Effect of HIF-1α on osteogenic-angiogenic coupling response in BMSCs sheets
10.12016/j.issn.2096-1456.202550104
- Author:
ZHANG Dan
1
;
HUANG Yinli
1
;
TENG Yonghui
1
;
HAN Chang
1
Author Information
1. Department of Orthodontics, Yinchuan Stomatology Hospital
- Publication Type:Journal Article
- Keywords:
hypoxia-inducible factor-1α;
bone marrow mesenchymal stem cells;
cell sheet;
prevascularization;
osteogenic-angiogenic coupling response;
bone tissue engineering;
alkaline phosphatase staining;
alizarin red staining;
osteopontin;
osteocalcin
- From:
Journal of Prevention and Treatment for Stomatological Diseases
2025;33(9):744-756
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the effect of HIF-1α on osteogenic-angiogenic coupling response in bone mesenchymal stem cells (BMSCs) and provide new concepts for engineered bone tissue in vitro.
Methods:With the approval of the hospital’s experimental animal ethics committee, BMSCs were harvested from Wistar rats. The lentivirus carrying hypoxia-inducible factor-1α (HIF-1α) and empty lentivirus were stably transfected into the third generations of BMSCs to form LV-HIF-1α-BMSCs and LV-BMSCs. Meanwhile, BMSCs without transfection of lentivirus were used as a blank control. Then, the effect of HIF-1α transfection was verified by qPCR and Western Blot. LV-HIF-1α-BMSCs were induced to differentiate into endothelium-like cells (iECs). The morphology was observed by optical microscopy, the differentiation rate was detected by cellular flow CD31, and the Transwell test was used to detect the migration ability. At the same time, LV-HIF-1α-BMSCs and LV-BMSCs were continuously cultured to form osteogenic cell sheets (OCTs), which were stained by alkaline phosphatase on day 14 and alizarin red staining on day 21, and counted for mineralization capacity. Finally, iECs were implanted into OCTs to form prevascularized osteogenic cell sheets (P-OCTs), immunofluorescence CD31 was performed to detect the formation of vascular networks, and the results were recorded on days 1, 3, 7, and 14. Meanwhile, osteopontin (OPN) and osteocalcin (OCN) were detected by western blot to verify their ability for osteogenic differentiation on days 1, 7, and 14.
Results:The optimal multiplicity of infection (MOI) for lentiviral transfection was 30, and the transfection efficiency was >80%. The results of qPCR and western blot showed that compared with the LV-BMSCs group and BMSCs group, the LV-HIF-1α-BMSCs group had stable and high expressions of HIF-1α (P<0.05). LV-HIF-1α-BMSCs showed an enhanced ability to differentiate into endothelial cells, with a differentiation rate as high as 91.81%. Transwell assay verified that HIF-1α could recruit iECs in vitro. Alkaline phosphatase staining and alizarin red staining confirmed that OCTs formed by LV-HIF-1α-BMSCs had a statistically significant osteogenic differentiation ability compared with LV -BMSCs control group (P<0.05). When iECs were implanted into the LV-HIF-1α-BMSCs group OCTs to form P-OCTs, iECs substantially proliferated and rapidly fused, and formation of the progressive lumen was revealed by immunofluorescent CD31 staining. The expressions of OPN and OCN were significantly enhanced compared with those of the LV-BMSCs control group; OCN was the highest on day 7, and OPN was the highest on day 1 (P<0.05).
Conclusion:BMSCs transfected by HIF-1α have good osteogenic-angiogenic effect after induction and differentiation, which provides experimental foundation for optimizing the construction of three-dimensional prevascularized bone tissue.
- Full text:202509051121313356HIF-1α对BMSCs膜片成骨-成血管耦联的效应研究.pdf
