| Objective:To explore the vascularization of tissue engineered bone repairing large bone defects.The preliminary experiments proved that bone marrow mesenchymal stem cells co-cultured with umbilical vein endothelial cells which through adenovirus transfected in vitro.It suggested that the co-culture of HUVECs and BMSCs could promote the proliferation and differentiation each other,and could be used to repair critical bone defects in vivo and to improve the necrosis caused by insufficient vascularization during repair.To further verify the effect of co-culture system in animals,in this experiment,the SD rat model of skull defect was used,and the BMSCs and HUVECs composited with HA-TCP scaffolds were transplanted into the defect.To observe the ability of this scaffolds to promote angiogenesis in vivo.To provide a theoretical basis and experimental basis for the construction of vascularized tissue engineering bone to repair large bone defects.Methods:1.Choose 4-week-old SD rats with 120-160 g weight,We get tibial and femoral bone marrow of SD rat in aseptic conditions,then separating and cultivating bone marrow mesenchymal stem cells(BMSCs)in vitro and identified.Cultivating HUVECs after recovering in vitro.2.Each group of cells were counted using blood count plate and seeded on the HA-TCPscaffold,then the cell/scaffold composites were divided in three groups: bone marrow mesenchymal stem cells-combined with bone-induced calcium phosphate bioceramic scaffold(BMSCs+HA-TCP),human umbilical vein endothelial cells-combined with bone-induced calcium phosphate bioceramic scaffold(HUVECs+HA-TCP),bone marrow mesenchymal stem cells + human umbilical vein endothelial cells-combined with bone-induced calcium phosphate Bioceramic scaffold(BMSCs+HUVECs+HA-TCP).Using scanning electron microscope observe the growth and attachment of the cells on HA-TCP scaffolds in each groups after culture.3.60 female SD rats(250-300g)were randomly divided into four groups of A,B,C and D with 15 rats in each group.Established a 10 mm diameter skull defect in the SD rat skull midline,and the blank HA-TCP scaffolds and BMSCs+HUVECs+HA-TCP,HUVECs+HA-TCP and BMSCs+HA-TCP scaffolds were transplanted into the skull defect corresponded to group A,B,C and D.And observe the growth of the scaffolds.Group A: transplantation of HA-TCP scaffolds cultured in the same medium;group B: transplantation of BMSCs+HUVECs+HA-TCP complex;C group: transplantation of HUVECs+HA-TCP complex;D group: transplantation of BMSCs+HA-TCP complex.4.Observation index:4,8,and 12 weeks after the operation,the A,B,C,and D rats were taken off as the observation target,respectively.(1)The new bone growth in the three dimensional direction of the skull defect were observed by Micro-CT.(2)Observation of the growth condition of the defect.(3)The samples were fixed, decalcified,embedded,sectioned to hematoxylin and eosin stain,and CD31/CD34 immunohistochemical staining were used to observe the number of new vessels in the cranial defect.It was used to detect microvessel density(MVD)and cumulative optical density(IOD).(4)Detection the samples of vascular related factors(CD31/CD34)by enzyme linked immunosorbent assay(Elisa),and Protein immunoblotting(Western Blot)for the detection of blood vessel related proteins(VEGF-A).Results:1.Bone marrow mesenchymal stem cells were successfully isolated and cultured,and then combined with scaffolds.Compound culture of human umbilical vein endothelial cells and scaffolds.And combined culture of bone marrow mesenchymal stem cells and human umbilical vein endothelial cells and scaffolds.2.The growth condition of the B group was better than that of the other groups,and the B group observed more punctate bleeding.3.At 4,8,and 12 weeks after operation,the Micro-CT observation B group new bone quantity was more than the other three groups,which indirectly reflected the more neovascularization in B group.After 4 weeks,immunohistochemical analysis of CD31 MVD value,Group A: 21.8±1.30,group B: 27.6 ±1.14,group C: 26±1.22,group D: 24.2±1.30.After 8 weeks,immunohistochemical analysis of CD31 MVD value,Group A: 25.8±1.79,group B: 38.2±1.48,group C: 32.4 ±2.30,group D: 31±1.87.After 12 weeks,immunohistochemical analysis of CD31 MVD value,Group A: 28.2±1.64,group B: 49.6±3.65,group C: 37±2.12,group D: 35.8 ±1.30.After 4 weeks,immunohistochemical analysis of CD34 MVD value,Group A: 20.6±2.07,group B: 27.8±1.09,group C: 25.4±0.55,group D: 22.8 ±1.30.After 8 weeks,immunohistochemical analysis of CD34 MVD value,Group A: 24±2.74,group B: 38.2 ±0.84,group C: 32 ±1.00,group D: 29.6±1.82.After 12 weeks,immunohistochemical analysis of CD34 MVD value,Group A: 28.8 ±0.84,group B: 49.6±1.14,group C: 36.8±1.30,group D: 35.2±0.84.After 4 weeks,immunohistochemical analysis of CD34 IOD(*103)value,Group A: 34.77±9.62,group B: 45.66±9.71,group C: 33.30 ±10.19,group D: 39.37 ±8.38.After 8 weeks,immunohistochemical analysis of CD34 IOD(*103)value,Group A:34.96±12.90,group B:53.65±9.86,group C:45.60±9.77,group D:46.19±10.26.After 12 weeks,immunohistochemical analysis of CD34 IOD(*103)value,Group A: 42.18±9.91,group B:77.20±7.99,group C:66.45±6.44,group D:53.57±8.70.Relative values show that B group>D group >C group >A group,and the increment of blood vessel in B group was the most with time.The increase of blood vessel in group C was faster than that in group D,and there were significant difference(P<0.05).5.At 4,8 and 12 weeks,the relative content of CD34 molecules in group B was 4.7± 0.4,9.6±0.2,11.0±1.2,respectively,and the results increased gradually,which were all higher than those in A,C and D groups at the same time,and there were significant difference(P<0.05).6.Western Blot detection into VEGF-A protein related blood vessels,VEGF-A protein and ?-actin reference gray level ratio in B group was higher than that of the other three groups.The ratios of 4,8,and 12 weeks were 1.23±0.09,1.34±0.10,1.43±0.12,respectively.There were significant differences between group B and A,C,D group(P<0.05).Conclusion:1.BMSCs and HUVECs can grow and crawl on porous HA-TCP scaffolds,respectively,and have no obvious rejection.2.BMSCs and HUVECs Co HA-TCP composite scaffold transplantation has strong ability of angiogenesis in calvarial defects of rat. |
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