连续低强度超声对骨软骨体外保存的影响△

Effect of continuous low- intensity ultrasound on the preservation of osteochondral tissue in vitro

摘要:

［目的］ 探究连续低强度超声（continuous low intensity ultrasound, cLIUS） 对骨软骨体外保存的影响。［方法］ 6 个月

龄猪股骨髁获取骨软骨标本60 个，随机分为两组，均于DMEM 培养液在4℃保存。静态组不做超声处理。超声组每天给予20

min 超声刺激，频率42 kHz，电功率50 W。于第14、28 d 检测细胞存活率、蛋白多糖表达、组织形态学、生物力学性能。

cLIUS 刺激2 h 后检测ERK 蛋白表达情况。［结果］ 第14 d，超声组细胞存活率显著高于静态组[(75.5±1.8)% vs (65.6±1.5)% , P<

0.05]；随时间推移，两组细胞存活率均显著下降（P<0.05）；第28 d 两组差异无统计学意义（P>0.05）。组织学染色显示，第

14 d，超声组组织形态结构优于静态组，且番红O 染色IOD [(224.7±4.1) vs (166.5±3.7) , P<0.05]、甲苯胺蓝染色IOD [(223.7±6.0)

vs (166.7±5.0) , P<0.05] 均显著高于静态组；第28 d 两组上述指标差异无统计学意义（P>0.05）。随时间推移，两组组织形态结

构紊乱，蛋白多糖显著流失（P<0.05）。第14 d，超声组杨氏模量显著优于静态组[(8.7±0.3) MPa vs (7.0±0.0) MPa, P<0.05]；第

28 d 两组差异无统计学意义（P>0.05）。随时间推移，两组杨氏模量显著下降（P<0.05）。超声刺激2 h 后，超声组ERK 蛋白表

达水平显著高于静态组[(0.9±0.1) vs (0.5±0.2), P<0.05]。［结论］ cLIUS 可以提高猪骨软骨的体外保存效果，延长保存时间，上调

ERK 蛋白的表达水平。

Abstract:

[Objective] To investigate the effect of continuous low-intensity ultrasound (cLIUS) on the preservation of osteochondral tis-sue in vitro. [Methods] Sixty osteochondral specimens were harvested from a 6-month-old pig femur condyles and were randomly dividedinto two groups, which were stored in DMEM medium at 4℃, those in the static group was not treated with ultrasound, while the samples inthe ultrasonic group was stimulated for 20 min every day at a frequency of 42 kHz and an electric power of 50W. The survival rate, proteo-glycan expression, histomorphology and biomechanical properties were detected at the 14 th and 28 th days. The expression of extracellularregulatory protein kinase (ERK) was detected 2 hours after cLIUS stimulation. [Results] The ultrasound group got significantly higher cellsurvival rate than the static group at 14 days [(75.5±1.8)% vs (65.6±1.5)%, P＜0.05], whereas which became not statistically significant be-tween the two group at 28 days. As time went by, the cell survival rate in both groups decreased significantly (P＜0.05). Histological stainingshowed that the ultrasound group was better than the static group, the former had higher the saffron O staining IOD [(224.7±4.1) vs (166.5±3.7), P＜0.05], toluidine blue staining IOD [(223.7±6.0) vs (166.7±5.0), P＜0.05] than the latter at 14 days, while which returned to be not sig-nificant difference between the two groups at 28 days (P＞0.05). The tissue morphology and structure of the two groups were disordered andproteoglycan was significantly lost over time (P＜0.05). In addition, the ultrasound group got significantly higher Young's modulus than thestatic group 14 days [(8.7±0.3) MPa vs (7.0±0.0) MPa, P＜0.05], which became not significantly different either at 28 days between the twogroups (P＞0.05). The Young's modulus of the two groups decreased significantly with time (P＜0.05). At 2 hours after ultrasonic stimulation,the ultrasonic group had significantly higher expression of ERK protein than the static group [(0.9±0.1) vs (0.5±0.2), P＜0.05]. [Conclusion]The cLIUS improves the preservation quality of porcine bone cartilage in vitro, prolong the preservation time and up-regulate the expres-sion of ERK protein.

Abstract:

[Objective] To investigate the effect of continuous low-intensity ultrasound (cLIUS) on the preservation of osteochondral tis-sue in vitro. [Methods] Sixty osteochondral specimens were harvested from a 6-month-old pig femur condyles and were randomly dividedinto two groups, which were stored in DMEM medium at 4℃, those in the static group was not treated with ultrasound, while the samples inthe ultrasonic group was stimulated for 20 min every day at a frequency of 42 kHz and an electric power of 50W. The survival rate, proteo-glycan expression, histomorphology and biomechanical properties were detected at the 14 th and 28 th days. The expression of extracellularregulatory protein kinase (ERK) was detected 2 hours after cLIUS stimulation. [Results] The ultrasound group got significantly higher cellsurvival rate than the static group at 14 days [(75.5±1.8)% vs (65.6±1.5)%, P＜0.05], whereas which became not statistically significant be-tween the two group at 28 days. As time went by, the cell survival rate in both groups decreased significantly (P＜0.05). Histological stainingshowed that the ultrasound group was better than the static group, the former had higher the saffron O staining IOD [(224.7±4.1) vs (166.5±3.7), P＜0.05], toluidine blue staining IOD [(223.7±6.0) vs (166.7±5.0), P＜0.05] than the latter at 14 days, while which returned to be not sig-nificant difference between the two groups at 28 days (P＞0.05). The tissue morphology and structure of the two groups were disordered andproteoglycan was significantly lost over time (P＜0.05). In addition, the ultrasound group got significantly higher Young's modulus than thestatic group 14 days [(8.7±0.3) MPa vs (7.0±0.0) MPa, P＜0.05], which became not significantly different either at 28 days between the twogroups (P＞0.05). The Young's modulus of the two groups decreased significantly with time (P＜0.05). At 2 hours after ultrasonic stimulation,the ultrasonic group had significantly higher expression of ERK protein than the static group [(0.9±0.1) vs (0.5±0.2), P＜0.05]. [Conclusion]The cLIUS improves the preservation quality of porcine bone cartilage in vitro, prolong the preservation time and up-regulate the expres-sion of ERK protein.