低强度脉冲超声对浅层关节软骨损伤的作用
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摘要
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2.5～3.3 kg。手术创建兔膝关节p-AC损伤,使用功率30 m W/cm2、脉冲通断比20%、脉冲频率1 kHz、超声固有频率1.5 MHz的LIPUS进行术后处理,每天20 min。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=0.002 3),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=0.000 036)。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/cm2, fixed frequency of 1.5 MHz, pulse frequency of 1 k Hz 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 and12 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 1 st, 3 rd and 5 th 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 1 st week(P = 0.046), the expression of proteoglycan(ACAN)(P =0.017) and SOX9(P = 0.001) were down-regulated at 3 rd 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.
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/cm2, fixed frequency of 1.5 MHz, pulse frequency of 1 k Hz 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 and12 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 1 st, 3 rd and 5 th 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 1 st week(P = 0.046), the expression of proteoglycan(ACAN)(P =0.017) and SOX9(P = 0.001) were down-regulated at 3 rd 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.
引文
[1]Jang KW.The effect of low-intensity pulsed ultrasound on chondrocyte migration and its potential for the repair of articular cartilage.Iowa:University of Iowa,2011.
[2]Naito K,Watari T,Muta T,et al.Low-intensity pulsed ultrasound(LIPUS)increases the articular cartilage typeⅡcollagen in a rat osteoarthritis model[J].J Orthop Res,2010,28(3):361-369.
[3]Uddin SM,Richbourgh B,Ding Y,et al.Chondro-protective effects of low intensity pulsed ultrasound[J].Osteoarthritis Cartilage,2016,24(11):1989-1998.
[4]Xia P,Shen S,Lin Q,et al.Low-intensity pulsed ultrasound treatment at an early osteoarthritis stage protects rabbit cartilage from damage via the integrin/focal adhesion kinase/mitogenactivated protein kinase signaling pathway[J].J Ultrasound Med,2015,34(11):1991-1999.
[5]Xia P,Ren S,Lin Q,et al.Low-intensity pulsed ultrasound affects chondrocyte extracellular matrix production via an integrin-mediated p38 MAPK signaling pathway[J].Ultrasound Med Biol,2015,41(6):1690-1700.
[6]Tan L,Ren Y,van Kooten TG,et al.Low-intensity pulsed ultrasound(LIPUS)and pulsed electromagnetic field(PEMF)treatments affect degeneration of cultured articular cartilage explants[J].Int Orthop,2015,39(3):549-557.
[7]Yuan LJ,Niu CC,Lin SS,et al.Effects of low-intensity pulsed ultrasound and hyperbaric oxygen on human osteoarthritic chondrocytes[J].J Orthop Surg Res,2014,9:5-5.
[8]Korstjens CM,van der Rijt RH,Albers GH,et al.Low-intensity pulsed ultrasound affects human articular chondrocytes in vitro[J].Med Biol Eng Comput,2008,46(12):1263-1270.
[9]Yilmaz V,Karadas魻,Dandinogˇlu T,et al.Efficacy of extracorporeal shockwave therapy and low-intensity pulsed ultrasound in a rat knee osteoarthritis model:A randomized controlled trial[J].Eur J Rheumatol,2017,4(2):104-108.
[10]Sekino J,Nagao M,Kato S,et al.Low-intensity pulsed ultrasound induces cartilage matrix synthesis and reduced MMP13 expression in chondrocytes[J].Biochem Biophys Res Commun,2018,506(1):290-297.
[11]Ji JB,Li XF,Liu L,et al.Effect of low intensity pulsed ultrasound on expression of TIMP-2 in serum and expression of MMP-13 in articular cartilage of rabbits with knee osteoarthritis[J].Asian Pac J Trop Med,2015,8(12):1043-1048.
[12]Ito A,Aoyama T,Yamaguchi S,et al.Low-intensity pulsed ultrasound inhibits messenger RNA expression of matrix metalloproteinase-13 induced by interleukin-1beta in chondrocytes in an intensity-dependent manner[J].Ultrasound Med Biol,2012,38(10):1726-1733.
[13]Xu SY,Zhang LM,Yao XM,et al.Effects and mechanism of low-intensity pulsed ultrasound on extracellular matrix in rabbit knee osteoarthritis[J].Zhongguo Gu Shang,2014,27(9):766-771.[徐守宇,张丽梅,姚新苗,等.低强度脉冲超声对兔膝骨关节炎软骨细胞外基质的影响及机制[J].中国骨伤,2014,27(9):766-771.]
[14]Gurkan I,Ranganathan A,Yang X,et al.Modification of osteoarthritis in the guinea pig with pulsed low-intensity ultrasound treatment[J].Osteoarthritis Cartilage,2010,18(5):724-733.
[15]Enomoto T,Akagi R,Ogawa Y,et al.Timing of intra-articular injection of synovial mesenchymal stem cells affects cartilage restoration in a partial thickness cartilage defect model in rats[J].Cartilage,2018 Jul 1:1947603518786542.doi:10.1177/1947603518786524.
[16]Redondo ML,Beer AJ,Yanke AB.Cartilage restoration:microfracture and osteochondral autograft transplantation[J].JKnee Surg,2018,31(3):231-238.
[17]Yu Y,Brouillette MJ,Seol D,et al.Use of recombinant human stromal cell-derived factor 1α-loaded fibrin/hyaluronic acid hydrogel networks to achieve functional repair of full-thickness bovine articular cartilage via homing of chondrogenic progenitor cells[J].Arthritis Rheumatol,2015,67(5):1274-1285.
[18]Seol D,McCabe DJ,Choe H,et al.Chondrogenic progenitor cells respond to cartilage injury[J].Arthritis Rheum,2012,64(11):3626-3637.
[19]Jang KW,Ding L,Seol D,et al.Low-intensity pulsed ultrasound promotes chondrogenic progenitor cell migration via focal adhesion kinase pathway[J].Ultrasound Med Biol,2014,40(6):1177-1186.
[20]Liu J,Song W,Yuan T,et al.A comparison between plateletrich plasma(PRP)and hyaluronate acid on the healing of cartilage defects[J].PLoS One,2014,9(5):e97293-e97293.
[21]Yang W,Cao Y,Zhang Z,et al.Targeted delivery of FGF2to subchondral bone enhanced the repair of articular cartilage defect[J].Acta Biomater,2018,69:170-182.
[22]Zhang Z,Li L,Yang W,et al.The effects of different doses of IGF-1 on cartilage and subchondral bone during the repair of full-thickness articular cartilage defects in rabbits[J].Osteoarthritis Cartilage,2017,25(2):309-320.
[23]Zhang Z,Yang W,Cao Y,et al.The functions of BMP3 in rabbit articular cartilage repair[J].Int J Mol Sci,2015,16(11):25934-25946.
[24]Zhao RL,Zhang XM,Jia LN,et al.(p)NNS-conjugated chitosan mediated IGF-1 and miR-140 overexpression in articular chondrocytes improves cartilage repair[J].Biomed Res Int,2019,2019:2761241-2761241.
[25]Takeuchi R,Ryo A,Komitsu N,et al.Low-intensity pulsed ultrasound activates the phosphatidylinositol 3 kinase/Akt pathway and stimulates the growth of chondrocytes in threedimensional cultures:a basic science study[J].Arthritis Res Ther,2008,10(4):R77-R77.
[26]Hoshiyama Y,Otsuki S,Oda S,et al.Chondrocyte clusters adjacent to sites of cartilage degeneration have characteristics of progenitor cells[J].J Orthop Res,2015,33(4):548-555.
[27]Zhang ZJ,Huckle J,Francomano CA,et al.The effects of pulsed low-intensity ultrasound on chondrocyte viability,proliferation,gene expression and matrix production[J].Ultrasound Med Biol,2003,29(11):1645-1651.
[28]Li X,Li J,Cheng K,Lin Q,et al.Effect of low-intensity pulsed ultrasound on MMP-13 and MAPKs signaling pathway in rabbit knee osteoarthritis[J].Cell Biochem Biophys,2011,61(2):427-434.
[2]Naito K,Watari T,Muta T,et al.Low-intensity pulsed ultrasound(LIPUS)increases the articular cartilage typeⅡcollagen in a rat osteoarthritis model[J].J Orthop Res,2010,28(3):361-369.
[3]Uddin SM,Richbourgh B,Ding Y,et al.Chondro-protective effects of low intensity pulsed ultrasound[J].Osteoarthritis Cartilage,2016,24(11):1989-1998.
[4]Xia P,Shen S,Lin Q,et al.Low-intensity pulsed ultrasound treatment at an early osteoarthritis stage protects rabbit cartilage from damage via the integrin/focal adhesion kinase/mitogenactivated protein kinase signaling pathway[J].J Ultrasound Med,2015,34(11):1991-1999.
[5]Xia P,Ren S,Lin Q,et al.Low-intensity pulsed ultrasound affects chondrocyte extracellular matrix production via an integrin-mediated p38 MAPK signaling pathway[J].Ultrasound Med Biol,2015,41(6):1690-1700.
[6]Tan L,Ren Y,van Kooten TG,et al.Low-intensity pulsed ultrasound(LIPUS)and pulsed electromagnetic field(PEMF)treatments affect degeneration of cultured articular cartilage explants[J].Int Orthop,2015,39(3):549-557.
[7]Yuan LJ,Niu CC,Lin SS,et al.Effects of low-intensity pulsed ultrasound and hyperbaric oxygen on human osteoarthritic chondrocytes[J].J Orthop Surg Res,2014,9:5-5.
[8]Korstjens CM,van der Rijt RH,Albers GH,et al.Low-intensity pulsed ultrasound affects human articular chondrocytes in vitro[J].Med Biol Eng Comput,2008,46(12):1263-1270.
[9]Yilmaz V,Karadas魻,Dandinogˇlu T,et al.Efficacy of extracorporeal shockwave therapy and low-intensity pulsed ultrasound in a rat knee osteoarthritis model:A randomized controlled trial[J].Eur J Rheumatol,2017,4(2):104-108.
[10]Sekino J,Nagao M,Kato S,et al.Low-intensity pulsed ultrasound induces cartilage matrix synthesis and reduced MMP13 expression in chondrocytes[J].Biochem Biophys Res Commun,2018,506(1):290-297.
[11]Ji JB,Li XF,Liu L,et al.Effect of low intensity pulsed ultrasound on expression of TIMP-2 in serum and expression of MMP-13 in articular cartilage of rabbits with knee osteoarthritis[J].Asian Pac J Trop Med,2015,8(12):1043-1048.
[12]Ito A,Aoyama T,Yamaguchi S,et al.Low-intensity pulsed ultrasound inhibits messenger RNA expression of matrix metalloproteinase-13 induced by interleukin-1beta in chondrocytes in an intensity-dependent manner[J].Ultrasound Med Biol,2012,38(10):1726-1733.
[13]Xu SY,Zhang LM,Yao XM,et al.Effects and mechanism of low-intensity pulsed ultrasound on extracellular matrix in rabbit knee osteoarthritis[J].Zhongguo Gu Shang,2014,27(9):766-771.[徐守宇,张丽梅,姚新苗,等.低强度脉冲超声对兔膝骨关节炎软骨细胞外基质的影响及机制[J].中国骨伤,2014,27(9):766-771.]
[14]Gurkan I,Ranganathan A,Yang X,et al.Modification of osteoarthritis in the guinea pig with pulsed low-intensity ultrasound treatment[J].Osteoarthritis Cartilage,2010,18(5):724-733.
[15]Enomoto T,Akagi R,Ogawa Y,et al.Timing of intra-articular injection of synovial mesenchymal stem cells affects cartilage restoration in a partial thickness cartilage defect model in rats[J].Cartilage,2018 Jul 1:1947603518786542.doi:10.1177/1947603518786524.
[16]Redondo ML,Beer AJ,Yanke AB.Cartilage restoration:microfracture and osteochondral autograft transplantation[J].JKnee Surg,2018,31(3):231-238.
[17]Yu Y,Brouillette MJ,Seol D,et al.Use of recombinant human stromal cell-derived factor 1α-loaded fibrin/hyaluronic acid hydrogel networks to achieve functional repair of full-thickness bovine articular cartilage via homing of chondrogenic progenitor cells[J].Arthritis Rheumatol,2015,67(5):1274-1285.
[18]Seol D,McCabe DJ,Choe H,et al.Chondrogenic progenitor cells respond to cartilage injury[J].Arthritis Rheum,2012,64(11):3626-3637.
[19]Jang KW,Ding L,Seol D,et al.Low-intensity pulsed ultrasound promotes chondrogenic progenitor cell migration via focal adhesion kinase pathway[J].Ultrasound Med Biol,2014,40(6):1177-1186.
[20]Liu J,Song W,Yuan T,et al.A comparison between plateletrich plasma(PRP)and hyaluronate acid on the healing of cartilage defects[J].PLoS One,2014,9(5):e97293-e97293.
[21]Yang W,Cao Y,Zhang Z,et al.Targeted delivery of FGF2to subchondral bone enhanced the repair of articular cartilage defect[J].Acta Biomater,2018,69:170-182.
[22]Zhang Z,Li L,Yang W,et al.The effects of different doses of IGF-1 on cartilage and subchondral bone during the repair of full-thickness articular cartilage defects in rabbits[J].Osteoarthritis Cartilage,2017,25(2):309-320.
[23]Zhang Z,Yang W,Cao Y,et al.The functions of BMP3 in rabbit articular cartilage repair[J].Int J Mol Sci,2015,16(11):25934-25946.
[24]Zhao RL,Zhang XM,Jia LN,et al.(p)NNS-conjugated chitosan mediated IGF-1 and miR-140 overexpression in articular chondrocytes improves cartilage repair[J].Biomed Res Int,2019,2019:2761241-2761241.
[25]Takeuchi R,Ryo A,Komitsu N,et al.Low-intensity pulsed ultrasound activates the phosphatidylinositol 3 kinase/Akt pathway and stimulates the growth of chondrocytes in threedimensional cultures:a basic science study[J].Arthritis Res Ther,2008,10(4):R77-R77.
[26]Hoshiyama Y,Otsuki S,Oda S,et al.Chondrocyte clusters adjacent to sites of cartilage degeneration have characteristics of progenitor cells[J].J Orthop Res,2015,33(4):548-555.
[27]Zhang ZJ,Huckle J,Francomano CA,et al.The effects of pulsed low-intensity ultrasound on chondrocyte viability,proliferation,gene expression and matrix production[J].Ultrasound Med Biol,2003,29(11):1645-1651.
[28]Li X,Li J,Cheng K,Lin Q,et al.Effect of low-intensity pulsed ultrasound on MMP-13 and MAPKs signaling pathway in rabbit knee osteoarthritis[J].Cell Biochem Biophys,2011,61(2):427-434.