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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: [email protected]

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:e61–e69 | 0278-4297 | www.aium.org

ORIGINAL RESEARCH

Objectives—To 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.

Methods—Thirty-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 β1, phosphorylatedFAK, and MAPKs (including extracellular signal-regulated kinase 1/2, MAPK 38, andc-Jun N-terminal kinase) was assessed by Western blot analysis.

Results—Cartilage 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 β1 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.

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

Key Words—focal 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.6–8

Integrin, a heterodimeric glycoprotein composed ofdifferent αand βsubunits, is one of the mechanical receptorson the cell surface,9 bridging signaling pathways from theextracellular matrix to cells.10 Of note, integrin α5β1 is themost common integrin on the adult chondrocyte cellmembrane, and integrin β1, 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.18

Furthermore, 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 β1, FAK, phosphorylated FAK, ERK1/2, phos-phorylated ERK1/2, p38, phosphorylated p38, JNK,phosphorylated JNK, and β-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 al—Low-Intensity Pulsed Ultrasound Treatment of Early Osteoarthritis

J Ultrasound Med 2015; 34:e61–e69e62


Surgical ProceduresAnterior cruciate ligament transection of the rabbit osteo -arthritis model was performed as described previously.19,20

Briefly, 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 relocated, and the wound was closed with 4-0 braidedabsorbable polyglactin 910 sutures. Penicillin and fentanylwere given to prevent bacterial infection and pain, respec-tively, after the incised skin was closed. Early and late osteo -arthritis was defined according to the cartilage change andMankin score as in a previous study, and early and late rabbitosteoarthritis models were established after 4 and 8 weeksof anterior cruciate ligament transection, respectively.20,21

The control groups underwent sham operations (the skinwas cut and the joint capsule exposed, but the anteriorcruciate ligament was not transected). The early and latecontrol groups were established 4 and 8 weeks after shamsurgery.

Postoperative InterventionFor the early and late treatment groups, low-intensitypulsed US was applied 4 and 8 weeks after anterior cruciateligament transection, respectively. Low-intensity pulsedUS (HT2009-1; Ito Corporation, Tokyo, Japan) was appliedas follows: free mode, on-off ratio of 20%, frequency of3 MHz, irradiation intensity of 40 mW/cm2, irradiationtime of 20 minutes, and treatment frequency of once perday at 6 d/wk for 6 consecutive weeks. For the sham earlyand late osteoarthritis groups, the exposure intensity,time, and duration were the same as for the early and latetreatment groups, but without US output.

Post-Therapeutic AnalysisRabbits were euthanized after US exposure for 6 weeks,and the knee joint in each animal was immediately opened.Improvement in the femoral condyle articular surface wasassessed by Mankin scores as described previously.22

Histopathologic Analysis The femoral condyle articular cartilage collected from theknee joints was fixed in neutral formalin, decalcified inEDTA for 3 weeks, embedded in paraffin, and divided into4-μm-thick sections with a microtome. All samples wereprocessed simultaneously. The knee joint cartilage specimenswere examined under a microscope for pathologic changes,

including surface irregularities, decreased hematoxylin-eosin staining of articular cartilage, and formation of cracks.Fibrosis, matrix distribution, cartilage loss, and chondro-cyte colonization were evaluated in a double-blind fashionby 2 independent experts using the Mankin score system(Table 1).

Western Blot AnalysisExpression of type II collagen, MMP-13, integrin β1, FAK,phosphorylated FAK, p38, phosphorylated p38, ERK1/2,phosphorylated ERK1/2, JNK, phosphorylated JNK, andβ-actin proteins was determined by Western blot analysis.Briefly, the rabbits were euthanized by overdose injectionsof sodium pentobarbital. Under sterile conditions, thefemoral condyle articular cartilage in the knee (≈50 g), waspulverized into powder in liquid nitrogen; then the lysisbuffer in the protein extraction kit (500 μL) was added, andsamples were centrifuged at 16,000g and 4°C for 10 minutes.The cell lysates (40 μg each) were separated by sodiumdodecyl sulfate–polyacrylamide gel electrophoresis, andthe separated proteins were electroblotted onto nitrocel-lulose membranes. After blocking with skim milk for 2hours, the membranes were incubated with primary anti-bodies against type II collagen (1:500), MMP-13 (1:500),integrin β1 (1:500), FAK (1:500), phosphorylated FAK(1:500), ERK1/2 (1:1000), phosphorylated ERK1/2

J Ultrasound Med 2015; 34:e61–e69 e63


Table 1. Mankin Scoring Scale

Subgroup 1: fibrillation

1. Even surface

2. Uneven surface

3. Fibrillated and fissured within superficial zone only

4. Fissures and erosions extending below surface zone, without

extending beyond radial zone

5. Fissures and erosions extending into deeper zone

Subgroup 2: matrix distribution

1. Normal staining

2. Moderate loss in staining

3. Severe loss in staining

4. No staining

Subgroup 3: chondrocyte loss

1. Loss extending into superficial zone

2. Loss extending into mid zone

3. Loss extending into radial zone

Subgroup 4: chondrocyte cloning

1. No clusters

2. Chondrocyte clusters in superficial zone

3. Chondrocyte clusters in superficial to mid zone (<4 cells)

4. Chondrocyte clusters of >4 cells located in superficial to mid

zone or chondrocyte clusters in deeper zone

Grading was performed separately for the medial femoral condyle,

lateral femoral condyle, medial tibial plateau, and lateral tibial plateau.

The minimum total score was 4.


(1:1000), JNK (1:1000), phosphorylated JNK (1:1000),p38 (1:1000), phosphorylated p38 (1:1000), and β-actin(1:500) at 4°C overnight. Next, the membranes were incu-bated with a peroxidase-conjugated secondary antibody(1:5000) at 37°C for 2 hours, and the protein bands weredeveloped by a specific chromogenic reaction accordingto the manufacturer’s instructions.

Statistical AnalysisAll data are expressed as mean ± standard deviation andwere analyzed with SPSS version 18.0 software (IBM Cor-poration, Armonk, NY). Differences between groups wereanalyzed by single-factor analysis of variance. ModifiedMankin scores were analyzed by a Wilcoxon signed ranktest. P < .05 was considered statistically significant.

Results

Pathologic Changes in Early and Late OsteoarthritisCartilage Before and After Low-Intensity Pulsed USExposureAfter 6 weeks of low-intensity pulsed US exposure,osteoarthritis cartilage was harvested for hematoxylin-eosinstaining and then observed under a microscope to comparethe morphologic differences and Mankin scores among thedifferent groups. The results showed that the cartilage sur-face in the early and late control groups was regular and even,with no obvious cartilage fibrosis and a well-stained extra-cellular matrix. Chondrocytes were evenly arrayed and wellorganized within the extracellular matrix. In the early osteo -arthritis group, the surface was slightly uneven and irregular;cartilage fibrosis was slight; staining was slightly to moder-ately lost; chondrocytes were disarranged; and the numberof the chondrocytes was reduced compared to the earlycontrol group. In the late osteoarthritis group, there weresome obvious disruptions on the surface of the cartilage, asevere loss of staining, severe cartilage fibrosis, and decreasedchondrocytes compared to the early osteoarthritis group.After low-intensity pulsed US exposure, a slightly unevencartilage surface and chondrocyte proliferation in the earlytreatment group were observed, but in the late treatmentgroup, cartilage damage was almost the same as that in thein late osteoarthritis group (Figure 1).

The Mankin score in the early treatment group wasmuch higher than that in the early control group (P < .05)but was obviously lower than that in the early osteoarthritisgroup (P < .05). There was no difference in Mankin scoresbetween the late treatment and late osteoarthritis groups(P > .05), but both scores were significantly higher thanthat in the late control group (P < .05; Figure 1).

Type II Collagen, MMP-13, and Integrin β1 Expressionand Phosphorylation Level of FAK Before and AfterLow-Intensity Pulsed US Exposure in Early and LateOsteoarthritis CartilageCompared to the early osteoarthritis group, type II collagenexpression in the early treatment group was significantlyincreased (P < .05), but MMP-13 expression was signifi-cantly decreased (P < .05). No significant difference in theexpression of the two proteins was found between the latetreatment and late osteoarthritis groups (P > .05). The inte-grin β1 and phosphorylated FAK levels were significantlyhigher in the early treatment group than the early osteo -arthritis and early control groups (P < .05), whereas theintegrin β1 and phosphorylated FAK levels in the late treat-ment and late osteoarthritis groups were significantlydecreased (P < .05). No significant difference in integrin β1or phosphorylated FAK expression was found betweenthe late treatment and late osteoarthritis groups (P > .05;Figure 2).

Phosphorylated ERK1/2, JNK, and p38 ExpressionBefore and After Low-Intensity Pulsed US Exposure in Early and Late Osteoarthritis CartilagePhosphorylated ERK1/2 and p38 expression in the earlytreatment group was significantly lower compared to theearly osteoarthritis group (P < .05). Compared to the latecontrol group, phosphorylated ERK1/2 and p38 expressionin the late osteoarthritis and late treatment groups wassignificantly increased (P < .05), but no significant differ-ence in the expression of the two phosphorylated proteinswas found between the late osteoarthritis and late treat-ment groups (P > .05). There was no significant differencein phosphorylated JNK expression between early andlate osteo arthritis cartilage before and after US exposure(P > .05; Figure 3).

Discussion

It is important to better understand the signaling pathwaywhereby a correct low-intensity pulsed US interventiontime for osteoarthritis therapy can be implemented;however, to our knowledge, there has been no study of theintegrin/FAK/MAPK signaling pathway by whichlow-intensity pulsed US acts on both early and late osteo -arthritis. In this study, different protective effects of low-intensity pulsed US on early and late osteoarthritis via theintegrin/FAK/MAPK signaling pathway in cartilagerepair was investigated using established early and lateosteoarthritis models by anterior cruciate ligament tran-section. Early low-intensity pulsed US treatment was




found to prevent cartilage damage, especially at the earlystage of osteoarthritis, by increasing type II collagen anddecreasing MMP-13 levels and upregulating integrin β1and phosphorylated FAK and downregulating phospho-rylated ERK1/2 and p38 in the signaling pathway.

Our findings demonstrate that the low-intensitypulsed US/integrin/FAK/MAPK mechanotransductionpathway plays an important role during the pathologicprocess of osteoarthritis by regulating extracellular matrixexpression in early and late osteoarthritis cartilage. Type II



Figure 1. Morphologic changes and Mankin scores for early- and late-stage osteoarthritis cartilage in each group. Morphologic changes were

revealed by hematoxylin-eosin staining and observed under a microscope. EC indicates early control group; EO, early osteoarthritis group; ET, early

treatment group; LC, late control group; LO, late osteoarthritis group; and LT, late treatment group (n = 6 per group). *P < .05.


collagen expression was found to be significantly increased,but MMP-13 expression was significantly decreased afterapplication of low-intensity pulsed US in early osteoarthritiscartilage. However, in late osteoarthritis cartilage, US appli-cation had no effects. Type II collagen is an important extra-cellular matrix component and maintains cartilage integrity23;MMP-13 is an important inflammatory factor in the MMPfamily that degrades the type II collagen components,2which destroys joint cartilage, causing osteoarthritis.1,3,24,25

Western blot results showed higher expression ofMMP-13, integrin β1, phosphorylated FAK, ERK1/2, JNK,and p38 in the early osteoarthritis group compared to theearly control group. In the late osteoarthritis group, inte-grin β1 and phosphorylated FAK expression was at a lowerlevel, and phosphorylated ERK1/2, JNK, and p38 expres-sion was at a higher level compared to the late control group.These results demonstrate that the variable expressionlevels of proteins in the integrin/FAK/MAPK signaling



Figure 2. Type II collagen, MMP-13, integrin β1, and phosphorylated

FAK expression before and after low-intensity pulsed US exposure in

early- and late-stage osteoarthritis cartilage. A, Western blot analysis of

type II collagen, MMP-13, integrin β1, FAK, and phosphorylated (p) FAK

with β-actin as a loading control. B, Quantitative analysis of type II col-

lagen. C, Quantitative analysis of MMP-13. D, Quantitative analysis of

integrin β1. E, Quantitative analysis of phosphorylated FAK. Abbrevia-

tions are as in Figure 1. *P < .05.


pathway are closely related to pathologic changes in earlyand late osteoarthritis cartilage. In fact, integrin β1, which isthe major subunit of integrin in chondrocyte membranes,26

plays an important role in transmitting mechanical andchemical signals through signaling pathways in cartilage.11

Integrin β1 is involved in the process that regulates prolif-eration, differentiation, extension, and migration of chon-drocytes, thus providing cartilage-protective effects.27,28

Focal adhesion kinase is a vital mediator in the inte-grin/FAK/MAPK mechanotransduction pathway.29

Phosphorylated FAK regulates proliferation and differen-tiation of chondrocytes and type II collagen expression.30

Furthermore, phosphorylated FAK inhibits MAPK signal-ing pathway proteins, including ERK1/2, JNK, and p38.31

Mitogen-activated protein kinases are associated withextracellular matrix synthesis and cartilage stabilization,32,33

and upregulation of the MAPK signaling pathway leads tocartilage dysfunction, accelerating the disease developmentprocess.34 Extracellular signal-regulated kinase 1/2, JNK,and p38 are related to chondrosteosis and play an impor-tant role in hypertrophy, calcification, and apoptosis ofchondrocytes.14,28 Phosphorylated ERK1/2, JNK, and p38have a negative impact on cartilage extracellular matrix reg-ulation and are extensively involved in signal transductionand cartilage degeneration.35

We found that exposure to early low-intensity pulsedUS treatment promoted integrin β1 and phosphorylatedFAK expression and downregulation of phosphorylated



Figure 3. Phosphorylated (p) ERK1/2, p38, and JNK expression

before and after low-intensity pulsed US exposure in early- and late-

stage osteoarthritis cartilage. A, Western blot analysis of total and

phosphorylated ERK1/2, p38, and JNK. B, Quantitative analysis

of phosphorylated ERK1/2 with total ERK1/2 as a loading control.

C, Quantitative analysis of phosphorylated p38 with total p38 as a

loading control. D, Quantitative analysis of phosphorylated JNK

with total JNK as a loading control. Abbreviations are as in Figure 1.

*P < .05.


ERK1/2 and p38 expression, leading to substantial pro-tective effects against cartilage damage. In contrast, latelow-intensity pulsed US treatment downregulated integrinβ1 and upregulated phosphorylated FAK, ERK1/2, andp38 expression slightly. These changes had no statisticalsignificance but suggested that late low-intensity pulsedUS treatment did not provide remarkable protective effectsagainst cartilage damage and may have had negative effectson late-stage osteoarthritis. These findings are in line withprevious studies in which low-intensity pulsed US wasfound to increase integrin β1 expression, induce phospho-rylated FAK, trigger ERK1/2 and p38 signaling pathways,and promote type II collagen synthesis.36,37

In conclusion, cartilage-protective effects from earlylow-intensity pulsed US treatment of osteoarthritis via theintegrin/FAK/MAPK signaling pathway is time sensitive,and optimal intervention timing may be important for pro-tecting or even repairing osteoarthritis cartilage. As low-intensity pulsed US is effective in protecting osteoarthritischondrocytes from degeneration by upregulation of thePI3K/Akt pathway,17 the question of whether low-intensitypulsed US functions by upregulation of the PI3K/Aktpathway and downregulation of the MAPK pathway at thesame time should be investigated in the future. Currently,low-intensity pulsed US is still not well accepted for treat-ing osteoarthritis in clinical practice. Our findings may pro-vide further evidence for the application of low-intensitypulsed US to clinical therapy, especially at an early stage ofosteoarthritis.

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