外源性转化生长因子-β_3联合兔牙髓干细胞对兔骨缺损处成骨细胞转化生长因子-β_3表达的影响

Influence of exogenous transforming growth factor-β_3 combined with dental pulp stem cells on the expression of transforming growth factor-β-3 of osteoblasts in bone defect

目的探讨转化生长因子-β_3(transforming growth factor-β_3,TGF-β3)联合体外培养的兔牙髓干细胞(dental pulp stem cells,DPSCs)对兔骨缺损处成骨细胞TGF-β_3表达的影响。方法 37只新西兰大白兔,取其中1只兔牙髓组织采用酶解组织块法分离培养获得DPSCs,体外培养,光镜下行细胞形态学观察,将DPSCs传代至第3代,冷冻储存待用。余36只新西兰大白兔随机分为PBS组、DPSCs组、TGF-β_3+DPSCs组,每组12只。在兔下颌前牙及磨牙区颊侧随机人工制备约8mm×2mm×3mm骨缺损区域,PBS组植入Bio-Oss粗颗粒骨粉0.30g+PBS 20μL,DPSCs组植入Bio-Oss粗颗粒骨粉0.30g+1×108/L DPSCs 20μL,TGF-β3+DPSCs组植入Bio-Oss粗颗粒骨粉0.30g+1×108/L DPSCs20μL+80ng/L TGF-β320μL。术后第4、8周3组分别处死6只兔,在骨缺损处行锥形束CT检查观察牙槽骨长度、宽度和高度丧失情况,采用茜素红染色观察骨缺损处骨愈合情况,采用免疫组织化学法观察种植体骨缺损处成骨细胞TGF-β_3表达水平。结果原代培养的兔DPSCs呈集落生长,多呈梭形,少数呈多角形或纺锤形;术后4周TGF-β_3+DPSCs组茜素红染色较PBS组、DPSCs组出现更多红色钙化结节,术后8周钙化结节进一步增多;术后4、8周,TGF-β_3+DPSCs组牙槽骨长度、宽度和高度丧失均少于DPSCs组和PBS组,DPSCs组少于PBS组(P<0.05);术后4周,TGF-β_3+DPSCs组成骨细胞TGF-β_3表达水平(0.33±0.02)明显高于DPSCs组(0.15±0.01)和PBS组(0.09±0.03)(P<0.05),DPSCs组与PBS组比较差异无统计学意义(P>0.05);术后8周,TGF-β_3+DPSCs组成骨细胞TGF-β_3表达水平(0.46±0.01)明显高于PBS组(0.29±0.02)(P<0.05),与DPSCs组(0.38±0.04)比较差异无统计学意义(P>0.05),DPSCs组与PBS组比较差异无统计学意义(P>0.05)。结论 DPSCs具有高度增殖的生物学特性,并具有成骨向分化潜能;外源性TGF-β_3联合DPSCs可增强内源性TGF-β_3的表达。

Objective To investigate the influence of exogenous transforming growth factor-β_3(TGF-β_3)combined with dental pulp stem cells(DPSCs)in vitro on the expression of TGF-β_3 of osteoblasts in bone defect in rabbits.Methods DPSCs were isolated by tissue enzymetic digestion method in 1out of 37 New Zealand rabbits,cultured in vitro,and the cell morphology was observed under light microscope.DPSCs were passed to the third generation,frozen and stored.The other 36 rabbits were randomly divided into PBS group,DPSCs group and TGF-β_3+DPSCs group,with 12 rabbits in each group.An alveolar bone defect of 8mm×2mm×3mm was made in the buccal side of rabbit's mandibular incisors and molars randomly.The bone defect was filled with 0.30 g of Bio-Oss powder mixed by 20μL of PBS in PBS group,was filled with 0.30 g of Bio-Oss powder+ 20μL of 1×108/L DPSCs in DPSCs group,and was filled with 0.30 g of Bio-Oss powder+ 20μL of 1×10~8/L DPSCs+ 20μL of 80ng/μL TGF-β_3in TGF-β_3+DPSCs group.Six rabbits were sacrificed in each group at the postoperative 4th and 8th week respectively.The length,width and height of the alveolar bone were measured by cone-beam CT in the bone defect area.ARS staining was used to observe the bone defect healing,and immunohistochemical method was used to detect the expression of TGF-β_3of osteoblasts in the bone defect.Results The primary cultured immature DPSCs in rabbits grew in clones,mostly showing fusiform and few showing polygon form.ARS staining showed more red calcified nodules in TGF-β_3 +DPSCs group than the other two groups at the postoperative 4th week,and the red calcified nodule was further increased at the postoperative 8th week.TGF-β_3+DPSCs group showed less loss in the length,width and height of the alveolar bone than DPSCs group and PBS group at the postoperative 4th and 8th week(P<0.05),and less in DPSCs group than PBS group(P<0.05).The expression of TGF-β_3of osteoblasts was significantly higher in TGF-β_3 +DPSCs group(0.33±0.02)than that in DPSCs group(0.15±0.01)and PBS group(0.09±0.03)(P<0.05),and there was no significant difference between DPSCs group and PBS group at the postoperative 4th week(P>0.05),and it was significantly higher in TGF-β_3+DPSCs group(0.46±0.01)than that in PBS group(0.29±0.02)(P<0.05),and there were no significant difference between TGF-β_3+DPSCs group and DPSCs group(0.38±0.04)(P>0.05)and between DPSCs group and PBS group at the postoperative 8th week(P>0.05).Conclusion DPSCs has strong proliferation ability and the potential osteogenic differentiation ability.Exogenous TGF-β_3 combined with DPSCs can effectively promote the expression of TGF-β_3 of osteoblast in bone defect.