低强度脉冲超声对浅层关节软骨损伤的作用

Effect of low-intensity pulsed ultrasound on partial-thickness articular cartilage defect

目的探讨低强度脉冲超声(LIPUS)对浅层关节软骨(p-AC)损伤的作用。方法选择健康日本大耳白兔16只,雌雄不限,体质量2.5~3.3kg。手术创建兔膝关节p-AC损伤,使用功率30mW/cm^2、脉冲通断比20%、脉冲频率1kHz、超声固有频率1.5MHz的LIPUS进行术后处理,每天20min。于LIPUS处理1周和3周,用逆转录-聚合酶链反应(RTPCR)检测损伤周围软骨组织mRNA的表达;处理6周和12周后,对损伤处进行组织学评估。分离、培养关节软骨细胞并进行LIPUS处理,分别在干预后第1、3、5天,检测软骨细胞的增殖及基因表达情况。结果在该实验参数下,6周和12周实验组和对照组均没有透明样软骨再生,但是对照组的白色纤维组织较多;与对照组相比,LIPUS下调了软骨组织中Ⅰ型胶原的基因表达,但是差异无统计学意义(P>0.05);在第1周时下调Ⅱ型胶原的表达(P=0.046);在第3周时下调蛋白多糖(ACAN)(P=0.017)、SOX9(P=0.001)的表达。组织学病理切片分析发现LIPUS没有促进p-AC修复;但是,与对照组相比,LIPUS对钙化软骨(CC)层与软骨下骨(SB)层有一定的保护作用。最后LIPUS在体外第5天时显著抑制软骨细胞增殖(P=0.0023),不能促进Ⅱ型胶原和ACAN的表达,但可以下调MMP-13的表达并在第5天时差异有显著统计学意义(P=0.000036)。结论在该实验条件下,LIPUS对p-AC损伤无明显的修复作用,但具有一定的组织保护和减弱组织退化的作用。

Objective To investigate the effect of low-intensity pulsed ultrasound(LIPUS) on partial-thickness articular cartilage( p-AC) defect.Methods A total of 16 healthy Japanese white rabbits(male or female,body weight of 2.5 - 3.3 kg) were selected. p-AC defects were surgically created in knees of the rabbits and treated by LIPUS.The experimental parameters of LIPUS was set as intensity of 30 mW/cm^2,fixed frequency of 1.5 MHz,pulse frequency of 1 kHz and duty cycle of 20 %.The defects were treated for 20 minutes per day after operation.After 1 week and 3 weeks LIPUS treatment,the mRNA expressions in cartilage adjacent defect was determined by reverse transcription-polymerase chain reaction(RT-PCR).After 6 weeks and 12 weeks treatment,the defects were assessed by histological evaluation.Meanwhile,the effects of LIPUS on the proliferation and gene expressions of the in vitro cultured chondrocyte were tested on 1st,3rd and 5th day after the treatment by LIPUS. Results Under the experimental parameters,there was no hyaline cartilage regeneration in experimental group and control group at 6-week and 12-week,while control group showed more white fibrous tissue.Compared with control group,LIPUS down-regulated gene expression of type Ⅰ collagen in cartilage tissue,but the difference was not statistically significant(P > 0.05).The expression of type Ⅱ collagen was down-regulated at 1st week(P = 0.046),the expression of proteoglycan(ACAN)(P = 0.017) and SOX9(P = 0.001) were down-regulated at 3rd week.The histological pathological analysis revealed that LIPUS did not promote p-AC repair.Compared with control group,LIPUS showed protective effect on calcified cartilage(CC) and subchondral bone(SB).LIPUS significantly inhibited chondrocyte proliferation at 5-day in vitro(P = 0.002 3),while did not promote the expression of type Ⅱ collagen and ACAN.LIPUS down-regulated the expression of MMP-13,and the differences were significant at 5-day(P = 0.000 036).Conclusion It is demonstrated that LIPUS has no obvious repair effect but tissue protection effect,and can reduce tissue degradation on p-AC defect under the set experiment parameters.