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Low-Intensity Pulsed Ultrasound Treatment at an Early OsteoarthritisStage Protects Rabbit Cartilage From Damage via the Integrin/FocalAdhesion Kinase/Mitogen-ActivatedProtein Kinase Signaling PathwayPeng Xia, MS, Shihao Shen, MS, Qiang Lin, MS, Kai Cheng, MS, Shasha Ren, MS, Mingxia Gao, MS,Xueping Li, PhD

Received October 10, 2014, from the Departmentof Rehabilitation Medicine, Nanjing First Hospital,Nanjing Medical University, Nanjing, China.Revision requested December 4, 2014. Revisedmanuscript accepted for publication February 10,2015.

We thank Medjaden Bioscience Limited(Wuhan, China) for assistance with manuscriptpreparation. This work was supported by theNational Science Foundation of China (grant81272151). Peng Xia and Shihao Shen contributedequally to this work.

Address correspondence to Xueping Li, PhD,Department of Rehabilitation Medicine, NanjingFirst Hospital, Nanjing Medical University,210006 Nanjing, China.

E-mail: lixueping6504@163.com

AbbreviationsAkt, protein kinase B; ERK, extracellularsignal-regulated kinase; FAK, focal adhesionkinase; JNK, c-Jun N-terminal kinase; MAPK,mitogen-activated protein kinase; MMP,matrix metalloproteinase; p38, mitogen- activated protein kinase 38; PI3K, phospha -tidylinositol 3-kinase; US, ultrasound

2015 by the American Institute of Ultrasound in Medicine | J Ultrasound Med 2015; 34:e61e69 | 0278-4297 | www.aium.org

ORIGINAL RESEARCH

ObjectivesTo investigate whether low-intensity pulsed ultrasound (US) has differentprotective effects on early and late rabbit osteoarthritis cartilage via the integrin/focaladhesion kinase (FAK)/mitogen-activated protein kinase (MAPK) signaling pathway.

MethodsThirty-six New Zealand White rabbits were divided into early control, earlyosteoarthritis, early treatment, late control, late osteoarthritis, and late treatment groups.The early and late osteoarthritis and treatment groups underwent anterior cruciate lig-ament transection. The remaining groups underwent sham operations with knee jointexposure. The early and late treatment groups were exposed to low-intensity pulsed US4 and 8 weeks after surgery. After 6 weeks of US exposure, pathologic changes on thearticular surface of the femoral condyle were assessed by modified Mankin scores.Expression of type II collagen, matrix metalloproteinase, integrin 1, phosphorylatedFAK, and MAPKs (including extracellular signal-regulated kinase 1/2, MAPK 38, andc-Jun N-terminal kinase) was assessed by Western blot analysis.

ResultsCartilage damage was less severe in the early treatment group than the earlyosteoarthritis group. The Mankin score was significantly lower in the early treatmentgroup than the early osteoarthritis group (P < .05). There was no significant differencein cartilage damage or Mankin score between the late treatment and late osteoarthritisgroups. There was a significant increase in type II collagen expression but a significantdecrease in matrix metalloproteinase 13 expression in the early treatment group com-pared to the early osteoarthritis group, whereas no significant difference was foundbetween the late treatment and late osteoarthritis groups. Integrin 1 and phosphory-lated FAK expression was significantly higher, and phosphorylated extracellular signal-regulated kinase 1/2 and phosphorylated MAPK 38 expression was significantly lowerin the early treatment group than the early osteoarthritis group.

ConclusionsOur findings indicate that low-intensity pulsed US protects cartilagefrom damage in early-stage osteoarthritis via the integrin/FAK/MAPK pathway.

Key Wordsfocal adhesion kinase; integrin; low-intensity pulsed ultrasound; mitogen-activated protein kinase; musculoskeletal ultrasound; osteoarthritis

doi:10.7863/ultra.14.10016steoarthritis is a multifactor-related degenerate disease whosemain feature is irreversible articular cartilage destruction.O

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Articular cartilage is composed of an extracellular matrixand a few chondrocytes. The extracellular matrix consists ofaffluent aggrecan and collagen, with type II collagen beingthe most abundant collagen and a typical marker of chon-drocytes.1 Matrix metalloproteinase 13 (MMP-13), animportant MMP, hydrolyzes the extracellular matrix,especially type II collagen.2 Extracellular matrix hydroly-sis is a crucial step leading to irreversible loss of the chon-drocyte extracellular matrix during cartilage degeneration.3Therefore, anabolism of the extracellular matrix is of impor-tance for maintenance of cartilage integrity.

Moderate mechanotransduction causes a series ofchanges in specific intracellular molecules and therebytheir downstream signaling pathways by stimulation ofmechanical loading, acceleration of chondrocyte differ-entiation and proliferation, and promotion of extracellu-lar matrix anabolism to maintain articular cartilageintegrity.4 Mechanical stress by low-intensity pulsedultrasound (US) produces a certain kind of mechanicalwave to activate osteoblasts and promote bone formationand strength.5 Low-intensity pulsed US promotes type IIcollagen synthesis for chondrocyte proliferation and carti-lage formation.68

Integrin, a heterodimeric glycoprotein composed ofdifferent and subunits, is one of the mechanical receptorson the cell surface,9 bridging signaling pathways from theextracellular matrix to cells.10 Of note, integrin 51 is themost common integrin on the adult chondrocyte cellmembrane, and integrin 1, mainly expressed on the chon-drocyte membrane, increases chondrocyte differentiationand maturation to promote cartilage formation and remod-eling via regulation of extracellular matrix synthesis.11

Integrin mediates the mechanotransduction pathwayby activating a crucial adhesive molecule, focal adhesionkinase (FAK), which activates FAK downstream signalingpathways, such as mitogen-activated protein kinase (MAPK)and phosphatidylinositol 3-kinase (PI3K)/protein kinaseB (Akt) pathways.12 The MAPK signaling pathway, whichincludes extracellular signal-regulated kinase 1/2 (ERK1/2),mitogen-activated protein kinase 38 (p38), and c-Jun N-terminal kinase (JNK), plays important roles in regulatingthe secretion of MMPs in chondrocytes.13 Upregulation ofthis signaling pathway affects the normal extracellularmatrix synthesis, causing cartilage damage.14,15 It was sug-gested in our previous studies16 that low-intensity pulsedUS can promote osteoarthritis cartilage repair by down-regulation of chondrocyte MMP-13 and p38 MAPK signal-ing pathways, and that low-intensity pulsed US protectsosteoarthritis chondrocytes by downregulation of theintegrin/Akt pathway.17

Although low-intensity pulsed US is effective in pro-tecting osteoarthritis chondrocytes from degeneration bydownregulation of either the MAPK16 or PI3K/Akt path-way17 and healing bone fractures, using low-intensity pulsedUS to repair cartilage damage is still limited clinically.18Furthermore, osteoarthritis is a chronic and progressivedisease; thus, choosing a correct stage for low-intensitypulsed US treatment is necessary. However, to our knowl-edge, there has been no study comparing the effect oflow-intensity pulsed US on early and late osteoarthritis. Also,the effect of low-intensity pulsed US on osteoarthritiscartilage via the integrin/FAK/MAPK signaling pathwayis not understood. The aim of this study was to investigatewhether low-intensity pulsed US has different protectiveeffects on early and late osteoarthritis via the integrin/FAK/MAPK signaling pathway in cartilage repair.

Materials and Methods

ReagentsPhosphate-buffered saline, a total protein extraction kit,and an enhanced chemiluminescence kit were purchasedfrom KeyGEN (Nanjing, China). Mouse antirabbitmonoclonal antibodies against type II collagen, MMP-13,integrin 1, FAK, phosphorylated FAK, ERK1/2, phos-phorylated ERK1/2, p38, phosphorylated p38, JNK,phosphorylated JNK, and -actin were purchased fromAcris (Herford, Germany). Goat antimouse (Fab)2 sec-ondary antibody was purchased from Santa Cruz Biotech-nology (Dallas, TX).

Experimental Animals and GroupingThirty-six 2-month-old healthy male New Zealand whiterabbits weighing 2.5 to 3.0 kg were purchased from theQinglongshan Experimental Animal Center (Nanjing,China). All animals were housed individually in cages undera 12-hour day and 12-hour night cycle with free access tofood and water. The experimental protocol was in accor-dance with the US National Institutes of Health guidelinesfor laboratory animals and approved by the NanjingMedical University Ethics Committee of Nanjing Hospital.

The rabbits were randomly and equally divided into 6groups, named early control, early osteoarthritis, early treat-ment, late control, late osteoarthritis, and late treatment.Rabbits in the early osteoarthritis, early treatment, lateosteoarthritis, and late treatment groups underwent anteriorcruciate ligament transection. The remaining groups under-went sham operations with knee joint exposure. The earlyand late treatment groups were exposed to low-intensitypulsed US 4 and 8 weeks after surgery.

Xia et alLow-Intensity Pulsed Ultrasound Treatment of Early Osteoarthritis

J Ultrasound Med 2015; 34:e61e69e62

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Surgical ProceduresAnterior cruciate ligament transection of the rabbit osteo -arthritis model was performed as described previously.19,20Briefly, rabbits were intravenously anesthetized with 3%sodium pentobarbital (1 mL/kg). The knee joint skin wasdisinfected with iodine after the fur was shaved, and a para-patellar skin incision was made on the medial side of the joint.The anterior cruciate ligament was transected with eyescissors, and a positive anterior drawer test result was usedto ensure complete transection of the ligament. The patellawas re

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