体外周期性压应力对兔胫骨骨折愈合过程成骨与破骨细胞增殖分化能力的影响

Effects of cyclic compressive stress in on the proliferation and differentiation of osteoblasts and osteoclasts during the healing of New Zealand rabbit tibia fracture

目的探究体外周期性压应力对兔胫骨骨折愈合过程成骨与破骨细胞增殖分化能力的影响。方法选取48只健康雄性4个月龄新西兰大白兔,所有样本均于右侧胫腓联合下0.5 cm处横行截骨构建兔胫骨骨折模型,行骨折复位钢板内固定术及高分子石膏外固定治疗。采用随机数字表法将48只新西兰大白兔分为实验组和对照组,每组24只。实验组于术后第8天开始施加大小15 N、频率1 Hz、叩击持续时间5 s、间隔3 s、30 min/次、1次/2 d的体外周期性压应力轴向应力刺激;对照组无应力刺激。分别于术后2、4、6、8周依次从2组各选6只新西兰大白兔,通过X线片检查骨折愈合情况,并计算比较2组Lane-Sandhu X线评分;术后2、4、6、8周2组分别处死6只新西兰大白兔,取骨折区骨组织标本,通过苏木精-伊红(HE)染色观察比较2组新生骨痂、骨髓腔、骨小梁等构成排列,以及新生骨组织中成骨细胞增生情况;术后2、4、6、8周分别取2组骨组织每组各3份切片,通过免疫组化染色检测2组新生骨组织中成骨细胞相关分子核心结合因子α1(Cbf-α1)、骨钙素、骨保护素(OPG)及核因子-κB受体活化因子配体(RANKL)的表达水平,并计算比较不同时间点2组OPG/RANKL比值。数据比较采用独立样本t检验。结果(1)X线片显示,术后2周,实验组和对照组骨折线均清晰可见,仅少量外骨痂生长;术后4周,实验组和对照组骨折间隙均明显变小,实验组骨折线较对照组模糊,骨痂量多于对照组;术后6周,实验组骨折线模糊,外骨痂生长致密,且有少量内骨痂生长,基本达到骨性愈合,对照组骨折线与第4周相比较模糊,骨折端骨痂量较实验组少;术后8周,对照组骨折线基本消失,实验组骨折线完全消失,且实验组骨痂量仍明显多于对照组。术后4、6、8周,实验组Lane-Sandhu评分分别为(5.17±1.07)、(7.33±0.94)、(9.17±1.07)分,均高于对照组[(3.50±0.76)、(5.83±1.07)、(7.33±1.25)分],比较差异均有统计学意义(t=2.84、2.36、2.50,P=0.02、0.04、0.03)。(2)HE染色观察显示,术后2周,实验组较对照组出现更多成骨细胞;术后4、6、8周,实验组较对照组更早形成骨髓腔及骨小梁组织,且骨组织较对照组更早成熟。(3)免疫组织化学染色观察显示:①实验组和对照组Cbf-α1术后2周均呈低表达,术后4周表达达到高峰,术后6~8周表达逐渐减少,且术后2、4、6、8周,实验组Cbf-α1表达均高于对照组;不同时间点实验组Cbf-α1的平均吸光度值分别为263.20±49.95、503.39±38.53、377.98±12.38、276.91±8.61,均高于对照组(123.05±14.60、359.51±58.98、339.14±18.98、224.54±23.94),比较差异均有统计学意义(t=4.67、3.54、2.97、3.57,P=0.01、0.03、0.04、0.03);②术后2、4、6、8周,实验组和对照组骨钙素表达量均逐渐增高,且实验组骨钙素的表达均高于对照组;术后2、4、6、8周,实验组骨钙素的平均吸光度值分别为45.28±4.96、283.80±49.01、450.06±61.42、619.00±105.40,均高于对照组(5.29±4.49、20.94±7.59、220.39±32.18、424.98±32.84),比较差异均有统计学意义(t=10.35、9.18、5.74、3.05,P<0.05);③实验组和对照组OPG术后2周均呈低表达,术后4周表达达到高峰,术后6、8周表达逐渐减少;术后2、4、6、8周,实验组OPG的表达均高于对照组;不同时间点实验组OPG的平均吸光度值分别为443.97±23.61、576.91±37.21、278.28±16.38、144.13±30.20,均高于对照组(374.66±26.30、454.50±49.95、233.17±21.35、62.82±4.16),比较差异均有统计学意义(t=3.40、3.40、2.90、4.62,P<0.05);④实验组和对照组RANKL表达量在术后2、4周均较低,术后6、8周表达量逐渐增多;术后2、4、6、8周,实验组RANKL的表达量均低于对照组;实验组不同时间点RANKL的平均吸光度值分别为203.34±18.16、186.63±19.50、261.78±28.33、441.06±17.89,均低于对照组(275.64±26.68、277.28±9.49、385.13±11.56、485.20±8.15),比较差异均有统计学意义(t=3.88、7.24、6.98、3.89,P<0.05);⑤术后2、4、6、8周,实验组OPG/RANKL的平均吸光度值比值分别为2.19±0.18、3.13±0.53、1.08±0.18、0.33±0.08,均大于对照组(1.37±0.21、1.64±0.22、0.61±0.07、0.13±0.01),比较差异均有统计学意义(t=5.14、4.50、4.14、4.50,P<0.05)。结论体外周期性压应力可通过促进骨折局部成骨细胞因子Cbf-α1、骨钙素、OPG的表达及抑制骨折局部破骨细胞因子RANKL的表达,提高OPG/RANKL的比值,从而延缓骨吸收促进兔胫骨骨折愈合。

Objective To investigate the effects of cyclic compressive stress in vitro on the proliferation and differentiation of osteoblasts and osteoclasts during fracture healing of New Zealand rabbit tibia fracture.Methods A total of 48 healthy male New Zealand rabbits at the age of 4 months were selected.All the samples were transversely osteotomized at 0.5 cm below the right joint of the tibia and fibula to construct a rabbit tibia fracture model,steel plate internal fixation and polymeric plaster external fixation treatment were used.Forty-eight New Zealand rabbits were divided into the experimental group and the control group according to the random number table method,24 in each group.On the 8th day after operation,the experimental group was applied external periodic compressive stress axial stress stimulation of 15 N,frequency 1 Hz,duration of slamming 5 s,interval 3 s,30 min/time,1 time/2 days;no stress stimulation was applied in the control group.At 2,4,6,and 8 weeks after operation,6 New Zealand white rabbits from each of the 2 groups were selected in turn,and the fracture healing was checked by Lane-Sandhu X-ray film,and the Lane-Sandhu X-ray scores of the 2 groups were calculated and compared.At 2,4,6,and 8 weeks after the operation,6 New Zealand rabbits were sacrificed in the 2 groups,and bone tissue samples were taken from the fracture area.The hematoxylin-eosin(HE)staining was used to observe and compare the composition and arrangement of new callus,bone marrow cavity,trabecular bone,and the proliferation of osteoblasts in the new bone tissue of the 2 groups.At 2,4,6,and 8 weeks after operation,3 slices of each group of bone tissues were taken from each of the 2 groups,and the expression levels of core-binding factorα1(Cbf-α1),osteocalcin,osteoprotegerin(OPG)and receptor activator of NF-κB ligand(RANKL)in the new callus of these 2 groups were compared by immunohistochemical staining,and the ratio of OPG/RANKL of the two groups at different time points were calculated and compared.Data were compared with independent sample t test.Results(1)X-ray film showed that the fracture lines of the experimental group and the control group were clearly visible 2 weeks after the operation,and only a small amount of external callus grew.At 4 weeks after the operation,the fracture gap between the experimental group and the control group was significantly smaller,and the fracture line of the experimental group was lower than that of the control group.At 6 weeks after the operation,the fracture line of the experimental group was blurred,the outer callus grew densely,and there was a small amount of internal callus growth,which basically achieved bony union,the fracture line of the control group was blurred compared with 4 weeks after the operation,and the volume of the fractured callus was higher Less.At 8 weeks after the operation,the fracture line in the control group basically disappeared,the fracture line in the experimental group disappeared completely,and the amount of callus in the experimental group was still significantly more than that in the control group.At 4,6 and 8 weeks after operation,the Lane-Sandhu scores of the experimental group were(5.17±1.07),(7.33±0.94),(9.17±1.07)points,which were higher than those of the control group[(3.50±0.76),(5.83±1.07),(7.33±1.25)points],the differences were statistically significant(t=2.84,2.36,2.50;P=0.02,0.04,0.03).(2)HE staining observation showed that the experimental group had more osteoblasts than the control group at 2 weeks after the operation;at 4,6,8 weeks after the operation,the bone marrow cavity and trabecular bone tissue were formed in the experimental group earlier than the control group,and the bone tissue matured earlier than the control group.(3)Immunohistochemical staining observations showed:①the expression levels of Cbf-α1 in the experimental group and the control group were both low at 2 weeks after the operation,reached a peak at 4 weeks after the operation,and gradually decreased at 6 to 8 weeks after the operation.At 2,4,6,and 8 weeks after the operation,the expression of Cbf-α1 in the experimental group was higher than that of the control group;the average absorbance values of Cbf-α1 in the experimental group at different time points were 263.20±49.95,503.39±38.53,377.98±12.38,276.91±8.61,respectively,all higher than the control group(123.05±14.60,359.51±58.98,339.14±18.98,224.54±23.94),the differences were statistically significant(t=4.67,3.54,2.97,3.57;P=0.01,0.03,0.04,0.03).②At 2,4,6,8 weeks after operation,the expression of osteocalcin in the experimental group and the control group gradually increased,and the expression of osteocalcin in the experimental group was higher than that of the control group.At 2,4,6,and 8 weeks after operation,the average absorbance values of osteocalcin in the experimental group were 45.28±4.96,283.80±49.01,450.06±61.42 and 619.00±105.40,which were higher than those of the control group(5.29±4.49,20.94±7.59,220.39±32.18,424.98±32.84),the differences were statistically significant(t=10.35,9.18,5.74,3.05;P<0.05).③The expression levels of OPG in the experimental group and the control group were both low expression at 2 weeks after operation,peaked at at 4 weeks after operation,,and gradually decreased from the 6 to 8 weeks after operation.At 2,4,6,and 8 weeks after operation,the expression of OPG in the experimental group was higher than that in the control group;the average absorbance values of OPG in the experimental group at different time points were 443.97±23.61,576.91±37.21,278.28±16.38,144.13±30.20,respectively,all higher than the control group(374.66±26.30,454.50±49.95,233.17±21.35,62.82±4.16),the differences were statistically significant(t=3.40,3.40,2.90,4.62;P<0.05).④The expression of RANKL in the experimental group and the control group were both low at 2 and 4 weeks after operation,and the expression increased gradually at 6 and 8 weeks after operation.At 2,4,6 and 8 weeks after operation,the expression of RANKL in the experimental group was lower than that in the control group;the average absorbance values of RANKL in the experimental group at different time points were 203.34±18.16,186.63±19.50,261.78±28.33,441.06±17.89,respectively,all lower than the control group(275.64±26.68,277.28±9.49,385.13±11.56,485.20±8.15),and the differences were statistically significant(t=3.88,7.24,6.98,3.89;P<0.05).⑤At 2,4,6,and 8 weeks after operation,the average absorbance ratios of OPG/RANKL in the experimental group were 2.19±0.18,3.13±0.53,1.08±0.18,0.33±0.08,which were all larger than those of the control group(1.37±0.21,1.64±0.22,0.61±0.07,0.13±0.01),the differences were statistically significant(t=5.14,4.50,4.14,4.50;P<0.05).Conclusion Cyclic compressive stress in vitro can promote the expression of local osteoblasts Cbf-α1,osteocalcin and OPG in fractures and inhibit the expression of local osteoclast factor RANKL in fractures,thereby increasing the ratio of OPG/RANKL,delaying bone resorption and promoting healing of New Zealand rabbit tibia fracture.