[Study on osteogenesis and angiogenesis of Pluronic F-127 composite gel loaded with transforming growth factor β 3 and bone marrow mesenchymal stem cells in rabbit maxillary sinus lift].

OBJECTIVE: To prepare Pluronic F-127 composite gel loaded with transforming growth factor β 3 (TGF-β 3) and bone marrow mesenchymal stem cells (BMSCs) and observe its osteogenesis and angiogenesis effects in vivo and in vitro.
METHODS: BMSCs were isolated from the tibial and femoral bone marrow of New Zealand white rabbits and passaged, and the 3rd generation cells were used for subsequent experiments after identification of osteogenic and adipogenic induction. Pluronic F-127 powder and TGF-β 3 were dissolved in L-DMEM medium to prepare Pluronic F-127 gel, TGF-β 3+Pluronic F-127 gel, BMSCs+Pluronic F-127 gel, and TGF-β 3+BMSCs+Pluronic F-127 gel. The 3rd generation of BMSCs were cultured with L-DMEM medium (group A), osteogenic induction medium (group B), osteogenic induction medium containing Pluronic F-127 gel (group C), and osteogenic induction medium containing TGF-β 3+Pluronic F-127 gel (group D), respectively. After 14 days of culturing, alkaline phosphatase (ALP) staining and Alizarin red staining were used to observe the osteogenesis. In addition, the BMSCs were cultured with L-DMEM medium containing Pluronic F-127 gel (experimental group) and L-DMEM medium (control group) for 1, 2, 3, and 4 days, respectively. And the cell proliferation was detected by MTT assay. Ten New Zealand white rabbits were taken to prepare the maxillary sinus lift models, and Pluronic F-127 gel (group A), TGF-β 3+Pluronic F-127 gel (group B), BMSCs+Pluronic F-127 gel (group C), and TGF-β 3+BMSCs+Pluronic F-127 gel (group D) were injected into the bone defects, respectively. On the 8th week, imaging examination and HE staining were used to observe the formation of new bone, immunohistochemical staining was used to observe the expression of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2) in bone tissue, and Western blot was used to detect the relative expressions of VEGF, oncostatin M (OSM), and BMP-4 proteins in bone tissue.
RESULTS: Osteogenic and adipogenic induction identified the isolated and cultured cells as BMSCs. In vitro staining showed that ALP activity and Alizarin red concentration in group D were significantly higher than those in other groups ( P<0.05). MTT assay showed that the absorbency ( A) value of the two groups increased gradually, and there was no significant difference between the groups at each time point ( P>0.05). In vivo experimental imaging examination showed that the bone mineral density and osteogenic continuity of group D were the best, and the proportion of new bone volume was superior to other groups ( P<0.05). HE staining showed that compared with other groups, bone trabeculae in group D were dense and arranged regularly, on which a large number of osteoblasts and osteoclasts were distributed, and a large number of new bone formation could be seen. Immunohistochemical staining showed the strong positive expressions of BMP-2 and VEGF in group D ( P<0.05); Western blot detection showed that the relative expressions of VEGF, OSM, and BMP-4 proteins in group D were significantly higher than those in other groups ( P<0.05).
CONCLUSION: The BMSCs in Pluronic F-127 composite gel loaded with TGF-β 3 and BMSCs can be induced to differentiate into osteoblasts, and the composite gel has no toxic effect on cells, and has obvious osteogenesis and angiogenesis in the maxillary sinus of rabbits.