负载TGF-β_(3)及BMSCs的Pluronic F-127复合凝胶在兔上颌窦提升中成骨及成血管作用的研究

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

目的制备负载TGF-β_(3)及BMSCs的Pluronic F-127复合凝胶,观察其体内、外成骨及成血管作用。方法取新西兰大白兔胫骨及股骨骨髓,分离培养BMSCs并传代,取第3代细胞经成骨、成脂诱导培养鉴定后用于后续实验。采用L-DMEM培养基溶解Pluronic F-127粉末、TGF-β_(3),分别制备Pluronic F-127凝胶、TGF-β_(3)+Pluronic F-127凝胶、BMSCs+Pluronic F-127凝胶、TGF-β_(3)+BMSCs+Pluronic F-127凝胶。取第3代BMSCs,分别采用L-DMEM培养基(A组)、成骨诱导液(B组)、含Pluronic F-127凝胶的成骨诱导液(C组)、含TGF-β_(3)+Pluronic F-127凝胶的成骨诱导液(D组)培养14 d后,ALP染色和茜素红染色观测成骨情况;另采用含Pluronic F-127凝胶的L-DMEM培养基(实验组)、L-DMEM培养基(对照组)培养1、2、3、4 d,MTT法检测细胞增殖情况。取10只新西兰兔制备上颌窦提升模型后,于每只兔骨缺损处注入Pluronic F-127凝胶(A组)、TGF-β_(3)+Pluronic F-127凝胶(B组)、BMSCs+Pluronic F-127凝胶(C组)、TGF-β_(3)+BMSCs+Pluronic F-127凝胶(D组),于第8周取材行影像学检查、HE染色观察新骨形成情况,免疫组织化学染色观察骨组织VEGF及BMP-2表达情况,Western blot检测骨组织VEGF、抑瘤素M(oncostatin M,OSM)及BMP-4蛋白表达。结果成骨、成脂诱导鉴定示分离培养细胞为BMSCs。体外实验染色显示D组ALP活性及茜素红浓度高于其他组(P<0.05);MTT法检测示随着时间延长,两组吸光度(A)值均逐渐升高,各时间点组间比较差异均无统计学意义(P>0.05)。体内实验影像学检查示D组成骨密度及成骨连续性最好,新骨体积占比优于其他组(P<0.05);HE染色示与其他组比较,D组骨小梁致密且排列规则,其上分布大量成骨细胞和破骨细胞,可见大量新骨形成;免疫组织化学染色示D组BMP-2、VEGF呈强阳性表达(P<0.05);Western blot检测D组VEGF、OSM及BMP-4蛋白相对表达量高于其他组(P<0.05)。结论负载TGF-β_(3)及BMSCs的Pluronic F-127复合凝胶中的BMSCs能被诱导分化为成骨细胞,并且复合凝胶对细胞无毒性,在兔上颌窦内有明显成骨及成血管效果。

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 3 rd 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 3 rd 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 8 th 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.