, newly formed osteoid was observed in skull defect tissue of the rats, with the highest amount of new bone formation. Conclusion CGF can promote the osteogenic differentiation and increase the richness of ECM in BMSCs sheets. CGF-containing composite CS can efficiently repair skull defects of the rats and serve as an ideal and safe material for promoting the bone regeneration.

Key words: Concentrated growth factor, Cell sheet, Bone marrow mesenchymal stem cell, Bone defect regeneration and repairment

CLC Number:

Jianhong SHI,Yuanye TIAN,Kai CHEN,Gao SUN,Guomin WU. Biological properties of concentrated growth factor combined with bone marrow mesenchymal stem cell sheet and its effect on bone defect repairment[J].Journal of Jilin University(Medicine Edition), 2024, 50(6): 1535-1546.

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References 25

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Gene	Primer sequence
GAPDH	F 5'-AAGTTCAACGGCACAGTCAAGG-3'
GAPDH	R 5'-GACATACTCAGCACCAGCATCAC-3'
ALP	F 5'-AGACTCTCAGGAGGCATAGACTTC-3'
ALP	R 5'-GCACAGGTTGGCGGCTTC-3'
RUNX2	F 5'-TCGTCAGCGTCCTATCAGTTCCR-3'
RUNX2	R 5'-CTTCCATCAGCGTCAACACCATC-3'
OCN	F 5'-GACCCTCTCTCTGCTCACTCTG-3'
OCN	R 5'-CACCACCTTACTGCCCTCCTG-3'
COL-1	F 5'-TGGTCCTGCTGGCAAGAATGG-3'
COL-1	R 5'-TCTGTCACCTTGTTCGCCTGTG-3'

Group	Activity of ALP	Mineralisation uplift value
Control	1.00±0.02	1.00±0.06
BMSC-CS	1.14±0.03^*	1.37±0.05^*
CGF/BMSC-CS	1.42±0.10^*△	2.00±0.17^*△

Group	Migration rate
Group	(t/h) 24	48
Control	19.29±1.40	91.56±2.00
BMSC-CS	36.90±1.94^*	95.39±1.28
CGF/BMSC-CS	47.61±3.15^*△	96.84±1.56^*

Group	ALP	COL-1	RUNX-2	OCN
Control	1.00±0.11	1.00±0.07	1.00±0.11	1.00±0.10
BMSC-CS	1.09±0.17	1.25±0.06^*	1.07±0.11	1.23±0.16^*
CGF/BMSC-CS	2.82±0.67^*△	1.81±0.19^*△	1.21±0.12^*	1.77±0.23^*△

Group	BV（V/mm³）	BV/TV (η/%)	Tb.Th (l/mm)	Tb.N（l/mm）
Control	0.20±0.08	2.09±1.06	0.15±0.04	0.13±0.05
BMSC-CS	3.11±0.95	11.71±2.80^*	0.17±0.04	0.70±0.09^*
CGF/BMSC-CS	17.23±1.20^*△	35.80±1.81^*△	0.53±0.02^*△	1.16±0.03^*△

Biological properties of concentrated growth factor combined with bone marrow mesenchymal stem cell sheet and its effect on bone defect repairment

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