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Effects and mechanotransduction pathways of therapeutic ultrasound on healthy and osteoarthritic chondrocytes: a systematic review of in vitro studies

  • S. Oliveira
    Correspondence
    Address correspondence and reprint requests to: S. Oliveira, CMEMS – UMinho, University of Minho, 4800-058 Guimarães, Portugal.
    Affiliations
    CMEMS – UMinho, University of Minho, 4800-058 Guimarães, Portugal

    LABBELS – Associate Laboratory, Braga, Guimarães, Portugal
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  • R. Andrade
    Affiliations
    Clínica Espregueira – FIFA Medical Centre of Excellence, Porto, Portugal

    Dom Henrique Research Centre, Porto, Portugal

    Porto Biomechanics Laboratory (LABIOMEP), Faculty of Sports, University of Porto, Porto, Portugal
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  • F.S. Silva
    Affiliations
    CMEMS – UMinho, University of Minho, 4800-058 Guimarães, Portugal

    LABBELS – Associate Laboratory, Braga, Guimarães, Portugal
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  • J. Espregueira-Mendes
    Affiliations
    Clínica Espregueira – FIFA Medical Centre of Excellence, Porto, Portugal

    Dom Henrique Research Centre, Porto, Portugal

    ICVS/3B's-PT Government Associate Laboratory, Braga/Guimarães, Portugal

    3B's Research Group-Biomaterials, Biodegradables and Biomimetics, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, University of Minho, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra, Barco, 4805-017 Guimarães, Portugal

    School of Medicine, University of Minho, Braga, Portugal
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  • B.B. Hinckel
    Affiliations
    Department of Orthopaedic Surgery, William Beaumont Hospital, Royal Oak, MI, USA
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  • A. Leal
    Affiliations
    CMEMS – UMinho, University of Minho, 4800-058 Guimarães, Portugal

    LABBELS – Associate Laboratory, Braga, Guimarães, Portugal

    Dom Henrique Research Centre, Porto, Portugal
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  • Ó. Carvalho
    Affiliations
    CMEMS – UMinho, University of Minho, 4800-058 Guimarães, Portugal

    LABBELS – Associate Laboratory, Braga, Guimarães, Portugal
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Open AccessPublished:December 05, 2022DOI:https://doi.org/10.1016/j.joca.2022.07.014

      Summary

      Objective

      To investigate the effects and mechanotransduction pathways of therapeutic ultrasound on chondrocytes.

      Method

      PubMed, EMBASE and Web of Science databases were searched up to 19th September 2021 to identify in vitro studies exploring ultrasound to stimulate chondrocytes for osteoarthritis (OA) treatment. Study characteristics, ultrasound parameters, in vitro setup, and mechanotransduction pathways were collected. Risk of bias was judged using the Risk of Bias Assessment for Non-randomized Studies (RoBANS) tool.

      Results

      Thirty-one studies were included comprising healthy and OA chondrocytes and explants. Most studies had high risk of performance, detection and pseudoreplication bias due to lack of temperature control, setup calibration, inadequate semi-quantitatively analyzes and independent experiments. Ultrasound was applied to the culture plate via acoustic gel, water bath or culture media. Regardless of the setup used, ultrasound stimulated the cartilage production and suppressed its degradation, although the effect size was nonsignificant. Ultrasound inhibited p38, c-Jun N-terminal kinases (JNK) and factor nuclear kappa B (NFκB) pathways in OA chondrocytes to reduce apoptosis, inflammation and matrix degradation, while triggered phosphoinositide-3-kinase/akt (PI3K/Akt), extracellular signal-regulated kinase (ERK), p38 and JNK pathways in healthy chondrocytes to promote matrix synthesis.

      Conclusion

      The included studies suggest that ultrasound application induces therapeutic effects on chondrocytes. However, these results should be interpreted with caution because high risk of performance, detection and pseudoreplication bias were identified. Future studies should explore the application of ultrasound on human OA chondrocytes cultures to potentiate the applicability of ultrasound towards cartilage regeneration of knee with OA.

      Keywords

      Introduction

      The articular cartilage is constantly subjected to mechanical loading. Chondrocytes sense mechanical deformation and respond by maintaining a healthy balance between synthesis and degradation of the cartilage matrix
      • Sanchez-adams J.
      • Leddy H.A.
      • Mcnulty A.L.
      • Guilak F.
      • Hill C.
      The mechanobiology of articular cartilage: bearing the burden of osteoarthritis.
      . Although mechanical stimulation is essential for cartilage health, excessive loading may result in cartilage degeneration and osteoarthritis (OA)
      • Qu P.
      • Qi J.
      • Han Y.
      • Zhou L.
      • Xie D.
      • Song H.
      • et al.
      Effects of rolling-sliding mechanical stimulation on cartilage preserved in vitro.
      . In arthritic joints, the catabolic activity of chondrocytes prevails, increasing the production of matrix-degrading proteases and pro-inflammatory cytokines
      • Loeser R.F.
      • Goldring S.R.
      • Scanzello C.R.
      • Goldring M.B.
      Osteoarthritis: a disease of the joint as an organ.
      . Chondrocytes further undergo hypertrophy-like changes, apoptosis and the calcified cartilage is intensified, being later replaced by bone
      • Van Der Kraan P.M.
      • Van Den Berg W.B.
      Chondrocyte hypertrophy and osteoarthritis: role in initiation and progression of cartilage degeneration ?.
      .
      Ultrasound is a form of acoustic mechanical energy at frequencies above human audibility. Commonly used for diagnostic imaging purposes, it can also be used to mechanically stimulate human biological tissues (therapeutic ultrasound)
      • Jiang X.
      • Savchenko O.
      • Li Y.
      • Qi S.
      • Yang T.
      • Zhang W.
      • et al.
      A review of low-intensity pulsed ultrasound for therapeutic applications.
      . Ultrasound generates mechanical vibrations that propagate in human body tissues
      • Khanna A.
      • Nelmes R.T.C.
      • Gougoulias N.
      • Maffulli N.
      • Gray J.
      The effects of LIPUS on soft-tissue healing: a review of literature.
      , inducing temperature increase, cavitation, acoustic radiation force and streaming
      • Haar G Ter
      Ultrasound bioeffects and safety.
      . Despite being approved only for bone fractures treatment by the Food and Drug Administration (FDA)
      • Schandelmaier S.
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      • Lytvyn L.
      • Heels-Ansdell D.
      • Siemieniuk R.A.C.
      • Agoritsas T.
      • et al.
      Low intensity pulsed ultrasound for bone healing: systematic review of randomized controlled trials.
      ,
      • Palanisamy P.
      • Alam M.
      • Li S.
      • Chow S.K.H.
      • Zheng Y.P.
      Low-intensity pulsed ultrasound stimulation for bone fractures healing: a review.
      , there is a growing interest in the use of therapeutic ultrasound to restore articular cartilage damage.
      Current evidence suggests that therapeutic ultrasound is a promising addition to the treatment of knee OA due to its potential effect on cartilage regeneration, pain relief and improved physical function
      • Wu Y.
      • Zhu S.
      • Lv Z.
      • Kan S.
      • Wu Q.
      • Song W.
      • et al.
      Effects of therapeutic ultrasound for knee osteoarthritis: a systematic review and meta-analysis.
      • Naito K.
      • Watari T.
      • Muta T.
      • Furuhata A.
      • Iwase H.
      • Igarashi M.
      • et al.
      Low-intensity pulsed ultrasound (LIPUS) increases the articular cartilage type II collagen in a rat osteoarthritis model.
      • Zahoor T.
      • Mitchell R.
      • Bhasin P.
      • Guo Y.
      • Paudel S.
      • Schon L.
      • et al.
      Effect of low-intensity pulsed ultrasound on joint injury and post-traumatic osteoarthritis: an animal study.
      . Nevertheless, ultrasound is not included in guidelines for knee OA management owing to inconsistent literature regarding its efficacy in in vivo or clinical studies
      • Uddin S.M.Z.
      • Komatsu D.E.
      Therapeutic potential low-intensity pulsed ultrasound for osteoarthritis: pre-clinical and clinical perspectives.
      • Rothenberg J.B.
      • Jayaram P.
      • Naqvi U.
      • Gober J.
      • Malanga G.A.
      The role of low-intensity pulsed ultrasound on cartilage healing in knee osteoarthritis: a review.
      • Kolasinski S.L.
      • Neogi T.
      • Hochberg M.C.
      • Oatis C.
      • Guyatt G.
      • Block J.
      • et al.
      2019 American College of Rheumatology/Arthritis Foundation guideline for the management of osteoarthritis of the hand, hip, and knee.
      • Geenen R.
      • Overman C.L.
      • Christensen R.
      • Åsenlöf P.
      • Capela S.
      • Huisinga K.L.
      • et al.
      EULAR recommendations for the health professional's approach to pain management in inflammatory arthritis and osteoarthritis.
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      • Bennell K.
      • Bierma-Zeinstra S.M.A.
      • et al.
      OARSI guidelines for the non-surgical management of knee, hip, and polyarticular osteoarthritis.
      • Loyola-Sánchez A.
      • Richardson J.
      • MacIntyre N.J.
      Efficacy of ultrasound therapy for the management of knee osteoarthritis: a systematic review with meta-analysis.
      . In vitro studies can add relevant knowledge since they explore the intracellular mechanisms by which ultrasound elicits therapeutic effects on chondrocytes in terms of proliferation, viability and matrix production. A well-defined and robust systematic review of in vitro studies on this topic is thus warranted as a starting point for a more comprehensive and rigorous implementation of ultrasound in both experimental and clinical settings.
      This systematic review aims to summarize and comprehensively analyze the effects of ultrasound on articular cartilage and chondrocytes in in vitro studies.

      Methods

      This systematic review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines
      • Page M.J.
      • McKenzie J.E.
      • Bossuyt P.M.
      • Boutron I.
      • Hoffmann T.C.
      • Mulrow C.D.
      • et al.
      The PRISMA 2020 statement: an updated guideline for reporting systematic reviews.
      . A protocol for this systematic review was not a priori registered.

      Search strategy

      The databases PubMed, EMBASE and Web of Science were searched from database inception up to 19th September 2021 to identify in vitro studies that assessed the effects of therapeutic ultrasound on chondrocytes for OA treatment. The search strategy is provided in Appendix 1. The reference list of the most significant studies was also screened to further identify potential other eligible studies.

      Study selection

      All records were exported to an Excel file (Microsoft® Office) and duplicates were removed by the software filter and then manually verified. Two authors (S.O. and R.A.) independently screened the titles and abstracts of all identified studies and assessed them for eligibility criteria of the most relevant studies full texts. Three other reviewers (B.B.H., A.L. and O.C.) were consulted in case of disagreement. The inclusion criteria were: 1) in vitro studies; 2) studies using chondrocytes or articular cartilage explants; 3) studies focusing on the effects of therapeutic ultrasound on chondrocytes or explants. The exclusion criteria were: 1) reviews or meta-analysis, conference proceedings or case reports; 2) non-English language; 3) in vivo studies; or 4) clinical studies. In vitro studies investigating the effect of ultrasound on the chondrogenic differentiation of stem cells were not included since our goal was to understand the ultrasound effects in restoring the normal function of chondrocytes. The Cohen's kappa coefficient was calculated using the IBM® SPSS® statistics software (version 26).

      Data collection and extraction

      Data from the included studies were extracted by one author (S.O.) and four other authors (R.A., B.B.H., A.L. and O.C.) independently reviewed the extracted data, each revised 25% of the studies. Study characteristics, measured outcomes, mechanotransduction pathways and ultrasound parameters were collected as described in the Supplementary Table S1. Quantitative data on outcome measures were extracted from the studies' figures using the software WebPlotDigitizer. Median, 25% and 75% percentiles, minimum and maximum values were calculated for each ultrasound parameter. We recorded all relevant information that each study reported to allow independent replication (Supplementary Text).

      Risk of bias

      Two authors (S.O. and R.A.) assessed the risk of bias using the Risk of Bias Assessment tool for Non-randomized Studies (RoBANS)
      • Kim S.
      • Park J.
      • Lee Y.
      • Seo H Ju
      • Sheen S Soo
      • Hahn S.
      • et al.
      Testing a tool for assessing the risk of bias for nonrandomized studies showed moderate reliability and promising validity.
      . It contains six domains comprising cell selection, confounding variables, exposure measurement, blinding of outcome assessment, incomplete outcome data and selective outcome reporting. We adapted these domains criteria to assess the risk of bias to in vitro studies
      • Oliveira S.
      • Andrade R.
      • Hinckel B.B.
      • Silva F.
      • Espregueira-mendes J.
      • Carvalho Ó.
      • et al.
      In vitro and in vivo effects of light therapy on cartilage regeneration for knee osteoarthritis: a systematic review.
      , and added the judgment of bias arising from the experimental conditions, funding and other biases. Supplementary Table S2 reports the criteria to classify the risk of bias domains as low, high or unclear risk of bias. Any disagreements were resolved by consensus.

      Data synthesis

      We quantitatively synthesized the results with standardized mean differences (SMDs) with 95% confidence intervals (CI). Due to the heterogeneity across studies regarding cell culture conditions, comparison conditions and outcome measures, a meta-analysis was not feasible; thus, the SMDs of each study were presented and the pooled SMD was not provided to avoid misleading and non-valid conclusions. The SMDs are provided as illustrative measures to enable comparisons between studies and are based on post-treatment values only. Data synthesis was performed separately for healthy and OA chondrocytes and grouped based on the ultrasound intensity. All main analyses were performed using the post-treatment values between control (cells without stimulation) and intervention (cells with stimulation).

      Results

      Search strategy

      The searches yielded a cumulative total of 3,827 records, from which 431 duplicates were removed. From 3,396 titles and abstracts, 103 full texts were analyzed, from which 31 studies met the eligibility criteria and were included in this systematic review. The reasons for exclusion are reported in the PRISMA flowchart (Fig. 1) and Supplementary Table S3. The Cohen's kappa coefficient was 0.876 (95% CI, 0.77 to 0.98) with an agreement of 94.9%.
      Fig. 1
      Fig. 1PRISMA 2020 flowchart of included and excluded studies.

      Risk of bias

      The judgment of risk of bias of each study is provided in Fig. 2. Only one study
      • Nishikori T.
      • Ochi M.
      • Uchio Y.
      • Maniwa S.
      • Kataoka H.
      • Kawasaki K.
      • et al.
      Effects of low-intensity pulsed ultrasound on proliferation and chondroitin sulfate synthesis of cultured chondrocytes embedded in Atelocollagen gel.
      (3%, k = 31) was judged to have a high risk of bias in the “Cell selection” domain due to the selection of chondrocytes from different anatomical sites. Almost half of the studies (48%, k = 31) were judged to have unclear risk of bias due to “Confounding” since they did not report the animal characteristics from which chondrocytes were isolated from or the cell density used, precluding us to assess the potential risk of differences between groups. The “Experimental conditions” domain was judged to have a high risk of bias in 84% of the studies (k = 31) due to the absence of temperature control and/or previous calibration of the setup. Nearly 65% of the studies (k = 31) were judged to have a high risk of performance and detection bias in the “Exposure measurement” and “Blinding outcome assessment” domains respectively; because semi-quantitative analyzes were not conducted by two independent observers or blindly, respectively. Almost one third of studies (32%, k = 31) were judged to have an unclear risk of bias since they did not clearly provide the number of experiments or replicas used, preventing us to ascertain if there was incomplete outcome data reporting. Only two studies (6%, k = 31) were judged to have high risk of reporting bias due to not reporting the results of all measured outcomes. The “Funding bias” domain was judged to have unclear risk of bias in 74% of studies (k = 31) owing to the absence of conflict of interest statements, while high risk of bias was judged in one study
      • Nishida T.
      • Kubota S.
      • Aoyama E.
      • Yamanaka N.
      • Lyons K.M.
      • Takigawa M.
      Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation.
      that reported important conflict of interests. Roughly 55% of studies (k = 31) were judged to have high risk of bias for pseudoreplication, arising from not clearly reporting if the experiments or replicas were independent. Only one study
      • Korstjens C.M.
      • Rijt R.H.H.
      • Albers G.H.R.
      • Semeins C.M.
      • Klein-Nulend J.
      Low-intensity pulsed ultrasound affects human articular chondrocytes in vitro.
      (3%, k = 31) has performed an a priori sample size calculation. Five studies (16%, k = 31) provided all relevant information to allow independent replication (Supplementary Table S4).
      Fig. 2
      Fig. 2Risk of bias plots. Traffic lights and weight summary plots for the included studies.

      Study characteristics

      In 61% of studies (k = 31), chondrocytes were isolated from animal sources. Primary chondrocytes were mainly cultured in a monolayer (65%, k = 31). Healthy chondrocytes were more commonly used (79%, k = 28), two studies utilized OA chondrocytes (7%, k = 28) and five studies included both (18%, k = 28). Chondrocyte cell lines were explored in four studies (14%, k = 28). Six studies (19%, k = 31) utilized healthy (66.6%, k = 6), OA (16.7%, k = 6), or both (16.7%, k = 6) explants cultures. In vitro models of OA were established in six studies (19%, k = 31) by administrating interleukin (IL)-1β (66.6%, k = 6) or hydrogen peroxide (16.7%, k = 6) to induce inflammation, or lipopolysaccharide (16.7%, k = 6) to induce oxidative stress. Study characteristics are summarized on the Supplementary Table S5 and described in Table I.
      Table IStudy characteristics
      Ref.Cell type & SourceCell culture modeStudy designOutcome measuresSignificant findings
      Zuo et al. (2021)Primary chondrocytes (healthy)

      New Zealand rabbits (knee joint)
      Monolayer

      +

      Incubation with LPS (1 μg/mL)
      G1: Control

      G2: Only LPS

      G3: LPS + PBNPs

      G4: LPS + US at 60 mW/cm2

      G5: LPS + PBNPs + US at 60 mW/cm2
      Cell apoptosis: colorimetric assay (CCK-8); flow cytometry; tunnel staining↑ Cell apoptosis [G2/G3/G4/G5 vs G1]

      ↓ Cell apoptosis [G3/G4/G5 vs G2; G5 vs G3/G4]
      ROS detection: Fluorescence microscopy↑ ROS production [G2/G3/G4/G5 vs G1]

      ↓ ROS production [G3/G4/G5 vs G2; G5 vs G3/G4]
      Protein quantification: Western blot↑ IL1-β [G2/G3/G4/G5 vs G1; G4 vs G3]

      ↓ IL1-β [G3/G4/G5 vs G2; G5 vs G3/G4]

      ↑ MMP-3 and ↑ MMP-13 [G2/G3/G4/G5 vs G1]

      ↓ MMP-3 [G3/G4/G5 vs G2; G4 vs G3; G5 vs G3/G4]

      ↓ MMP-13 [G3/G4/G5 vs G2; G5 vs G3/G4]
      Chen et al. (2020)Primary chondrocytes (healthy and OA)

      Human (knee joint)
      3D culture: alginate beadsG1: Control

      G2: 20 nM pure rapa

      G3: 20 nM Liposome-rapa

      G4: 20 nM Liposome-rapa + US at 500 mW/cm2

      G5: 2 nM pure rapa

      G6: 2 nM Liposome-rapa

      G7: 2 nM Liposome-rapa + US at 500 mW/cm2

      G8: US at 60 mW/cm2
      Cell proliferation: BrdU assay↓ Cell proliferation [G2 vs G1 for healthy cells]

      No effect on OA cells.
      Gene quantification: RT-PCR↑ Aggrecan [G3/G4/G7 vs G1 for OA cells]

      ↑ COL II [G2/G3/G4/G5/G6/G7 vs G1 for OA cells]

      ↓ MMP-13 and IL-6 [G2/G3/G4/G5/G6/G7 vs G1 for OA cells]
      Protein quantification: ELISA↑ COL II [G4/G6/G7 vs G1 for healthy cells]

      ↑ COL II [G7 vs G1 for OA cells]

      ↓ MMP-13 [G2/G3/G4/G5/G6/G7 vs G1 for OA cells]

      ↓ IL-6 [G4/G7 vs G1 for OA cells]
      PGs quantification: DMMB assay↑ PGs [G4/G7 vs G1 for healthy cells]

      ↑ PGs [G3/G4/G7 vs G1 for OA cells]
      Guan et al. (2020)Primary chondrocytes (healthy)

      Mice (knee joint)
      Monolayer

      +

      Incubation with IL-1β (10 ng/mL)
      G1: Control

      G2: US at 30 mW/cm2

      G3: IL-1β

      G4: US at 30 mW/cm2 + IL-1β
      Gene quantification: RT-PCR↓ VEGF [G4 vs G3]

      ↑ COL II [G2 vs G1; G4 vs G3]

      ↓ MMP-13 and COL X [G2 vs G1; G4 vs G3]
      Protein quantification: ELISA↓ VEGF [G4 vs G3]
      Ding et al. (2020)Primary chondrocytes (OA)

      Human (knee joint)
      MonolayerG1: Control

      G2: US at 30 mW/cm2

      G3: US at 60 mW/cm2

      G4: US at 90 mW/cm2
      Cell proliferation and apoptosis: flow cytometerNo effect on cell proliferation.

      ↓ Cell apoptosis [G2/G3/G4 vs G1]
      Gene quantification: RT-PCR↑ COL II [G2/G3/G4 vs G1; G2 vs G3/G4]

      ↑ Aggrecan [G2/G3/G4 vs G1; G3 vs G2/G4]

      ↑ SOX9 [G2/G3/G4 vs G1]
      Protein quantification: ELISA, Western Blot↑ COL II [G2/G3/G4 vs G1]

      ↑ Aggrecan [G2/G3/G4 vs G1]

      ↑ SOX9 [G2/G3/G4 vs G1]
      Sekino et al. (2018)ATDC5 cell line

      Mouse
      MonolayerG1: Control

      G2: US at 30 mW/cm2

      G3: US at 60 mW/cm2
      Gene quantification: RT-PCR↓ SOX9 [G2/G3 vs G1 at day 3 and 7; G3 vs G1 at day 5]

      ↑ COL II [G3 vs G1 at day 5]

      ↑ COL X [G2/G3 vs G1 at day 7]

      ↑ Aggrecan [G2/G3 vs G1 at day 5; G3 vs G1 at day 7]

      ↓ MMP-13 [G2/G3 vs G1 at day 3 and 7]

      ↓ ADAMTS-5 [G2/G3 vs G1 at day 3]

      ↑ ADAMTS-5 [G3 vs G1 at day 7]
      Protein quantification: Western Blot↑ COL II [G3 vs G1 at day 5]

      ↑ COL X [G2/G3 vs G1 at day 7]

      ↑ Aggrecan [G2/G3 vs G1 at day 5]

      ↓ MMP-13 [G2/G3 vs G1 at day 7]
      PGs quantification: Alcian blue stainingNo effect.
      Nishida et al. (2017)HCS-2/8 cell line

      Human
      MonolayerG1: Control

      G2: US at 60 mW/cm2
      Gene quantification: RT-PCR↑ COL II [G2 vs G1]

      ↑ Aggrecan [G2 vs G1]

      ↑ MMP-13 [G2 vs G1]

      ↑ CCN2 [G2 vs G1]

      No effect on SOX9.
      Protein quantification: Western Blot↑ CCN2 [G2 vs G1]
      Uddin et al. (2016)Cartilage explants (healthy)

      Human (knee joint)
      Ex vivo

      +

      Incubation with IL-1β (10 ng/mL)
      G1: Control

      G2: IL-1β

      G3: US at 30 mW/cm2 + IL-1β

      G4: US at 30 mW/cm2
      Cell viability: MTT↑ Cell viability [G3/G4 vs G1 at day 5 for C-28/I2 cells]
      Gene quantification: RT-PCR↓ COL II, ↓ Aggrecan, ↓ COMP [G2 vs G1 for C-28/I2 cells]

      ↑ COL II, ↑ Aggrecan, ↑ COMP [G3 vs G2; G4 vs G1 for C-28/I2 cells]

      ↑ MMP-13, ↑ ADAMTS4, ↑ ADAMTS5 [G2 vs G1 for C-28/I2 cells]

      ↓ MMP-13, ↓ ADAMTS4, ↓ ADAMTS5 [G3 vs G2; G4 vs G1 for C-28/I2 cells]
      C-28/I2 cell line

      Human
      Monolayer

      +

      Incubation with IL-1β (10 ng/mL)
      PGs quantification: DMMB assay↑ PGs release [G2 vs G1 for explants]

      ↓ PGs release [G3 vs G2; G4 vs G1 for explants]
      Histology: Picrosirius Red Staining (COL) and Safranin O Staining (PGs)↑ PGs [G3 vs G2; G4 vs G1 for C-28/I2 cells]

      ↓ PGs [G2 vs G1 for C-28/I2 cells]

      ↑ COL [G3 vs G2; G4 vs G1 for C-28/I2 cells]

      ↓ COL [G2 vs G1 for C-28/I2 cells]

      ↑ PGs [G3 vs G2; G4 vs G1 for explants]

      ↓ PGs [G2 vs G1 for explants]
      Ji et al. (2015)Primary chondrocytes (healthy and OA)

      New Zealand rabbits (knee joint)
      MonolayerG1: Control (healthy cells)

      G2: OA cells

      G3: OA cells + US at 40 mW/cm2
      Gene quantification: RT-PCR↓ TIMP-2 [G2 vs G1]

      ↑ TIMP-2 [G3 vs G2]

      ↑ MMP-13 [G2 vs G1]

      ↓ MMP-13 [G3 vs G2]
      Protein quantification: Western Blot↓ TIMP-2 [G2 vs G1]

      ↑ TIMP-2 [G3 vs G2]

      ↑ MMP-13 [G2 vs G1]

      ↓ MMP-13 [G3 vs G2]
      Tan et al. (2015)Cartilage explants (Healthy)

      Pigs and human
      Ex vivoG1: Control

      G2: US at 30 mW/cm2

      G3: PEMF
      Gene quantification: RT-PCR↓ COL II, ↓ Aggrecan [G2/G3 vs G1]

      ↑ COL II, ↑ Aggrecan [G3 vs G2]

      ↑ COL I, ↑ COL X [G2/G3 vs G1; G3 vs G1]
      Histology: Safranin O Staining, thionine staining, Mankin scoreG2 and G3: No fibrillated surface zone; cell proliferation; new tissue formation and formation of cell clusters.

      ↓ Mankin score [G2 vs G1/G3]
      Xia et al. (2015)Primary chondrocytes (healthy and OA)

      Sprague–Dawley rats (knee joint)
      MonolayerG1: Control

      G2: US at 20 mW/cm2

      G3: US at 30 mW/cm2

      G4: US at 40 mW/cm2

      G5: US at 50 mW/cm2
      Protein quantification: Western BlotHealthy cells:

      ↑ COL II [G2/G3/G4/G5 vs G1; G4 vs G2/G3/G5]

      ↓ MMP-13 [G2/G3/G4/G5 vs G1; G4 vs G2/G5]

      OA cells:

      ↑ COL II [G2/G3/G4/G5 vs G1; G3 vs G4/G5]

      ↓ MMP-13 [G2/G3/G4/G5 vs G1; G3 vs G2/G4/G5]
      Cheng et al. (2014)Primary chondrocytes (healthy and OA)

      New Zealand rabbits (knee joint)
      MonolayerG1: Control (normal cells)

      G2: OA cells

      G3: OA cells + US at 40 mW/cm2
      Gene quantification: RT-PCR↓ COL II [G2/G3 vs G1; G2 vs G3]

      ↓ Aggrecan [G2/G3 vs G1; G2 vs G3]

      ↑ MMP-13, ↑ MMP-1 [G2/G3 vs G1] G2 vs G3]

      ↓ MMP-13, ↓ MMP-1 [G2 vs G3]
      Protein quantification: Western Blot↓ COL II [G2/G3 vs G1; G2 vs G3]

      ↓ Aggrecan [G2/G3 vs G1; G2 vs G3]

      ↑ MMP-13, ↑ MMP-1 [G2/G3 vs G1]

      ↓ MMP-13, ↓ MMP-1 [G2 vs G3]
      Yuan et al. (2014)Primary chondrocytes (OA)

      Human (articular cartilage)
      MonolayerG1: Control

      G2: HBO

      G3: US at 30 mW/cm2

      G4: HBO + US at 30 mW/cm2
      Gene quantification: RT-PCR↓ NOS [G2 vs G1]

      ↑ NOS [G3 vs G1]

      ↑ COL II [G2/G3/G4 vs G1]

      ↑ Aggrecan [G2/G3/G4 vs G1]
      Protein quantification: ELISA↓ MMP-3 [G2 vs G1]

      ↑ TIMP-1 [G2/G3/G4 vs G1]
      Ito et al. (2012)Primary Chondrocytes (healthy)

      Wistar rats (knee joint)
      Monolayer

      +

      Incubation with IL-1β (100 pg/mL or 1 ng/mL)
      G1: Control

      G2: US (7.5, 30 or 120 mW/cm2)

      G3: IL-1β

      G4: US (7.5, 30 or 120 mW/cm2) + IL-1β
      Gene quantification: RT-PCR↓ MMP-13 [G2 vs G1 at 120 mW/cm2; G2 vs G3 at 7.5, 30 and 120 mW/cm2]

      ↑ TIMP-1 [G2 vs G1 at 30 mW/cm2]

      No effect on TIMP-2.
      Articular cartilage explants (healthy)

      Pigs
      Ex vivoG1: Control

      G2: US at 27 mW/cm2

      G3: US at 67 mW/cm2
      ↓ MMP-13 [G3 vs G1/G2; G2 vs G1]

      ↓ MMP-1 [G3 vs G1/G2]

      ↓ TIMP-1 [G3 vs G1]

      No effect on TIMP-2.
      Hasanova et al. (2011)Primary chondrocytes (healthy)

      Calves (shoulder joint)
      3D culture: chitosan scaffoldsG1: Control

      G2: US at 0.14 mW/cm2 once

      G3: US at 0.14 mW/cm2 twice

      G4: US at 0.14 mW/cm2 4 times

      G5: US at 0.14 mW/cm2 8 times
      Cell viability: MTT

      Cell proliferation: total cell count
      ↑ Cell viability [G4 vs G1]

      ↑ Cell proliferation [G4/G5 vs G1]
      Gene quantification: RT-PCR↑ COL I, ↑ COL II [G4/G5 vs G1]

      No effect on Aggrecan and MMP-3.

      ↑ SOX5, ↑ SOX9 [G5 vs G1]

      ↑COX2 [G2 vs G1]
      Protein quantification: Western Blot↑ COL I, ↑ COL II, ↑ Aggrecan [G4/G5 vs G1]
      Vaughan et al. (2010)Primary chondrocytes (healthy)

      Steers (metacarpophalangeal joint)
      3D culture: agaroseG1: Control

      G2: US at 30 mW/cm2

      G3: US at 100 mW/cm2
      Cell viability: Live and dead stainingNo effect for cells in monolayer and 3D culture.
      MonolayerPGs quantification: DMMB assay,35SO4 incorporation↑ PGs [G3 vs G1 at day 1 and 16 for cells in 3D culture]

      No effects on PGs for cells in monolayer.
      Tien et al. (2008)Primary chondrocytes (healthy)

      Human child with ablated polydactylia (articular cartilage)
      3D culture: agaroseG1: Control

      G2: US at 18 mW/cm2

      G3: US at 48 mW/cm2

      G4: US at 72 mW/cm2

      G5: US at 98 mW/cm2
      Cell proliferation: DNA quantificationNo effects on cell proliferation.
      Protein quantification: Western Blot, ELISA↑ Aggrecan [G2/G3/G4/G5 vs G1]

      ↑ COL II [G2/G3 vs G1; G4 and G5 were not analyzed]
      Korstjens et al. (2008)Primary chondrocytes (healthy and OA)

      Human (knee joint)
      MonolayerG1: Control

      G2: US at 30 mW/cm2
      PGs quantification:35SO4 incorporation, autoradiographyMonolayer:

      ↑ PGs [G2 vs G1 for both healthy and OA cells]

      Ex vivo:

      No effects on PGs by35SO4 incorporation.

      ↑ PGs [G2 vs G1 for OA explants] by autoradiography.
      Articular cartilage explants (healthy and OA)

      Human (knee joint)
      Ex vivoHistology: toluidine blue stainingEx vivo:

      ↓ Cell nests 1–3 [G2 vs G1 in deep layer for healthy and OA explants]

      ↑ Cell nests 4–6 [G2 vs G1 in superficial layer for healthy explants, in deep layer for healthy and OA explants]
      Noriega et al. (2007)Primary chondrocytes (healthy)

      Humans with amputated limbs (articular cartilage)
      3D culture: chitosan scaffoldsG1: Control

      G2: US at 1.5 MHz

      G3: US at 5 MHz

      G4: US at 8.5 MHz
      Cell proliferation: DNA↑ Cell proliferation [G3 vs G1]
      Cell viability: MTT, live and dead staining↓ Cell viability [G4 vs G1] (MTT data not shown)
      PGs quantification: DMMB assayData not shown.
      COL quantification: 3H-proline incorporation↑ COL [G3/G4 vs G1] (data not shown)
      Gene quantification: RT-PCR↑ COL, ↑ Aggrecan [G3/G4 vs G1]
      Park et al. (2007)Primary chondrocytes (healthy)

      Pigs (knee joint)
      Monolayer

      +

      Incubation with IL-1β (5 ng/mL)
      G1: Control

      G2: IL-1β

      G3: US for 10 min + IL-1β

      G4: US for 20 min + IL-1β

      G5: US for 30 min + IL-1β

      G6: US for 50min + IL-1β
      Cell viability: total cell count, trypan blueNo effect.
      PGs quantification: DMMB assay↑ PGs [G6/G1 vs G2]

      No effect between G1, G2 and G6.
      Gene quantification: RT-PCRG6 was compared with G1 and G2:

      ↓ COL II/I [G6 vs G2]

      No effect on Aggrecan.

      ↓ MMP-1 [G6 vs G2]

      No effect on MMP-13 and TGF-β1 and β3.
      Histology: Safranin-O (PGs) and Sirius-red (COL) staining↑ PGs [G6 vs G2; G1 vs G2]

      ↑ COL [G6 vs G2]
      Choi et al. (2007)C-28/I2 cell line

      Human
      MonolayerG1: Control

      G2: US at 200 mW/cm2
      Gene quantification: RT-PCR↑ COL II [G2 vs G1] after 3 h of incubation

      ↑ Aggrecan [G2 vs G1] after 3 h of incubation
      Min et al. (2006)Articular cartilage explants (OA)

      Human
      Ex vivoG1: Control

      G2: US at 40 mW/cm2

      G3: US at 200 mW/cm2

      G4: US at 500 mW/cm2

      G5: US at 700 mW/cm2
      Cell proliferation: 3H-thymidine incorporationNo effect.
      PGs quantification35SO4 incorporation↑ PGs [G3 vs G1]

      ↓ PGs [G4/G5 vs G1]
      DMMB assay↑ PGs [G3 vs G1]

      ↓ PGs [G5 vs G1]
      COL quantification: 3H-proline incorporation↑ COL [G3 vs G1]

      ↓ COL [G4/G5 vs G1]
      Histology: Safranin O/fast green (PGs) and immunostaining (COL II and X)

      Only G3 was analyzed.
      ↑ PGs, ↑ COL II [G3 vs G1]

      ↓ COL X [G3 vs G1]
      Kopakkala-Tani et al. (2006)Primary chondrocytes (healthy)

      Pigs (articular cartilage)
      MonolayerG1: Control

      G2: US at 580 mW/cm2
      PGs quantification:35SO4 incorporation↑ PGs [G2 vs G1 from day 2 onwards]
      Protein quantification:

      Western Blot
      No effect on heat shock protein (Hsp70).
      Choi et al. (2006)Primary chondrocytes (healthy)

      Human (knee joint)
      3D culture: alginate beadsG1: Control

      G2: US at 100 mW/cm2

      G3: US at 200 mW/cm2

      G4: US at 300 mW/cm2
      Cell proliferation: 3H-thymidine incorporationNo effect.
      Cell viability: Trypan blue↑ Cell viability [G3 vs G1]
      Collagen quantification: 3H-proline incorporation↑ COL [G3 vs G1]
      PGs quantification:35SO4 incorporation↑ PGs [G3 vs G1]
      Gene quantification: Northern blot analysis↑ COL II [G3/G4 vs G1/G2]

      No effect on TIMP-1.

      ↓ MMP-1 [G2/G3/G4 vs G1]
      Protein quantification: Western blotNo effect on COL II, TIMP-1 and MMP-1.
      Histology: Alcian blue and Safranin O staining (PGs)↑ Cell size and number [G3 vs G1/G2/G4]
      Morphological analysis: TEM

      Only G3 was analyzed.
      ↑ Rough endoplasmic reticulum and mitochondria and well organized.
      Mukai et al. (2005)Primary chondrocytes (healthy)

      Wister rats (articular cartilage)
      Aggregates cultureG1: Control

      G2: US at 30 mW/cm2
      Cell proliferation: DNA quantification↑ Cell proliferation [G2 vs G1]
      Gene quantification: Northern blot analysis↑ COL II, ↑ Aggrecan [G2 vs G1]

      ↓ COL X [G2 vs G1]

      ↑ TGF-β1 [G2 vs G1]
      Protein quantificationELISA↑ TGF-β1 [G2 vs G1]
      ALP activity↓ ALP [G2 vs G1]
      Zhang et al. (2003)Primary chondrocytes (healthy)

      Leghorn chick embryos (sternum)
      3D culture: alginate beadsG1: Control

      G2: US at 2 mW/cm2

      G3: US at 30 mW/cm2
      Cell viability: Live and death stainingNo effect.
      Cell proliferation: DNA quantification↑ Cell proliferation [G2 vs G1 at day 3]

      ↓ Cell proliferation [G3 vs G1 at day 1, 3 and 7]
      Protein quantification: Immunohistochemistry↑ COL II staining [G2 vs G3]

      No effect on Aggrecan.
      Gene quantification: RT-PCR↓ COL II [G2/G3 vs G1 at day 1]

      ↑ COL II [G2/G3 vs G1 at day 7; G2 vs G3 at day 7]

      ↓ Aggrecan [G2/G3 vs G1 at day 1 and 3]

      No effect on Aggrecan at day 5 and 7.

      ↑ COL X [G2/G3 vs G1 at day 1; G2 vs G3 at day 1]

      ↓ COL X [G3 vs G1 at day 5 and 7]
      Zhang et al. (2002)Cartilage explants (healthy)

      Leghorn chick embryos (sternum)
      Ex vivoG1: Control

      G2: US at 30 mW/cm2
      Protein quantification: ImmunohistochemistryProximal area (endochondral ossification):

      ↑ COL II [G2 vs G1 at days 3, 5 and 7]

      ↑ Aggrecan [G2 vs G1 at days 1, 3, 5 and 7]

      ↑ COL X [G2 vs G1 at days 1 and 3]

      Distal area (articular cartilage):

      ↑ COL II [G2 vs G1 at days 3, 5 and 7]

      ↑ Aggrecan [G2 vs G1 at days 1, 3, 5 and 7]
      Parvizi et al. (2002)Primary chondrocytes (healthy)

      Long Evans rats (articular cartilage)
      MonolayerG1: Control

      G2: US at 50 mW/cm2
      PGs quantification:35SO4 incorporation↑ PGs [G2 vs G1]
      Nishikori et al. (2001)Primary chondrocytes (healthy)

      Japanese white rabbits (hip, knee, and shoulder joints)
      3D culture: Atelocollagen (3% COL I)G1: Control

      G2: US at 30 mW/cm2
      Cell proliferation: Trypan blueNo effect.
      Protein quantification: ImmunohistochemistryNo effect.
      CS quantification: HPLC analysis↑ CS [G2 vs G1]
      Stiffness of 3D culture↑ Stiffness [G2 vs G1]
      Parvizi et al. (1999)Primary chondrocytes (healthy)

      Japanese white rabbits (articular cartilage)
      MonolayerG1: Control

      G2: US at 230 kPa

      G3: US at 360 kPa
      Cell proliferation: 3H-thymidine incorporationNo effect.
      PGs quantification:35SO4 incorporation↑ PGs [G2/G3 vs G1]
      Gene quantification: Northern blot analysisNo effect on COL II/I.

      ↑ Aggrecan [G2/G3 vs G1]
      NR: Not reported; G: Group; LPS: Lipopolysaccharide; PBNPs: Prussian blue nanoparticles; US: Ultrasound; ROS: Reactive oxidative stress; IL: Interleukin; MMP: Metalloproteinase; OA: Osteoarthritis; 3D: Three-dimensional; Rapa: Rapamycin; RT-PCR: Real-time polymerase chain reaction; BrdU: Bromodeoxyuridine; ELISA: Enzyme-linked immunosorbent assay; DMMB: Dimethylmethylene blue; COL: Collagen; PGs: Proteoglycans; VEGF: Vascular endothelial growth factor; SOX: SRY-box transcription factor; ADAMTS: A Disintegrin and Metalloproteinase with Thrombospondin motifs; CCN2: CCN family protein 2; MTT: (4,5-dimethyl-thiazol-2yl)-2,5-diphenyltetrazolium bromide; COMP: Cartilage oligomeric matrix protein; TIMP: Tissue inhibitor of metalloproteinase; NOS: Nitric oxide synthase; HBO: Hyperbaric oxygen; COX: Cyclooxygenase; SO4: Sulfate; TGF: Transforming Growth Factor; CS: Chondroitin sulfate.
      Note: Two studies, Whitney et al. (2012) and Kim et al. (2014), were not included since they only analyzed the mechanotransduction process in the cells exposed to US.

      Stimulation parameters

      The range of ultrasound parameters is provided in the Supplementary Tables S6 and S7. Ultrasound was applied with a median center frequency of 1.5 MHz (range, 1 to 8.5) at a median power density of 40 mW/cm2 (range, 0.14 to 700) for a median stimulation time of 20 min (range, 0.5 to 60) and a median number of sessions of 6 (range, 1 to 80) once daily. In pulsed mode, ultrasound waves were repeated at a median frequency of 1 kHz (range, 0.1 to 1) and median duty cycle of 20% (range, 20 to 40) with a pulse duration of 200 μs. The ultrasound equipment commonly used was the SAFHS® (23%, k = 31).

      In vitro setup

      Three different setups were employed to apply ultrasound to chondrocytes (Fig. 3). More than half of studies applied the ultrasound in direct contact with the culture plate (55%, k = 31) using acoustic gel [Fig. 3(A)]. Almost 20% of studies (k = 31) immersed the ultrasonic transducer directly into the culture media [Fig. 3(B)], while others immersed the transducer in water bath [Fig. 3(C)] and placed the culture plates on the water surface (16%, k = 31). Only 39% of the studies (k = 31) reported the distance between the ultrasound transducer and the cells (Supplementary Tables S6 and S7).
      Fig. 3
      Fig. 3Ultrasound setup explored in the included studies: (A) ultrasonic transducer in direct contact with culture plate; (B) ultrasonic transducer immersed in culture media; and (C) ultrasonic transducer immersed in water bath.

      In vitro effects

      Chondrocyte viability, proliferation or apoptosis (Supplementary Table S5) were assessed in 48% of the studies (k = 31). Viability was either statistically significantly increased
      • Uddin S.M.Z.
      • Richbourgh B.
      • Ding Y.
      • Hettinghouse A.
      • Komatsu D.E.
      • Qin Y.X.
      • et al.
      Chondro-protective effects of low intensity pulsed ultrasound.
      • Hasanova G.I.
      • Noriega S.E.
      • Mamedov T.G.
      • Thakurta Guha S.
      • Turner J.A.
      • Subramanian A.
      The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds.
      • Choi B.H.
      • Woo J.I.
      • Min B.H.
      • Park S.R.
      Low-intensity ultrasound stimulates the viability and matrix gene expression of human articular chondrocytes in alginate bead culture.
      or unaffected in healthy chondrocytes after ultrasound application
      • Vaughan N.M.
      • Grainger J.
      • Bader D.L.
      • Knight M.M.
      The potential of pulsed low intensity ultrasound to stimulate chondrocytes matrix synthesis in agarose and monolayer cultures.
      • Zhang Z.J.
      • Huckle J.
      • Francomano C.A.
      • Spencer R.G.S.
      The effects of pulsed low-intensity ultrasound on chondrocyte viability, proliferation, gene expression and matrix production.
      • Park K.
      • Hoffmeister B.
      • Han D.K.
      • Hasty K.
      Therapeutic ultrasound effects on interleukin-1β stimulated cartilage construct in vitro.
      , but statistically reduced with increasing center frequency
      • Noriega S.
      • Mamedov T.
      • Turner J.A.
      • Subramanian A.
      Intermittent applications of continuous ultrasound on the viability, proliferation, morphology, and matrix production of chondrocytes in 3D matrices.
      . Apoptosis was statistically significantly decreased in both healthy
      • Zuo D.
      • Tan B.
      • Jia G.
      • Wu D.
      • Yu L.
      • Jia L.
      A treatment combined prussian blue nanoparticles with low-intensity pulsed ultrasound alleviates cartilage damage in knee osteoarthritis by initiating PI3K/Akt/mTOR pathway.
      and OA
      • Ding W.
      • Du D.
      • Chen S.
      LIPUS promotes synthesis and secretion of extracellular matrix and reduces cell apoptosis in human osteoarthritis through upregulation of SOX9 expression.
      cells. Proliferation of healthy chondrocytes statistically enhanced
      • Hasanova G.I.
      • Noriega S.E.
      • Mamedov T.G.
      • Thakurta Guha S.
      • Turner J.A.
      • Subramanian A.
      The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds.
      ,
      • Zhang Z.J.
      • Huckle J.
      • Francomano C.A.
      • Spencer R.G.S.
      The effects of pulsed low-intensity ultrasound on chondrocyte viability, proliferation, gene expression and matrix production.
      ,
      • Noriega S.
      • Mamedov T.
      • Turner J.A.
      • Subramanian A.
      Intermittent applications of continuous ultrasound on the viability, proliferation, morphology, and matrix production of chondrocytes in 3D matrices.
      ,
      • Mukai S.
      • Ito H.
      • Nakagawa Y.
      • Akiyama H.
      • Miyamoto M.
      • Nakamura T.
      Transforming growth factor-β1 mediates the effects of low-intensity pulsed ultrasound in chondrocytes.
      following intervention, while seven studies
      • Choi B.H.
      • Woo J.I.
      • Min B.H.
      • Park S.R.
      Low-intensity ultrasound stimulates the viability and matrix gene expression of human articular chondrocytes in alginate bead culture.
      ,
      • Ding W.
      • Du D.
      • Chen S.
      LIPUS promotes synthesis and secretion of extracellular matrix and reduces cell apoptosis in human osteoarthritis through upregulation of SOX9 expression.
      ,
      • Chen C.H.
      • Kuo S.M.
      • Tien Y.C.
      • Shen P.C.
      • Kuo Y.W.
      • Huang H.H.
      Steady augmentation of anti-osteoarthritic actions of rapamycin by liposome-encapsulation in collaboration with low-intensity pulsed ultrasound.
      • Tien Y.C.
      • Lin S.D.
      • Chen C.H.
      • Lu C.C.
      • Su S.J.
      • Chih T.T.
      Effects of pulsed low-intensity ultrasound on human child chondrocytes.
      • Min B.H.
      • Woo J.I.
      • Cho H.S.
      • Choi B.H.
      • Park S.J.
      • Choi M.J.
      • et al.
      Effects of low-intensity ultrasound (LIUS) stimulation on human cartilage explants.
      • Parvizi J.
      • Wu C.C.
      • Lewallen D.G.
      • Greenleaf J.F.
      • Bolander M.E.
      Low-intensity ultrasound stimulates proteoglycan synthesis in rat chondrocytes by increasing aggrecan gene expression.
      did not observe statistically significant effects in both healthy and OA chondrocytes (Table I). The effect sizes for each outcome and study are reported in Table II and the forest plots are provided on Supplementary Fig. S1.
      Table IIData synthesis of the included studies
      OutcomeStudiesInterventionControlType of cellsCell cultureTime after treatmentIntensity (mW/cm2)SMD (95% CI)
      ACAN (RT-PCR)Hasanova et al. (2011)UltrasoundHealthy chondrocytes without stimulationPrimary3D10 days0.14

      (1× daily)
      −0.54 (−2.76 to 1.69)
      Hasanova et al. (2011)UltrasoundHealthy chondrocytes without stimulationPrimary3D10 days0.14

      (2× daily)
      1.05 (−1.79 to 3.90)
      Hasanova et al. (2011)UltrasoundHealthy chondrocytes without stimulationPrimary3D10 days0.14

      (4× daily)
      0.25 (−1.77 to 2.26)
      Hasanova et al. (2011)UltrasoundHealthy chondrocytes without stimulationPrimary3D10 days0.14

      (8× daily)
      0.49 (−1.69 to 2.68)
      Uddin et al. (2016)UltrasoundHealthy chondrocytes without stimulationC-28/12 cell lineMonolayerNR301.28 (−1.91 to 4.47)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer3 days305.08 (−5.07 to 15.22)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer5 days304.41 (−4.46 to 13.28)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer7 days30−0.52 (−2.73 to 1.69)
      Mukai et al. (2005)UltrasoundHealthy chondrocytes without stimulationPrimaryAggregates15 days301.17 (−1.85 to 4.20)
      Nishida et al. (2017)UltrasoundHealthy chondrocytes without stimulationHCS cell lineMonolayer30 min600.74 (−1.70 to 3.19)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer3 days600.56 (−1.69 to 2.81)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer5 days604.05 (−4.13 to 12.23)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer7 days601.02 (−1.78 to 3.81)
      COL II (RT-PCR)Hasanova et al. (2011)UltrasoundHealthy chondrocytes without stimulationPrimary3D10 days0.14

      (1× daily)
      0.12 (−1.85 to 2.10)
      Hasanova et al. (2011)UltrasoundHealthy chondrocytes without stimulationPrimary3D10 days0.14

      (2× daily)
      1.30 (−1.92 to 4.52)
      Hasanova et al. (2011)UltrasoundHealthy chondrocytes without stimulationPrimary3D10 days0.14

      (4× daily)
      2.96 (−3.16 to 9.09)
      Hasanova et al. (2011)UltrasoundHealthy chondrocytes without stimulationPrimary3D10 days0.14

      (8× daily)
      3.86 (−3.95 to 11.67)
      Uddin et al. (2016)UltrasoundHealthy chondrocytes without stimulationC-28/12 cell lineMonolayerNR303.12 (−3.30 to 9.53)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer3 days300.89 (−1.73 to 3.52)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer5 days300.68 (−1.73 to 3.50)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer7 days30−0.28 (−2.32 to 1.75)
      Mukai et al. (2005)UltrasoundHealthy chondrocytes without stimulationPrimaryAggregates15 days300.88 (−1.73 to 3.50)
      Guan et al. (2020)UltrasoundHealthy chondrocytes without stimulationPrimaryMonolayer2 h301.78 (−2.22 to 5.78)
      Nishida et al. (2017)UltrasoundHealthy chondrocytes without stimulationHCS cell lineMonolayer30 min602.23 (−2.56 to 7.02)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer3 days600.56 (−1.69 to 2.81)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer5 days605.48 (−5.44 to 16.40)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer7 days60−1.11 (−4.03 t 1–82)
      COL X (RT-PCR)Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer3 days301.55 (−2.06 to 5.16)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer5 days30−0.15 (−2.13 to 1.83)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer7 days30−1.06 (−3.92 to 1.80)
      Mukai et al. (2005)UltrasoundHealthy chondrocytes without stimulationPrimaryAggregates15 days30−1.28 (−4.45 to 1.90)
      Guan et al. (2020)UltrasoundHealthy chondrocytes without stimulationPrimaryMonolayer2 h30−1.44 (−4.88 to 2.00)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer3 days601.97 (−2.36 to 6.31)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer5 days600.22 (−1.79 to 2.23)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer7 days600.98 (−1.77 to 3.73)
      MMP-13 (RT-PCR)Ito et al. (2012)UltrasoundHealthy chondrocytes without stimulationPrimaryMonolayer1 h7.5−0.66 (−3.02 to 1.69)
      Uddin et al. (2016)UltrasoundHealthy chondrocytes without stimulationC-28/12 cell lineMonolayerNR30−1.38 (−4.72 to 1.96)
      Ito et al. (2012)UltrasoundHealthy chondrocytes without stimulationPrimaryMonolayer1 h30−0.65 (−2.98 to 1.69)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer3 days30−0.78 (−3.27 to 1.71)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer5 days30−6.82 (−20.33 to 6.69)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer7 days300.00 (−1.96 to 1.96)
      Guan et al. (2020)UltrasoundHealthy chondrocytes without stimulationPrimaryMonolayer2 h30−1.06 (−3.91 to 1.79)
      Nishida et al. (2017)UltrasoundHealthy chondrocytes without stimulationHCS cell lineMonolayer30 min600.51 (−1.69 to 2.71)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer3 days60−6.42 (−19.15 to 6.31)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer5 days601.22 (−1.87 to 4.30)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer7 days60−1.54 (−5.14 to 2.06)
      Ito et al. (2012)UltrasoundHealthy chondrocytes without stimulationPrimaryMonolayer1 h120−1.86 (−6.00 to 2.28)
      GAGs (DMMB, Alcian blue and35SO4 incorporation assays)Uddin et al. (2016)UltrasoundHealthy chondrocytes without stimulationC-28/12 cell lineMonolayerNR30−1.19 (−4.24 to 1.86)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer14 days300.95 (−1.75 to 3.64)
      Sekino et al. (2018)UltrasoundHealthy chondrocytes without stimulationATDC 5 cell lineMonolayer14 days600.50 (−1.69 to 2.70)
      Chen et al. (2020)UltrasoundHealthy chondrocytes without stimulationPrimary3D7 days5000.62 (−1.69 to 2.93)
      Tani et al. (2006)UltrasoundHealthy chondrocytes without stimulationPrimaryMonolayer1 day5800.00 (−1.96 to 1.96)
      Tani et al. (2006)UltrasoundHealthy chondrocytes without stimulationPrimaryMonolayer2 days5800.79 (−1.71 to 3.30)
      Tani et al. (2006)UltrasoundHealthy chondrocytes without stimulationPrimaryMonolayer3 days5802.70 (−2.94 to 8.34)
      Tani et al. (2006)UltrasoundHealthy chondrocytes without stimulationPrimaryMonolayer4 days5801.52 (−2.04 to 5.08)
      Tani et al. (2006)UltrasoundHealthy chondrocytes without stimulationPrimaryMonolayer5 days5803.47 (−3.61 to 10.55)
      Viability (MTT, trypan blue, live and dead analysis)Hasanova et al. (2011)UltrasoundHealthy chondrocytes without stimulationPrimary3D10 days0.14

      (1× daily)
      1.16 (−1.84 to 4.15)
      Hasanova et al. (2011)UltrasoundHealthy chondrocytes without stimulationPrimary3D10 days0.14

      (2× daily)
      −0.31 (−2.37 to 1.74)
      Hasanova et al. (2011)UltrasoundHealthy chondrocytes without stimulationPrimary3D10 days0.14

      (4× daily)
      1.24 (−1.88 to 4.36)
      Hasanova et al. (2011)UltrasoundHealthy chondrocytes without stimulationPrimary3D10 days0.14

      (8× daily)
      0.04 (−1.92 to 2.00)
      Zhang et al. (2003)UltrasoundHealthy chondrocytes without stimulationPrimary3D1 day21.58 (−2.08 to 5.24)
      Zhang et al. (2003)UltrasoundHealthy chondrocytes without stimulationPrimary3D3 days20.57 (−1.69 to 2.82)
      Zhang et al. (2003)UltrasoundHealthy chondrocytes without stimulationPrimary3D5 days2−1.10 (−4.02 to 1.82)
      Zhang et al. (2003)UltrasoundHealthy chondrocytes without stimulationPrimary3D7 days2−3.49 (−10.62 to 3.63)
      Zhang et al. (2003)UltrasoundHealthy chondrocytes without stimulationPrimary3D1 day30−0.54 (−2.76 to 1.69)
      Zhang et al. (2003)UltrasoundHealthy chondrocytes without stimulationPrimary3D3 days302.12 (−2.47 to 6.71)
      Zhang et al. (2003)UltrasoundHealthy chondrocytes without stimulationPrimary3D5 days30−0.34 (−2.42 to 1.73)
      Zhang et al. (2003)UltrasoundHealthy chondrocytes without stimulationPrimary3D7 days30−2.42 (−7.55 to 2.71)
      Choi et al. (2006)UltrasoundHealthy chondrocytes without stimulationPrimary3D15 days1001.20 (−1.86 to 4.27)
      Choi et al. (2006)UltrasoundHealthy chondrocytes without stimulationPrimary3D15 days2002.38 (−2.68 to 7.44)
      Choi et al. (2006)UltrasoundHealthy chondrocytes without stimulationPrimary3D15 days3002.60 (−2.86 to 8.05)
      Proliferation (DNA quantification and BrdU assay)Tien et al. (2008)UltrasoundHealthy chondrocytes without stimulationPrimary3D10 days18−0.23 (−2.24 to 1.78)
      Noriega et al. (2007)UltrasoundHealthy chondrocytes without stimulationPrimary3D10 days<30

      1.5 MHz
      −0.29 (−2.34 to 1.75)
      Noriega et al. (2007)UltrasoundHealthy chondrocytes without stimulationPrimary3D10 days<30

      5 MHz
      1.97 (−2.36 to 6.30)
      Noriega et al. (2007)UltrasoundHealthy chondrocytes without stimulationPrimary3D10 days<30

      8.5 MHz
      0.31 (−1.74 to 2.37)
      Mukai et al. (2005)UltrasoundHealthy chondrocytes without stimulationPrimaryAggregates15 days302.21 (−2.55 to 6.97)
      Tien et al. (2008)UltrasoundHealthy chondrocytes without stimulationPrimary3D10 days48−0.11 (−2.09 to 1.86)
      Tien et al. (2008)UltrasoundHealthy chondrocytes without stimulationPrimary3D10 days72−0.18 (−2.17 to 1.81)
      Tien et al. (2008)UltrasoundHealthy chondrocytes without stimulationPrimary3D10 days98−0.04 (−2.01 to 1.92)
      Chen et al. (2020)UltrasoundHealthy chondrocytes without stimulationPrimary3D7 days5001.36 (−1.95 to 4.68)
      COL II (RT-PCR)Uddin et al. (2016)Ultrasound + IL-1βHealthy chondrocytes + IL-1β without stimulationC-28/12 cell lineMonolayerNR301.92 (−2.32 to 6.16)
      Guan et al. (2020)Ultrasound + IL-1βHealthy chondrocytes + IL-1β without stimulationPrimaryMonolayer2 h305.68 (−5.62 to 16.98)
      Park et al. (2007)Ultrasound + IL-1βHealthy chondrocytes + IL-1β without stimulationPrimaryMonolayer5 days50−0.61 (−2.91 to 1.69)
      MMP-13 (RT-PCR)Ito et al. (2012)Ultrasound + IL-1βHealthy chondrocytes + IL-1β without stimulationPrimaryMonolayer1 h7.5−1.18 (−4.22 to 1.85)
      Uddin et al. (2016)Ultrasound + IL-1βHealthy chondrocytes + IL-1β without stimulationC-28/12 cell lineMonolayerNR30−1.59 (−5.26 to 2.09)
      Guan et al. (2020)Ultrasound + IL-1βHealthy chondrocytes + IL-1β without stimulationPrimaryMonolayer2 h30−1.87 (−6.03 to 2.29)
      Ito et al. (2012)Ultrasound + IL-1βHealthy chondrocytes + IL-1β without stimulationPrimaryMonolayer1 h30−3.05 (−9.35 to 3.24)
      Park et al. (2007)Ultrasound + IL-1βHealthy chondrocytes + IL-1β without stimulationPrimaryMonolayer5 days500.21 (−1.79 to 2.21)
      Ito et al. (2012)Ultrasound + IL-1βHealthy chondrocytes + IL-1β without stimulationPrimaryMonolayer1 h120−2.83 (−8.72 to 3.06)
      ACAN (RT-PCR)Cheng et al. (2014)UltrasoundOA chondrocytes without stimulationPrimaryMonolayer6 days401.86 (−2.28 to 6.00)
      Yuan et al. (2014)UltrasoundOA chondrocytes without stimulationPrimaryMonolayer2 days401.95 (−2.35 to 6.25)
      Chen et al. (2020)UltrasoundOA chondrocytes without stimulationPrimary3D7 days5001.56 (−2.07 to 5.20)
      MMP-13 (RT-PCR)Cheng et al. (2014)UltrasoundOA chondrocytes without stimulationPrimaryMonolayer6 days40−0.87 (−3.46 to 1.73)
      Ji et al. (2015)UltrasoundOA chondrocytes without stimulationPrimaryMonolayer7 days40−0.90 (−3.53 to 1.74)
      Chen et al. (2020)UltrasoundOA chondrocytes without stimulationPrimary3D7 days500−2.63 (−8.14 to 2.88)
      COL II (Western blot)Xia et al. (2015)UltrasoundOA chondrocytes without stimulationPrimaryMonolayer6 days207.14 (−6.99 to 21.26)
      Ding et al. (2020)UltrasoundOA chondrocytes without stimulationPrimaryMonolayer6 days301.68 (−2.15 to 5.51)
      Xia et al. (2015)UltrasoundOA chondrocytes without stimulationPrimaryMonolayer6 days307.85 (−7.66 to 23.36)
      Cheng et al. (2014)UltrasoundOA chondrocytes without stimulationPrimaryMonolayer6 days401.69 (−2.16 to 5.55)
      Xia et al. (2015)UltrasoundOA chondrocytes without stimulationPrimaryMonolayer6 days403.85 (−3.95 to 11.65)
      Xia et al. (2015)UltrasoundOA chondrocytes without stimulationPrimaryMonolayer6 days506.77 (−6.64 to 20.18)
      Ding et al. (2020)UltrasoundOA chondrocytes without stimulationPrimaryMonolayer6 days602.29 (−2.60 to 7.18)
      Ding et al. (2020)UltrasoundOA chondrocytes without stimulationPrimaryMonolayer6 days901.72 (−2.18 to 5.61)
      GAGs (DMMB, and35SO4 incorporation assays)Korstjens et al. (2008)UltrasoundOA chondrocytes without stimulationPrimaryExplants6 days30−0.07 (−2.04 to 1.89)
      Min et al. (2006)UltrasoundOA chondrocytes without stimulationPrimaryExplants7 days400.04 (−1.92 to 2.00)
      Min et al. (2006)UltrasoundOA chondrocytes without stimulationPrimaryExplants7 days2001.16 (−1.84 to 4.16)
      Min et al. (2006)UltrasoundOA chondrocytes without stimulationPrimaryExplants7 days500−1.94 (−6.20 to 2.33)
      Chen et al. (2020)UltrasoundOA chondrocytes without stimulationPrimary3D7 days5000.90 (−1.74 to 3.54)
      Min et al. (2006)UltrasoundOA chondrocytes without stimulationPrimaryExplants7 days700−2.43 (−7.57 to 2.72)
      Proliferation (Flow cytometer, BrdU assay and 3H-thymidine incorporation)Ding et al. (2020)UltrasoundOA chondrocytes without stimulationPrimaryMonolayer6 days30−0.04 (−2.01 to 1.92)
      Min et al. (2006)UltrasoundOA chondrocytes without stimulationPrimaryExplants7 days400.02 (−1.94 to 1.98)
      Ding et al. (2020)UltrasoundOA chondrocytes without stimulationPrimaryMonolayer6 days60−0.28 (−2.31 to 1.76)
      Ding et al. (2020)UltrasoundOA chondrocytes without stimulationPrimaryMonolayer6 days90−0.08 (−2.05 to 1.88)
      Min et al. (2006)UltrasoundOA chondrocytes without stimulationPrimaryExplants7 days200−0.01 (−1.97 to 1.95)
      Min et al. (2006)UltrasoundOA chondrocytes without stimulationPrimaryExplants7 days500−0.03 (−1.99 to 1.93)
      Chen et al. (2020)UltrasoundOA chondrocytes without stimulationPrimary3D7 days5001.06 (−1.79 to 3.91)
      Min et al. (2006)UltrasoundOA chondrocytes without stimulationPrimaryExplants7 days7000.03 (−1.93 to 1.99)
      ACAN: Aggrecan; RT-PCR: Real time Polymerase Chain Reaction; 3D: three-dimensional; COL II: Collagen type II; COL X: Collagen type X; MMP-13: Metalloproteinase 13; GAGs: Glycosaminoglycans; DMMB: Dimethylmethylene Blue; 35SO4: Sulfate; MTT: (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide); BrdU: 5′-bromo-2′-deoxyuridine; IL-1β: Interleukin 1β; OA: Osteoarthritic.
      About 84% of the studies (k = 31) measured the effect of ultrasound on extracellular matrix (ECM) synthesis (Supplementary Table S5). The expression of ECM proteins including collagen (COL) type II
      • Nishida T.
      • Kubota S.
      • Aoyama E.
      • Yamanaka N.
      • Lyons K.M.
      • Takigawa M.
      Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation.
      ,
      • Uddin S.M.Z.
      • Richbourgh B.
      • Ding Y.
      • Hettinghouse A.
      • Komatsu D.E.
      • Qin Y.X.
      • et al.
      Chondro-protective effects of low intensity pulsed ultrasound.
      • Hasanova G.I.
      • Noriega S.E.
      • Mamedov T.G.
      • Thakurta Guha S.
      • Turner J.A.
      • Subramanian A.
      The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds.
      • Choi B.H.
      • Woo J.I.
      • Min B.H.
      • Park S.R.
      Low-intensity ultrasound stimulates the viability and matrix gene expression of human articular chondrocytes in alginate bead culture.
      ,
      • Zhang Z.J.
      • Huckle J.
      • Francomano C.A.
      • Spencer R.G.S.
      The effects of pulsed low-intensity ultrasound on chondrocyte viability, proliferation, gene expression and matrix production.
      ,
      • Noriega S.
      • Mamedov T.
      • Turner J.A.
      • Subramanian A.
      Intermittent applications of continuous ultrasound on the viability, proliferation, morphology, and matrix production of chondrocytes in 3D matrices.
      ,
      • Ding W.
      • Du D.
      • Chen S.
      LIPUS promotes synthesis and secretion of extracellular matrix and reduces cell apoptosis in human osteoarthritis through upregulation of SOX9 expression.
      • Mukai S.
      • Ito H.
      • Nakagawa Y.
      • Akiyama H.
      • Miyamoto M.
      • Nakamura T.
      Transforming growth factor-β1 mediates the effects of low-intensity pulsed ultrasound in chondrocytes.
      • Chen C.H.
      • Kuo S.M.
      • Tien Y.C.
      • Shen P.C.
      • Kuo Y.W.
      • Huang H.H.
      Steady augmentation of anti-osteoarthritic actions of rapamycin by liposome-encapsulation in collaboration with low-intensity pulsed ultrasound.
      ,
      • Tien Y.C.
      • Lin S.D.
      • Chen C.H.
      • Lu C.C.
      • Su S.J.
      • Chih T.T.
      Effects of pulsed low-intensity ultrasound on human child chondrocytes.
      ,
      • Min B.H.
      • Woo J.I.
      • Cho H.S.
      • Choi B.H.
      • Park S.J.
      • Choi M.J.
      • et al.
      Effects of low-intensity ultrasound (LIUS) stimulation on human cartilage explants.
      ,
      • Guan M.
      • Zhu Y.
      • Liao B.
      • Tan Q.
      • Qi H.
      • Zhang B.
      • et al.
      Low-intensity pulsed ultrasound inhibits VEGFA expression in chondrocytes and protects against cartilage degeneration in experimental osteoarthritis.
      • Sekino J.
      • Nagao M.
      • Kato S.
      • Sakai M.
      • Abe K.
      • Nakayama E.
      • et al.
      Low-intensity pulsed ultrasound induces cartilage matrix synthesis and reduced MMP13 expression in chondrocytes.
      • Xia P.
      • Ren S.
      • Lin Q.
      • Cheng K.
      • Shen S.
      • Gao M.
      • et al.
      Low-intensity pulsed ultrasound affects chondrocyte extracellular matrix production via an integrin-mediated p38 MAPK signaling pathway.
      • Cheng K.
      • Xia P.
      • Lin Q.
      • Shen S.
      • Gao M.
      • Ren S.
      • et al.
      Effects of low-intensity pulsed ultrasound on integrin-FAK-PI3K/Akt mechanochemical transduction in rabbit osteoarthritis chondrocytes.
      • Yuan L.J.
      • Niu C.C.
      • Lin S.S.
      • Yang C.Y.
      • Chan Y.S.
      • Chen W.J.
      • et al.
      Effects of low-intensity pulsed ultrasound and hyperbaric oxygen on human osteoarthritic chondrocytes.
      • Choi B.H.
      • Choi M.H.
      • Kwak M.G.
      • Min B.H.
      • Woo Z.H.
      • Park S.R.
      Mechanotransduction pathways of low-intensity ultrasound in C-28/I2 human chondrocyte cell line.
      • Zhang Z.J.
      • Huckle J.
      • Francomano C.A.
      • Spencer R.G.S.
      The influence of pulsed low-intensity ultrasound on matrix production of chondrocytes at different stages of differentiation: an explant study.
      , aggrecan (ACAN)
      • Nishida T.
      • Kubota S.
      • Aoyama E.
      • Yamanaka N.
      • Lyons K.M.
      • Takigawa M.
      Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation.
      ,
      • Uddin S.M.Z.
      • Richbourgh B.
      • Ding Y.
      • Hettinghouse A.
      • Komatsu D.E.
      • Qin Y.X.
      • et al.
      Chondro-protective effects of low intensity pulsed ultrasound.
      ,
      • Noriega S.
      • Mamedov T.
      • Turner J.A.
      • Subramanian A.
      Intermittent applications of continuous ultrasound on the viability, proliferation, morphology, and matrix production of chondrocytes in 3D matrices.
      ,
      • Ding W.
      • Du D.
      • Chen S.
      LIPUS promotes synthesis and secretion of extracellular matrix and reduces cell apoptosis in human osteoarthritis through upregulation of SOX9 expression.
      • Mukai S.
      • Ito H.
      • Nakagawa Y.
      • Akiyama H.
      • Miyamoto M.
      • Nakamura T.
      Transforming growth factor-β1 mediates the effects of low-intensity pulsed ultrasound in chondrocytes.
      • Chen C.H.
      • Kuo S.M.
      • Tien Y.C.
      • Shen P.C.
      • Kuo Y.W.
      • Huang H.H.
      Steady augmentation of anti-osteoarthritic actions of rapamycin by liposome-encapsulation in collaboration with low-intensity pulsed ultrasound.
      ,
      • Tien Y.C.
      • Lin S.D.
      • Chen C.H.
      • Lu C.C.
      • Su S.J.
      • Chih T.T.
      Effects of pulsed low-intensity ultrasound on human child chondrocytes.
      ,
      • Parvizi J.
      • Wu C.C.
      • Lewallen D.G.
      • Greenleaf J.F.
      • Bolander M.E.
      Low-intensity ultrasound stimulates proteoglycan synthesis in rat chondrocytes by increasing aggrecan gene expression.
      ,
      • Sekino J.
      • Nagao M.
      • Kato S.
      • Sakai M.
      • Abe K.
      • Nakayama E.
      • et al.
      Low-intensity pulsed ultrasound induces cartilage matrix synthesis and reduced MMP13 expression in chondrocytes.
      ,
      • Cheng K.
      • Xia P.
      • Lin Q.
      • Shen S.
      • Gao M.
      • Ren S.
      • et al.
      Effects of low-intensity pulsed ultrasound on integrin-FAK-PI3K/Akt mechanochemical transduction in rabbit osteoarthritis chondrocytes.
      • Yuan L.J.
      • Niu C.C.
      • Lin S.S.
      • Yang C.Y.
      • Chan Y.S.
      • Chen W.J.
      • et al.
      Effects of low-intensity pulsed ultrasound and hyperbaric oxygen on human osteoarthritic chondrocytes.
      • Choi B.H.
      • Choi M.H.
      • Kwak M.G.
      • Min B.H.
      • Woo Z.H.
      • Park S.R.
      Mechanotransduction pathways of low-intensity ultrasound in C-28/I2 human chondrocyte cell line.
      • Zhang Z.J.
      • Huckle J.
      • Francomano C.A.
      • Spencer R.G.S.
      The influence of pulsed low-intensity ultrasound on matrix production of chondrocytes at different stages of differentiation: an explant study.
      and proteoglycans (PGs)
      • Korstjens C.M.
      • Rijt R.H.H.
      • Albers G.H.R.
      • Semeins C.M.
      • Klein-Nulend J.
      Low-intensity pulsed ultrasound affects human articular chondrocytes in vitro.
      ,
      • Uddin S.M.Z.
      • Richbourgh B.
      • Ding Y.
      • Hettinghouse A.
      • Komatsu D.E.
      • Qin Y.X.
      • et al.
      Chondro-protective effects of low intensity pulsed ultrasound.
      ,
      • Choi B.H.
      • Woo J.I.
      • Min B.H.
      • Park S.R.
      Low-intensity ultrasound stimulates the viability and matrix gene expression of human articular chondrocytes in alginate bead culture.
      ,
      • Vaughan N.M.
      • Grainger J.
      • Bader D.L.
      • Knight M.M.
      The potential of pulsed low intensity ultrasound to stimulate chondrocytes matrix synthesis in agarose and monolayer cultures.
      ,
      • Park K.
      • Hoffmeister B.
      • Han D.K.
      • Hasty K.
      Therapeutic ultrasound effects on interleukin-1β stimulated cartilage construct in vitro.
      ,
      • Chen C.H.
      • Kuo S.M.
      • Tien Y.C.
      • Shen P.C.
      • Kuo Y.W.
      • Huang H.H.
      Steady augmentation of anti-osteoarthritic actions of rapamycin by liposome-encapsulation in collaboration with low-intensity pulsed ultrasound.
      ,
      • Min B.H.
      • Woo J.I.
      • Cho H.S.
      • Choi B.H.
      • Park S.J.
      • Choi M.J.
      • et al.
      Effects of low-intensity ultrasound (LIUS) stimulation on human cartilage explants.
      ,
      • Parvizi J.
      • Wu C.C.
      • Lewallen D.G.
      • Greenleaf J.F.
      • Bolander M.E.
      Low-intensity ultrasound stimulates proteoglycan synthesis in rat chondrocytes by increasing aggrecan gene expression.
      ,
      • Kopakkala-Tani M.
      • Leskinen J.J.
      • Karjalainen H.M.
      • Karjalainen T.
      • Hynynen K.
      • Töyräs J.
      • et al.
      Ultrasound stimulates proteoglycan synthesis in bovine primary chondrocytes.
      ,
      • Parvizi J.
      • Parpura V.
      • Greenleaf J.F.
      • Bolander M.E.
      Calcium signaling is required for ultrasound-stimulated aggrecan synthesis by rat chondrocytes.
      was statistically significantly increased in nearly all studies after ultrasound in both healthy and OA chondrocytes, even in IL-1β-treated cultures (Table I)
      • Uddin S.M.Z.
      • Richbourgh B.
      • Ding Y.
      • Hettinghouse A.
      • Komatsu D.E.
      • Qin Y.X.
      • et al.
      Chondro-protective effects of low intensity pulsed ultrasound.
      ,
      • Guan M.
      • Zhu Y.
      • Liao B.
      • Tan Q.
      • Qi H.
      • Zhang B.
      • et al.
      Low-intensity pulsed ultrasound inhibits VEGFA expression in chondrocytes and protects against cartilage degeneration in experimental osteoarthritis.
      . Although most studies reported an increase in ECM production, statistically significant decrease
      • Park K.
      • Hoffmeister B.
      • Han D.K.
      • Hasty K.
      Therapeutic ultrasound effects on interleukin-1β stimulated cartilage construct in vitro.
      ,
      • Tan L.
      • Ren Y.
      • van Kooten T.G.
      • Grijpma D.W.
      • Kuijer R.
      Low-intensity pulsed ultrasound (LIPUS) and pulsed electromagnetic field (PEMF) treatments affect degeneration of cultured articular cartilage explants.
      or no effect
      • Hasanova G.I.
      • Noriega S.E.
      • Mamedov T.G.
      • Thakurta Guha S.
      • Turner J.A.
      • Subramanian A.
      The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds.
      ,
      • Zhang Z.J.
      • Huckle J.
      • Francomano C.A.
      • Spencer R.G.S.
      The effects of pulsed low-intensity ultrasound on chondrocyte viability, proliferation, gene expression and matrix production.
      ,
      • Park K.
      • Hoffmeister B.
      • Han D.K.
      • Hasty K.
      Therapeutic ultrasound effects on interleukin-1β stimulated cartilage construct in vitro.
      ,
      • Parvizi J.
      • Wu C.C.
      • Lewallen D.G.
      • Greenleaf J.F.
      • Bolander M.E.
      Low-intensity ultrasound stimulates proteoglycan synthesis in rat chondrocytes by increasing aggrecan gene expression.
      ,
      • Sekino J.
      • Nagao M.
      • Kato S.
      • Sakai M.
      • Abe K.
      • Nakayama E.
      • et al.
      Low-intensity pulsed ultrasound induces cartilage matrix synthesis and reduced MMP13 expression in chondrocytes.
      in the expression of these proteins were also reported. Similarly, the hypertrophic marker COL X statistically significantly increased
      • Sekino J.
      • Nagao M.
      • Kato S.
      • Sakai M.
      • Abe K.
      • Nakayama E.
      • et al.
      Low-intensity pulsed ultrasound induces cartilage matrix synthesis and reduced MMP13 expression in chondrocytes.
      ,
      • Tan L.
      • Ren Y.
      • van Kooten T.G.
      • Grijpma D.W.
      • Kuijer R.
      Low-intensity pulsed ultrasound (LIPUS) and pulsed electromagnetic field (PEMF) treatments affect degeneration of cultured articular cartilage explants.
      or decreased
      • Zhang Z.J.
      • Huckle J.
      • Francomano C.A.
      • Spencer R.G.S.
      The effects of pulsed low-intensity ultrasound on chondrocyte viability, proliferation, gene expression and matrix production.
      ,
      • Guan M.
      • Zhu Y.
      • Liao B.
      • Tan Q.
      • Qi H.
      • Zhang B.
      • et al.
      Low-intensity pulsed ultrasound inhibits VEGFA expression in chondrocytes and protects against cartilage degeneration in experimental osteoarthritis.
      , whereas the chondrocyte fibrocartilage marker, COL I, remained unaltered after ultrasound application
      • Hasanova G.I.
      • Noriega S.E.
      • Mamedov T.G.
      • Thakurta Guha S.
      • Turner J.A.
      • Subramanian A.
      The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds.
      ,
      • Tan L.
      • Ren Y.
      • van Kooten T.G.
      • Grijpma D.W.
      • Kuijer R.
      Low-intensity pulsed ultrasound (LIPUS) and pulsed electromagnetic field (PEMF) treatments affect degeneration of cultured articular cartilage explants.
      . The effect sizes for each outcome and study are reported in Table II and the forest plots are provided on Supplementary Fig. S1.
      ECM degradation markers were assessed in 13 studies (42%, k = 31) (Supplementary Table S5). Most of these studies exhibited statistically significant reduction in the metalloproteinases (MMPs) expression that degrade ECM, namely, MMP-1
      • Choi B.H.
      • Woo J.I.
      • Min B.H.
      • Park S.R.
      Low-intensity ultrasound stimulates the viability and matrix gene expression of human articular chondrocytes in alginate bead culture.
      ,
      • Park K.
      • Hoffmeister B.
      • Han D.K.
      • Hasty K.
      Therapeutic ultrasound effects on interleukin-1β stimulated cartilage construct in vitro.
      ,
      • Cheng K.
      • Xia P.
      • Lin Q.
      • Shen S.
      • Gao M.
      • Ren S.
      • et al.
      Effects of low-intensity pulsed ultrasound on integrin-FAK-PI3K/Akt mechanochemical transduction in rabbit osteoarthritis chondrocytes.
      , MMP-3
      • Zuo D.
      • Tan B.
      • Jia G.
      • Wu D.
      • Yu L.
      • Jia L.
      A treatment combined prussian blue nanoparticles with low-intensity pulsed ultrasound alleviates cartilage damage in knee osteoarthritis by initiating PI3K/Akt/mTOR pathway.
      , MMP-13
      • Uddin S.M.Z.
      • Richbourgh B.
      • Ding Y.
      • Hettinghouse A.
      • Komatsu D.E.
      • Qin Y.X.
      • et al.
      Chondro-protective effects of low intensity pulsed ultrasound.
      ,
      • Zuo D.
      • Tan B.
      • Jia G.
      • Wu D.
      • Yu L.
      • Jia L.
      A treatment combined prussian blue nanoparticles with low-intensity pulsed ultrasound alleviates cartilage damage in knee osteoarthritis by initiating PI3K/Akt/mTOR pathway.
      ,
      • Chen C.H.
      • Kuo S.M.
      • Tien Y.C.
      • Shen P.C.
      • Kuo Y.W.
      • Huang H.H.
      Steady augmentation of anti-osteoarthritic actions of rapamycin by liposome-encapsulation in collaboration with low-intensity pulsed ultrasound.
      ,
      • Guan M.
      • Zhu Y.
      • Liao B.
      • Tan Q.
      • Qi H.
      • Zhang B.
      • et al.
      Low-intensity pulsed ultrasound inhibits VEGFA expression in chondrocytes and protects against cartilage degeneration in experimental osteoarthritis.
      ,
      • Sekino J.
      • Nagao M.
      • Kato S.
      • Sakai M.
      • Abe K.
      • Nakayama E.
      • et al.
      Low-intensity pulsed ultrasound induces cartilage matrix synthesis and reduced MMP13 expression in chondrocytes.
      ,
      • Cheng K.
      • Xia P.
      • Lin Q.
      • Shen S.
      • Gao M.
      • Ren S.
      • et al.
      Effects of low-intensity pulsed ultrasound on integrin-FAK-PI3K/Akt mechanochemical transduction in rabbit osteoarthritis chondrocytes.
      ,
      • Ji J.B.
      • Li X.F.
      • Liu L.
      • Wang G.Z.
      • Yan X.F.
      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.
      • Xia P.
      • Shen S.
      • Lin Q.
      • Cheng K.
      • Ren S.
      • Gao M.
      • et al.
      Low-intensity pulsed ultrasound treatment at an early osteoarthritis stage protects rabbit cartilage from damage via the integrin/focal adhesion kinase/mitogen-activated protein kinase signaling pathway.
      • Ngai S.P.C.
      • Spencer L.M.
      • Jones A.Y.M.
      • Alison J.A.
      • Vemulpad S.
      Acu-TENS reduces breathlessness during exercise in people with chronic obstructive pulmonary disease.
      • Ito A.
      • Zhang X.
      • Yamaguchi S.
      • Aoyama T.
      • Akiyama H.
      • Kuroki H.
      Low-intensity pulsed ultrasound inhibits messenger RNA expression of matrix metalloproteinase-13 induced by interleukin-1 beta in intensity-dependent manner on chondrocytes.
      and the aggrecanase ADAMTS
      • Uddin S.M.Z.
      • Richbourgh B.
      • Ding Y.
      • Hettinghouse A.
      • Komatsu D.E.
      • Qin Y.X.
      • et al.
      Chondro-protective effects of low intensity pulsed ultrasound.
      after ultrasound application, while the tissue inhibitors, TIMPs, were statistically significantly increased
      • Yuan L.J.
      • Niu C.C.
      • Lin S.S.
      • Yang C.Y.
      • Chan Y.S.
      • Chen W.J.
      • et al.
      Effects of low-intensity pulsed ultrasound and hyperbaric oxygen on human osteoarthritic chondrocytes.
      ,
      • Ji J.B.
      • Li X.F.
      • Liu L.
      • Wang G.Z.
      • Yan X.F.
      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.
      ,
      • Ito A.
      • Zhang X.
      • Yamaguchi S.
      • Aoyama T.
      • Akiyama H.
      • Kuroki H.
      Low-intensity pulsed ultrasound inhibits messenger RNA expression of matrix metalloproteinase-13 induced by interleukin-1 beta in intensity-dependent manner on chondrocytes.
      (Table I). Both statistically significant increased
      • Nishida T.
      • Kubota S.
      • Aoyama E.
      • Yamanaka N.
      • Lyons K.M.
      • Takigawa M.
      Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation.
      ,
      • Sekino J.
      • Nagao M.
      • Kato S.
      • Sakai M.
      • Abe K.
      • Nakayama E.
      • et al.
      Low-intensity pulsed ultrasound induces cartilage matrix synthesis and reduced MMP13 expression in chondrocytes.
      and unaltered
      • Choi B.H.
      • Woo J.I.
      • Min B.H.
      • Park S.R.
      Low-intensity ultrasound stimulates the viability and matrix gene expression of human articular chondrocytes in alginate bead culture.
      ,
      • Park K.
      • Hoffmeister B.
      • Han D.K.
      • Hasty K.
      Therapeutic ultrasound effects on interleukin-1β stimulated cartilage construct in vitro.
      ,
      • Yuan L.J.
      • Niu C.C.
      • Lin S.S.
      • Yang C.Y.
      • Chan Y.S.
      • Chen W.J.
      • et al.
      Effects of low-intensity pulsed ultrasound and hyperbaric oxygen on human osteoarthritic chondrocytes.
      expression were also noticed for these degradation markers. For vascular endothelial growth factor A, VEGFA
      • Guan M.
      • Zhu Y.
      • Liao B.
      • Tan Q.
      • Qi H.
      • Zhang B.
      • et al.
      Low-intensity pulsed ultrasound inhibits VEGFA expression in chondrocytes and protects against cartilage degeneration in experimental osteoarthritis.
      , and inflammation, IL-1β and IL-6
      • Zuo D.
      • Tan B.
      • Jia G.
      • Wu D.
      • Yu L.
      • Jia L.
      A treatment combined prussian blue nanoparticles with low-intensity pulsed ultrasound alleviates cartilage damage in knee osteoarthritis by initiating PI3K/Akt/mTOR pathway.
      ,
      • Chen C.H.
      • Kuo S.M.
      • Tien Y.C.
      • Shen P.C.
      • Kuo Y.W.
      • Huang H.H.
      Steady augmentation of anti-osteoarthritic actions of rapamycin by liposome-encapsulation in collaboration with low-intensity pulsed ultrasound.
      , ultrasound exposure statistically significantly decreased their expression, whereas the oxidation markers have both statistically significantly increased
      • Yuan L.J.
      • Niu C.C.
      • Lin S.S.
      • Yang C.Y.
      • Chan Y.S.
      • Chen W.J.
      • et al.
      Effects of low-intensity pulsed ultrasound and hyperbaric oxygen on human osteoarthritic chondrocytes.
      or decreased
      • Zuo D.
      • Tan B.
      • Jia G.
      • Wu D.
      • Yu L.
      • Jia L.
      A treatment combined prussian blue nanoparticles with low-intensity pulsed ultrasound alleviates cartilage damage in knee osteoarthritis by initiating PI3K/Akt/mTOR pathway.
      . The effect sizes for each outcome and study are reported in Table II and the forest plots are provided on Supplementary Fig. S1.

      Mechanotransduction pathways

      Eleven studies
      • Nishida T.
      • Kubota S.
      • Aoyama E.
      • Yamanaka N.
      • Lyons K.M.
      • Takigawa M.
      Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation.
      ,
      • Uddin S.M.Z.
      • Richbourgh B.
      • Ding Y.
      • Hettinghouse A.
      • Komatsu D.E.
      • Qin Y.X.
      • et al.
      Chondro-protective effects of low intensity pulsed ultrasound.
      ,
      • Hasanova G.I.
      • Noriega S.E.
      • Mamedov T.G.
      • Thakurta Guha S.
      • Turner J.A.
      • Subramanian A.
      The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds.
      ,
      • Zuo D.
      • Tan B.
      • Jia G.
      • Wu D.
      • Yu L.
      • Jia L.
      A treatment combined prussian blue nanoparticles with low-intensity pulsed ultrasound alleviates cartilage damage in knee osteoarthritis by initiating PI3K/Akt/mTOR pathway.
      ,
      • Guan M.
      • Zhu Y.
      • Liao B.
      • Tan Q.
      • Qi H.
      • Zhang B.
      • et al.
      Low-intensity pulsed ultrasound inhibits VEGFA expression in chondrocytes and protects against cartilage degeneration in experimental osteoarthritis.
      ,
      • Sekino J.
      • Nagao M.
      • Kato S.
      • Sakai M.
      • Abe K.
      • Nakayama E.
      • et al.
      Low-intensity pulsed ultrasound induces cartilage matrix synthesis and reduced MMP13 expression in chondrocytes.
      ,
      • Cheng K.
      • Xia P.
      • Lin Q.
      • Shen S.
      • Gao M.
      • Ren S.
      • et al.
      Effects of low-intensity pulsed ultrasound on integrin-FAK-PI3K/Akt mechanochemical transduction in rabbit osteoarthritis chondrocytes.
      ,
      • Choi B.H.
      • Choi M.H.
      • Kwak M.G.
      • Min B.H.
      • Woo Z.H.
      • Park S.R.
      Mechanotransduction pathways of low-intensity ultrasound in C-28/I2 human chondrocyte cell line.
      ,
      • Xia P.
      • Shen S.
      • Lin Q.
      • Cheng K.
      • Ren S.
      • Gao M.
      • et al.
      Low-intensity pulsed ultrasound treatment at an early osteoarthritis stage protects rabbit cartilage from damage via the integrin/focal adhesion kinase/mitogen-activated protein kinase signaling pathway.
      ,
      • Kim E.J.
      • Kim G.Y.
      Effect of low intensity pulsed ultrasound in activating the mitogen-activated protein kinase signaling pathway and inhibition inflammation cytokine synthesis in chondrocytes.
      ,
      • Whitney N.P.
      • Lamb A.C.
      • Louw T.M.
      • Subramanian A.
      Integrin-mediated mechanotransduction pathway of low-intensity continuous ultrasound in human chondrocytes.
      investigated the signaling pathways involved in transducing the ultrasound effects (Table III). The mitogen-activated protein kinases (MAPK) subfamilies were the most commonly explored (Supplementary Table S5), including extracellular signal-related kinases (ERK1/2; 27%, k = 11), c-Jun amino-terminal kinases (JNK; 27%, k = 11) and p-38 (45%, k = 11), along with integrin-mediated pathways (45%, k = 11).
      Table IIIIntracellular signaling pathways
      Signaling pathwayExperimental designOutcome measuresSignificant findingsGeneral remarksRef.
      Integrin-mediatedG1: Control (healthy cells)

      G2: US in healthy cells

      G3: OA cells

      G4: US in OA cells

      G5: Control (healthy cells) + GRGDSP

      G6: US in healthy cells + GRGDSP

      G7: OA cells + GRGDSP

      G8: US in OA cells + GRGDSP

      GRGDSP (integrin inhibitor)
      Protein quantification:

      Integrin β1

      MMP-13

      COL II

      (Western blot)
      ↑ Integrin β1 [G2/G3/G4 vs G1; G4 vs G3]

      ↓ MMP-13 [G2 vs G1; G4 vs G3; G5 vs G1; G7 vs G3]

      ↑ COL II [G2 vs G1; G4 vs G3; G5 vs G1; G7 vs G3]

      No effect between G5 vs G6 and G7 vs G8 for MMP-13 and COL II.
      ↑ ECM production and ↓ MMP-13 expression after US treatment through integrin-p38 MAPK signaling pathway.Xia et al. (2015)
      G1: Control (healthy cells)

      G2: OA cells

      G3: OA cells + US
      Gene and Protein quantification:

      Integrin β1

      Protein quantification: p-FAK/FAK

      (RT-PCR and Western blot)
      ↑ Integrin β1 [G2/G3 vs G1; G3 vs G4]

      ↑ p-FAK/FAK [G2/G3 vs G1; G3 vs G4]
      The integrin-FAK signaling may be involved in ↓ COL II and ↓ Aggrecan production and ↓ MMPs after US treatment.Cheng et al. (2014)
      G1: Control

      G2: US at once

      G3: US at twice

      G4: US at 4 times

      G5: US at 8 times
      Gene and Protein quantification:

      Integrins β1, α2, α5 and αV

      (RT-PCR and Western blot)
      ↑ Integrin β1 [G2/G3/G4/G5 vs G1]

      ↓ Integrin α2 [G2/G3/G4/G5 vs G1]

      ↑ Integrin α5 [G2/G3/G4/G5 vs G1]

      No effect on integrin αV.
      Integrin subunits may be involved in the regulation of chondrocyte function in response to US treatment.Hasanova et al. (2011)
      G1: Control

      G2: US

      G3: US + GRGDSP (inhibitor of integrin)

      G4: US + PP1 (inhibitor of Src)

      G5: US + PD98059 (inhibitor of MAPK/Erk kinase)
      Protein quantification: p-FAK/FAK

      p-Src/Src

      p-p130Cas/p130Cas

      p-CrkII/CrkII

      p-Erk1/2/Erk1/2

      (Western blot)
      ↑ p-FAK/FAK [G2 vs G1]

      ↑ p-Src/Src [G2 vs G1]

      ↑ p-p130Cas/p130Cas [G2 vs G1]

      ↑ p-CrkII/CrkII [G2 vs G1]

      ↑ p-Erk1/2/Erk1/2 [G2 vs G1]

      ↓ p-Erk1/2/Erk1/2 [G3/G4/G5 vs G2]
      US treatment activated the integrin/MAPK pathways involving FAK, Src, p130Cas, and CrkII.Whitney et al. (2012)
      G1: Control

      G2: US

      G3: US + GRGDSP (inhibitor of integrins)
      Gene quantification:

      COL II

      Aggrecan

      (RT-PCR)
      ↑ COL II [G2 vs G1]

      ↓ COL II [G3 vs G2]

      ↑ Aggrecan [G2 vs G1]

      ↓ Aggrecan [G3 vs G2]
      US treatment activated integrin to ↑ COL II and ↑ Aggrecan.Choi et al. (2007)
      ERK1/2 MAPKG1: Control

      G2: US at 30 mW/cm2

      G3: US at 60 mW/cm2
      Protein quantification: p-ERK1/2/ERK1/2

      (Western blot)
      ↑ ERK1/2/p-ERK1/2 [G2/G3 vs G1; G3 vs G2]ERK1/2 pathway was involved in chondrocyte differentiation and ↑ ECM synthesis after US treatment.Sekino et al. (2018)
      G1: Control

      G2: US

      G3: 5 min after US

      G4: 10 min after US

      G5: 15 min after US

      G6: 30 min after US

      G7: 60 min after US

      Pre-treatment with PD98059 (ERK inhibitor)
      Protein quantification: p-ERK1/2/ERK1/2

      CCN2

      (Western blot)
      ↑ ERK1/2/p-ERK1/2 [G2/G3/G4 vs G1]

      ↓ ERK1/2/p-ERK1/2 [G5/G6/G7 vs G1]

      ↓ CCN2 production in G2 after PD98059
      ERK1/2 pathway was involved in the ↑ CCN2 production by US treatment.Nishida et al. (2017)
      G1: Control

      G2: US

      G3: US + PD98059 and U0126 (ERK inhibitors)
      Protein quantification: p-ERK/ERK

      (Western blot)
      ↑ p-ERK/ERK [G2 vs G1]

      ↓ p-ERK/ERK [G3 vs G1]
      US treatment activated the ERK proteins of MAPK family.Choi et al. (2007)
      JNK MAPKG1: Control

      G2: Only LPS

      G3: LPS + PBNPs + US
      Protein quantification: p-JNK/JNK

      p-c-Jun/c-Jun protein

      (Western blot)
      ↑ p-JNK/JNK [G2 vs G1]

      ↓ p-JNK/JNK [G3 vs G1/G2]
      JNK/c-Jun pathway may be responsible for ↓ inflammation and ↓ MMPs after PBNPs/LIPUS treatment.Zuo et al. (2021)
      G1: Control

      G2: US

      G3: IL-1β

      G4: US + IL-1β
      Protein quantification: p-JNK/JNK

      (Western blot)
      ↑ JNK/p-JNK [G3 vs G1]

      No effect in G4.
      JNK pathway was not involved in US treatment effects.Guan et al. (2020)
      G1: Control

      G2: US
      Protein quantification: p-JNK/JNK

      (Western blot)
      ↑ p-JNK/JNK [G2 vs G1]US treatment activated the JNK proteins of MAPK family.Choi et al. (2007)
      p38 MAPKG1: Control

      G2: US

      G3: IL-1β

      G4: US + IL-1β

      G5: IL-1β + SB203580 (p38 MAPK inhibitor)

      G6: US + IL-1β + SB203580
      Protein quantification: p-p38/p38

      (Western blot)

      Gene quantification:

      VEGFA, following MAPK inhibition

      (RT-PCR)
      ↑ p38 MAPK/p-p38 MAPK [G3 vs G1]

      ↓ p38 MAPK/p-p38 MAPK [G4 vs G3]

      ↓ VEGFA [G5 vs G3]

      No effect on G6.
      US ↓ VEGFA expression by inhibiting the p38 MAPK pathway.Guan et al. (2020)
      G1: Control

      G2: US

      G3: 5 min after US

      G4: 10 min after US

      G5: 15 min after US

      G6: 30 min after US

      G7: 60 min after US

      Pre-treatment with SB203580 (p38 MAPK inhibitor)
      Protein quantification: p-p38/p38

      CCN2

      (Western blot)
      ↑ p38 MAPK/p-p38 MAPK [G2 vs G1]

      ↓ p38 MAPK/p-p38 MAPK [G3/G4/G5/G6/G7 vs G2]

      ↓ CCN2 production in G2 after SB203580
      The p38 MAPK pathway was involved in ↑ CCN2 production by US treatment.Nishida et al. (2017)
      G1: Control (healthy cells)

      G2: US in healthy cells

      G3: OA cells

      G4: US in OA cells

      G5: Control (healthy cells) + SB203580

      G6: US in healthy cells + SB203580

      G7: OA cells + SB203580

      G8: US in OA cells + SB203580

      SB203580 (p38 inhibitor)
      Protein quantification: p-p38/p38

      MMP-13

      COL II

      (Western blot)
      ↑ p-p38/p38 [G2 vs G1]

      ↓ p-p38/p38 [G4 vs G3]

      ↓ MMP-13 [G2 vs G1; G4 vs G3; G5 vs G1; G7 vs G3; G8 vs G7]

      ↑ COL II [G2 vs G1; G4 vs G3; G5 vs G1; G7 vs G3; G8 vs G7]

      No effect between G5 and G6 for MMP-13 and COL II.
      ↑ ECM production and ↓ MMP-13 expression after US treatment through integrin-p38 MAPK signaling pathway.Xia et al. (2015)
      G1: Control

      G2: US

      G3: H2O2

      G4: H2O2 + US

      G5: H2O2 + US + SB203580

      SB203580 (p38 inhibitor)
      Protein quantification: p-p38/p38

      (Western blot)
      ↑ p-p38/p38 [G2/G3 vs G1; G4 vs G2/G3]

      ↓ p-p38/p38 [G5 vs G4]
      The p38 MAPK pathway ↓ oxidative stress-induced chondrocyte damage after US treatment.Kim et al. (2014)
      G1: Control

      G2: US
      Protein quantification: p-p38/p38

      (Western blot)
      No effect on p-p38/p38.US treatment did not have any effect on p-38.Choi et al. (2007)
      Ca2+ channelsG1: Control

      G2: US

      G3: Ionomycin (calcium ionophore)

      G4: US + ionomycin
      Fluorescence microscopy:

      Fluo-4 probe to observe Ca2+ influx
      ↑ Ca2+ influx [G2 vs G1; G3 vs G2/G4]

      ↓ Ca2+ influx [G4 vs G3]
      US treatment activates Ca2+ influx, ↑ CCN2 levels.Nishida et al. (2017)
      G1: Control

      G2: US at 50 mW/cm2
      Fluorescence microscopy:

      Photofluor fura-2 AM to observe Ca2+ influx
      ↑ Ca2+ influx [G2 vs G1]US treatment increased the Ca2+ influx which was associated with increased proteoglycans synthesis.Parvizi et al. (2002)
      Stretch-activated ion channels (SACs)G1: Control

      G2: US

      G3: US + gadolinium (SACs inhibitor)
      Gene quantification:

      COL II

      Aggrecan

      (RT-PCR)
      ↑ COL II [G2 vs G1]

      ↓ COL II [G3 vs G2]

      ↑ Aggrecan [G2 vs G1]

      ↓ Aggrecan [G3 vs G2]
      US treatment activated SACs to ↑ COL II and ↑ Aggrecan.Choi et al. (2007)
      Immunocytochemistry:

      COL II and actin
      ↑ COL II [G2 vs G1]

      ↓ Actin [G3 vs G2]
      PI3K/AktG1: Control

      G2: Only LPS

      G3: LPS + PBNPs + US

      G4: LPS + PBNPs + US + Wortmannin (inhibitor of PI3K)
      Protein quantification: p-PI3K/PI3K

      p-Akt/Akt

      p-mTOR/mTOR

      (Western blot)
      ↓ p-PI3K/PI3K [G2 vs G1; G4 vs G1/G2/G3]

      ↑ p-PI3K/PI3K [G3 vs G1/G2]

      ↓ p-Akt/Akt [G2 vs G1; G4 vs G1/G2/G3]

      ↑ p-Akt/Akt [G3 vs G1/G2]

      ↓ p-mTOR/mTOR [G2 vs G1; G4 vs G1/G2/G3]

      ↑ p-mTOR/mTOR [G3 vs G1/G2]
      PI3K-Akt-mTOR pathway may be responsible for ↓ ROS induced by LPS and ↓ cell apoptosis after PBNPs/LIPUS treatment.Zuo et al. (2021)
      Cell apoptosis (Flow cytometry; tunnel staining)↑ Cell apoptosis [G4 vs G3]
      ROS detection (Fluorescence microscopy)↑ ROS production [G4 vs G3]
      G1: Control (healthy cells)

      G2: OA cells

      G3: OA cells + US
      Protein quantification: p-PI3K/PI3K

      p-Akt/Akt

      (Western blot)
      ↑ p-PI3K/PI3K [G2/G3 vs G1; G3 vs G1]

      ↑ p-Akt/Akt [G2/G3 vs G1; G3 vs G1]
      The PI3K/Akt pathway may be involved in ↑ COL II and ↑ Aggrecan production and ↓ MMPs after US treatment.Cheng et al. (2014)
      RhoA/ROCKG1: Control

      G2: US

      G3: US + Y27632/NSC23766 (RhoA/ROCK inhibitor)
      Protein quantification:

      CCN2

      (Western blot)
      No effect on CCN2 production in any group.RhoA/ROCK pathway was not involved in the production of CCN2 by US treatment.Nishida et al. (2017)
      NFκBG1: Control

      G2: IL-1β

      G3: US + IL-1β

      G4: US
      Protein quantification: p-NFκB-p65/NFκB-p65

      p-IκBα/IκBα

      (Western blot)
      ↑ NFκB-p65/pNFκB-p65 [G2 vs G1/G3]

      ↑ IκBα/pIκBα [G2 vs G1/G3]
      US treatment ↓ IL-1β activation by inhibiting the NFκB pathway.Uddin et al. (2016)
      G: Group; PI3K/Akt: Phosphoinositide-3-kinase/akt; LPS: Lipopolysaccharide; PBNPs: Prussian blue nanoparticles; US: Ultrasound; mTOR: mammalian target of rapamycin; JNK: c-Jun N-terminal kinases; ROS: Reactive oxidative stress; IL: Interleukin; MMP: Metalloproteinase; OA: Osteoarthritis; RT-PCR: Real-time polymerase chain reaction; COL: Collagen; VEGF: Vascular endothelial growth factor;; CCN2: CCN family protein 2; MAPK: mitogen-activated protein kinase; RhoA/ROCK: Rho-associated protein kinase; Erk1/2: Extracellular signal-regulated kinase 1/2; Ca2+: Calcium; NFκB: Factor nuclear kappa B; IκBα: inhibitor of NFκB; FAK: Focal adhesion kinase.
      Integrin-mediated pathways were triggered by ultrasound application
      • Hasanova G.I.
      • Noriega S.E.
      • Mamedov T.G.
      • Thakurta Guha S.
      • Turner J.A.
      • Subramanian A.
      The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds.
      ,
      • Xia P.
      • Ren S.
      • Lin Q.
      • Cheng K.
      • Shen S.
      • Gao M.
      • et al.
      Low-intensity pulsed ultrasound affects chondrocyte extracellular matrix production via an integrin-mediated p38 MAPK signaling pathway.
      ,
      • Cheng K.
      • Xia P.
      • Lin Q.
      • Shen S.
      • Gao M.
      • Ren S.
      • et al.
      Effects of low-intensity pulsed ultrasound on integrin-FAK-PI3K/Akt mechanochemical transduction in rabbit osteoarthritis chondrocytes.
      ,
      • Choi B.H.
      • Choi M.H.
      • Kwak M.G.
      • Min B.H.
      • Woo Z.H.
      • Park S.R.
      Mechanotransduction pathways of low-intensity ultrasound in C-28/I2 human chondrocyte cell line.
      ,
      • Whitney N.P.
      • Lamb A.C.
      • Louw T.M.
      • Subramanian A.
      Integrin-mediated mechanotransduction pathway of low-intensity continuous ultrasound in human chondrocytes.
      , statistically significantly increasing ECM synthesis and reducing its degradation
      • Hasanova G.I.
      • Noriega S.E.
      • Mamedov T.G.
      • Thakurta Guha S.
      • Turner J.A.
      • Subramanian A.
      The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds.
      ,
      • Xia P.
      • Ren S.
      • Lin Q.
      • Cheng K.
      • Shen S.
      • Gao M.
      • et al.
      Low-intensity pulsed ultrasound affects chondrocyte extracellular matrix production via an integrin-mediated p38 MAPK signaling pathway.
      ,
      • Cheng K.
      • Xia P.
      • Lin Q.
      • Shen S.
      • Gao M.
      • Ren S.
      • et al.
      Effects of low-intensity pulsed ultrasound on integrin-FAK-PI3K/Akt mechanochemical transduction in rabbit osteoarthritis chondrocytes.
      ,
      • Choi B.H.
      • Choi M.H.
      • Kwak M.G.
      • Min B.H.
      • Woo Z.H.
      • Park S.R.
      Mechanotransduction pathways of low-intensity ultrasound in C-28/I2 human chondrocyte cell line.
      . Integrin subunit β1 was statistically significantly increased following ultrasound in both healthy and OA chondrocytes
      • Hasanova G.I.
      • Noriega S.E.
      • Mamedov T.G.
      • Thakurta Guha S.
      • Turner J.A.
      • Subramanian A.
      The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds.
      ,
      • Xia P.
      • Ren S.
      • Lin Q.
      • Cheng K.
      • Shen S.
      • Gao M.
      • et al.
      Low-intensity pulsed ultrasound affects chondrocyte extracellular matrix production via an integrin-mediated p38 MAPK signaling pathway.
      ,
      • Cheng K.
      • Xia P.
      • Lin Q.
      • Shen S.
      • Gao M.
      • Ren S.
      • et al.
      Effects of low-intensity pulsed ultrasound on integrin-FAK-PI3K/Akt mechanochemical transduction in rabbit osteoarthritis chondrocytes.
      , being larger in OA chondrocytes
      • Xia P.
      • Ren S.
      • Lin Q.
      • Cheng K.
      • Shen S.
      • Gao M.
      • et al.
      Low-intensity pulsed ultrasound affects chondrocyte extracellular matrix production via an integrin-mediated p38 MAPK signaling pathway.
      ,
      • Cheng K.
      • Xia P.
      • Lin Q.
      • Shen S.
      • Gao M.
      • Ren S.
      • et al.
      Effects of low-intensity pulsed ultrasound on integrin-FAK-PI3K/Akt mechanochemical transduction in rabbit osteoarthritis chondrocytes.
      . Ultrasound application statistically significantly increased the integrin subunit α5 expression and decreased the subunit α2 expression, depending on the regime employed, while it had no statistically significant effect on the subunit αv
      • Hasanova G.I.
      • Noriega S.E.
      • Mamedov T.G.
      • Thakurta Guha S.
      • Turner J.A.
      • Subramanian A.
      The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds.
      . The focal adhesion kinase (FAK) was phosphorylated following integrins activation, triggering other intracellular signaling pathways
      • Whitney N.P.
      • Lamb A.C.
      • Louw T.M.
      • Subramanian A.
      Integrin-mediated mechanotransduction pathway of low-intensity continuous ultrasound in human chondrocytes.
      .
      The ERK1/2 pathway was activated after ultrasound application in three studies
      • Nishida T.
      • Kubota S.
      • Aoyama E.
      • Yamanaka N.
      • Lyons K.M.
      • Takigawa M.
      Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation.
      ,
      • Sekino J.
      • Nagao M.
      • Kato S.
      • Sakai M.
      • Abe K.
      • Nakayama E.
      • et al.
      Low-intensity pulsed ultrasound induces cartilage matrix synthesis and reduced MMP13 expression in chondrocytes.
      ,
      • Choi B.H.
      • Choi M.H.
      • Kwak M.G.
      • Min B.H.
      • Woo Z.H.
      • Park S.R.
      Mechanotransduction pathways of low-intensity ultrasound in C-28/I2 human chondrocyte cell line.
      , by statistically significantly increasing the phosphorylation of ERK1/2 proteins (p-ERK1/2) in an intensity-dependent manner
      • Sekino J.
      • Nagao M.
      • Kato S.
      • Sakai M.
      • Abe K.
      • Nakayama E.
      • et al.
      Low-intensity pulsed ultrasound induces cartilage matrix synthesis and reduced MMP13 expression in chondrocytes.
      . The ERK activation led to statistically significant ECM and connective tissue growth factor (CCN2) production, involved in the regeneration processes of healthy chondrocytes
      • Nishida T.
      • Kubota S.
      • Aoyama E.
      • Yamanaka N.
      • Lyons K.M.
      • Takigawa M.
      Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation.
      ,
      • Sekino J.
      • Nagao M.
      • Kato S.
      • Sakai M.
      • Abe K.
      • Nakayama E.
      • et al.
      Low-intensity pulsed ultrasound induces cartilage matrix synthesis and reduced MMP13 expression in chondrocytes.
      .
      The JNK pathway was activated in OA chondrocytes subjected to IL-1β
      • Guan M.
      • Zhu Y.
      • Liao B.
      • Tan Q.
      • Qi H.
      • Zhang B.
      • et al.
      Low-intensity pulsed ultrasound inhibits VEGFA expression in chondrocytes and protects against cartilage degeneration in experimental osteoarthritis.
      and oxidative stress
      • Zuo D.
      • Tan B.
      • Jia G.
      • Wu D.
      • Yu L.
      • Jia L.
      A treatment combined prussian blue nanoparticles with low-intensity pulsed ultrasound alleviates cartilage damage in knee osteoarthritis by initiating PI3K/Akt/mTOR pathway.
      by statistically significantly increasing the expression of phosphorylated JNK (p-JNK). Ultrasound exposure did not change the p-JNK expression in IL-1β-treated chondrocytes
      • Guan M.
      • Zhu Y.
      • Liao B.
      • Tan Q.
      • Qi H.
      • Zhang B.
      • et al.
      Low-intensity pulsed ultrasound inhibits VEGFA expression in chondrocytes and protects against cartilage degeneration in experimental osteoarthritis.
      . For the oxidative-stressed chondrocytes, ultrasound was combined with prussian blue nanoparticles (PBNPs), statistically significantly reducing the p-JNK along with inflammatory and degradation markers
      • Zuo D.
      • Tan B.
      • Jia G.
      • Wu D.
      • Yu L.
      • Jia L.
      A treatment combined prussian blue nanoparticles with low-intensity pulsed ultrasound alleviates cartilage damage in knee osteoarthritis by initiating PI3K/Akt/mTOR pathway.
      . In healthy chondrocytes, ultrasound statistically significantly enhanced p-JNK, improving ECM protein synthesis
      • Choi B.H.
      • Choi M.H.
      • Kwak M.G.
      • Min B.H.
      • Woo Z.H.
      • Park S.R.
      Mechanotransduction pathways of low-intensity ultrasound in C-28/I2 human chondrocyte cell line.
      .
      The p38 pathway was implicated in anabolic activities of chondrocytes
      • Nishida T.
      • Kubota S.
      • Aoyama E.
      • Yamanaka N.
      • Lyons K.M.
      • Takigawa M.
      Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation.
      ,
      • Guan M.
      • Zhu Y.
      • Liao B.
      • Tan Q.
      • Qi H.
      • Zhang B.
      • et al.
      Low-intensity pulsed ultrasound inhibits VEGFA expression in chondrocytes and protects against cartilage degeneration in experimental osteoarthritis.
      ,
      • Xia P.
      • Ren S.
      • Lin Q.
      • Cheng K.
      • Shen S.
      • Gao M.
      • et al.
      Low-intensity pulsed ultrasound affects chondrocyte extracellular matrix production via an integrin-mediated p38 MAPK signaling pathway.
      ,
      • Choi B.H.
      • Choi M.H.
      • Kwak M.G.
      • Min B.H.
      • Woo Z.H.
      • Park S.R.
      Mechanotransduction pathways of low-intensity ultrasound in C-28/I2 human chondrocyte cell line.
      ,
      • Kim E.J.
      • Kim G.Y.
      Effect of low intensity pulsed ultrasound in activating the mitogen-activated protein kinase signaling pathway and inhibition inflammation cytokine synthesis in chondrocytes.
      . Ultrasound application statistically significantly increased the p38 phosphorylation (p-p38) in healthy chondrocytes and decreased it in OA chondrocytes. Notwithstanding these different mechanisms, COL II and CCN2 expressions were statistically significantly increased, while MMP-1
      • Nishida T.
      • Kubota S.
      • Aoyama E.
      • Yamanaka N.
      • Lyons K.M.
      • Takigawa M.
      Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation.
      ,
      • Xia P.
      • Shen S.
      • Lin Q.
      • Cheng K.
      • Ren S.
      • Gao M.
      • et al.
      Low-intensity pulsed ultrasound treatment at an early osteoarthritis stage protects rabbit cartilage from damage via the integrin/focal adhesion kinase/mitogen-activated protein kinase signaling pathway.
      and oxidative stress
      • Kim E.J.
      • Kim G.Y.
      Effect of low intensity pulsed ultrasound in activating the mitogen-activated protein kinase signaling pathway and inhibition inflammation cytokine synthesis in chondrocytes.
      statistically significantly declined in healthy and OA chondrocytes after ultrasound. IL-1β-treated chondrocytes had statistically significantly increased p-p38 and VEGFA quantification, but ultrasound reverted these expressions
      • Guan M.
      • Zhu Y.
      • Liao B.
      • Tan Q.
      • Qi H.
      • Zhang B.
      • et al.
      Low-intensity pulsed ultrasound inhibits VEGFA expression in chondrocytes and protects against cartilage degeneration in experimental osteoarthritis.
      . No statistically significant effect of ultrasound application on this pathway was observed for the C-28/I2 cell line
      • Choi B.H.
      • Choi M.H.
      • Kwak M.G.
      • Min B.H.
      • Woo Z.H.
      • Park S.R.
      Mechanotransduction pathways of low-intensity ultrasound in C-28/I2 human chondrocyte cell line.
      .
      Calcium (Ca2+) channels and stretch-activated ion channels (SACs) were activated following ultrasound application
      • Nishida T.
      • Kubota S.
      • Aoyama E.
      • Yamanaka N.
      • Lyons K.M.
      • Takigawa M.
      Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation.
      ,
      • Choi B.H.
      • Choi M.H.
      • Kwak M.G.
      • Min B.H.
      • Woo Z.H.
      • Park S.R.
      Mechanotransduction pathways of low-intensity ultrasound in C-28/I2 human chondrocyte cell line.
      ,
      • Parvizi J.
      • Parpura V.
      • Greenleaf J.F.
      • Bolander M.E.
      Calcium signaling is required for ultrasound-stimulated aggrecan synthesis by rat chondrocytes.
      . Such activation was linked to increased expression of CCN2, COL II, aggrecan and PGs.
      Other pathways were explored, yet less frequently. Ultrasound activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway
      • Zuo D.
      • Tan B.
      • Jia G.
      • Wu D.
      • Yu L.
      • Jia L.
      A treatment combined prussian blue nanoparticles with low-intensity pulsed ultrasound alleviates cartilage damage in knee osteoarthritis by initiating PI3K/Akt/mTOR pathway.
      ,
      • Cheng K.
      • Xia P.
      • Lin Q.
      • Shen S.
      • Gao M.
      • Ren S.
      • et al.
      Effects of low-intensity pulsed ultrasound on integrin-FAK-PI3K/Akt mechanochemical transduction in rabbit osteoarthritis chondrocytes.
      in both healthy and OA chondrocytes, being larger in OA chondrocytes
      • Cheng K.
      • Xia P.
      • Lin Q.
      • Shen S.
      • Gao M.
      • Ren S.
      • et al.
      Effects of low-intensity pulsed ultrasound on integrin-FAK-PI3K/Akt mechanochemical transduction in rabbit osteoarthritis chondrocytes.
      . Oxidative stress, cell apoptosis or MMPs production were statistically significantly decreased, while ECM production was statistically significantly increased
      • Zuo D.
      • Tan B.
      • Jia G.
      • Wu D.
      • Yu L.
      • Jia L.
      A treatment combined prussian blue nanoparticles with low-intensity pulsed ultrasound alleviates cartilage damage in knee osteoarthritis by initiating PI3K/Akt/mTOR pathway.
      ,
      • Cheng K.
      • Xia P.
      • Lin Q.
      • Shen S.
      • Gao M.
      • Ren S.
      • et al.
      Effects of low-intensity pulsed ultrasound on integrin-FAK-PI3K/Akt mechanochemical transduction in rabbit osteoarthritis chondrocytes.
      . The rho-associated protein kinase (RhoA/ROCK) pathway did not influence the ultrasound-induced CCN2 synthesis
      • Nishida T.
      • Kubota S.
      • Aoyama E.
      • Yamanaka N.
      • Lyons K.M.
      • Takigawa M.
      Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation.
      . The factor nuclear kappa B (NFκB) pathway
      • Uddin S.M.Z.
      • Richbourgh B.
      • Ding Y.
      • Hettinghouse A.
      • Komatsu D.E.
      • Qin Y.X.
      • et al.
      Chondro-protective effects of low intensity pulsed ultrasound.
      was activated in IL-1β-treated chondrocytes, statistically significantly increasing the degradation of ECM. Ultrasound suppressed these effects, promoting the ECM synthesis over its degradation
      • Uddin S.M.Z.
      • Richbourgh B.
      • Ding Y.
      • Hettinghouse A.
      • Komatsu D.E.
      • Qin Y.X.
      • et al.
      Chondro-protective effects of low intensity pulsed ultrasound.
      .

      Discussion

      The main finding of this systematic review is that all in vitro setups (i.e., ultrasound transducer in contact with well plate, culture media or water bath) positively influenced several intracellular signaling pathways, resulting in cartilage synthesis and suppression of its degradation, inflammation and apoptosis, both in healthy and OA chondrocytes.
      Ultrasound exerted a stimulatory effect, in most studies, by improving the expression of chondrogenic markers (i.e., SOX9, COL II, ACAN, PGs) and inhibiting degradation, and inflammatory and apoptosis markers on both healthy and OA chondrocytes. Even in in vitro OA models, ultrasound application reverted the catabolic activities on chondrocytes. Ultrasound did not seem to greatly influence the viability and proliferation due to the number of studies reporting no effect on those outcomes, neither has reverted the hypertrophic and fibrocartilage chondrocyte phenotypes. This is in line with our SMDs results which could be caused by the lack of studies calculating a priori the sample size required to detect statistically significant differences. Nevertheless, the studies' findings may suggest a potential effect of ultrasound in restoring articular cartilage in vitro, as also suggested by animal and clinical studies
      • Uddin S.M.Z.
      • Komatsu D.E.
      Therapeutic potential low-intensity pulsed ultrasound for osteoarthritis: pre-clinical and clinical perspectives.
      ,
      • Rothenberg J.B.
      • Jayaram P.
      • Naqvi U.
      • Gober J.
      • Malanga G.A.
      The role of low-intensity pulsed ultrasound on cartilage healing in knee osteoarthritis: a review.
      .
      The ultrasound-induced effects varied depending on stimulation protocols. Ultrasound used at intensities ranging from 2 to 200 mW/cm2 had a stimulatory effect on cartilage synthesis. Higher intensities of up to 700 mW/cm2 showed the opposite effect, decreasing the matrix production compared to lower dosages and/or control groups
      • Choi B.H.
      • Woo J.I.
      • Min B.H.
      • Park S.R.
      Low-intensity ultrasound stimulates the viability and matrix gene expression of human articular chondrocytes in alginate bead culture.
      ,
      • Min B.H.
      • Woo J.I.
      • Cho H.S.
      • Choi B.H.
      • Park S.J.
      • Choi M.J.
      • et al.
      Effects of low-intensity ultrasound (LIUS) stimulation on human cartilage explants.
      . Center frequencies of 3 or 5 MHz also stimulated the COL II and aggrecan
      • Hasanova G.I.
      • Noriega S.E.
      • Mamedov T.G.
      • Thakurta Guha S.
      • Turner J.A.
      • Subramanian A.
      The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds.
      ,
      • Noriega S.
      • Mamedov T.
      • Turner J.A.
      • Subramanian A.
      Intermittent applications of continuous ultrasound on the viability, proliferation, morphology, and matrix production of chondrocytes in 3D matrices.
      ,
      • Cheng K.
      • Xia P.
      • Lin Q.
      • Shen S.
      • Gao M.
      • Ren S.
      • et al.
      Effects of low-intensity pulsed ultrasound on integrin-FAK-PI3K/Akt mechanochemical transduction in rabbit osteoarthritis chondrocytes.
      ,
      • Xia P.
      • Shen S.
      • Lin Q.
      • Cheng K.
      • Ren S.
      • Gao M.
      • et al.
      Low-intensity pulsed ultrasound treatment at an early osteoarthritis stage protects rabbit cartilage from damage via the integrin/focal adhesion kinase/mitogen-activated protein kinase signaling pathway.
      ,
      • Whitney N.P.
      • Lamb A.C.
      • Louw T.M.
      • Subramanian A.
      Integrin-mediated mechanotransduction pathway of low-intensity continuous ultrasound in human chondrocytes.
      production and chondrocytes proliferation
      • Hasanova G.I.
      • Noriega S.E.
      • Mamedov T.G.
      • Thakurta Guha S.
      • Turner J.A.
      • Subramanian A.
      The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds.
      ,
      • Noriega S.
      • Mamedov T.
      • Turner J.A.
      • Subramanian A.
      Intermittent applications of continuous ultrasound on the viability, proliferation, morphology, and matrix production of chondrocytes in 3D matrices.
      , while higher frequencies of 8.5 MHz may have limited cell viability
      • Noriega S.
      • Mamedov T.
      • Turner J.A.
      • Subramanian A.
      Intermittent applications of continuous ultrasound on the viability, proliferation, morphology, and matrix production of chondrocytes in 3D matrices.
      . Increasing the number of sessions
      • Hasanova G.I.
      • Noriega S.E.
      • Mamedov T.G.
      • Thakurta Guha S.
      • Turner J.A.
      • Subramanian A.
      The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds.
      or the stimulation time
      • Park K.
      • Hoffmeister B.
      • Han D.K.
      • Hasty K.
      Therapeutic ultrasound effects on interleukin-1β stimulated cartilage construct in vitro.
      contributed to greater COL II and PGs production and lower MMPs expression.
      Ultrasound induces mechanical deformations in chondrocytes, activating their intracellular pathways
      • Vaca-González J.J.
      • Guevara J.M.
      • Moncayo M.A.
      • Castro-Abril H.
      • Hata Y.
      • Garzón-Alvarado D.A.
      Biophysical stimuli: a review of electrical and mechanical stimulation in hyaline cartilage.
      . We observed that these intracellular pathways were distinct between healthy and OA chondrocytes.
      In healthy chondrocytes (Fig. 4), following ultrasound application, SACs and Ca2+ channels were activated, increasing the intracellular Ca2+ which improved COL II and aggrecan synthesis
      • Nishida T.
      • Kubota S.
      • Aoyama E.
      • Yamanaka N.
      • Lyons K.M.
      • Takigawa M.
      Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation.
      ,
      • Choi B.H.
      • Choi M.H.
      • Kwak M.G.
      • Min B.H.
      • Woo Z.H.
      • Park S.R.
      Mechanotransduction pathways of low-intensity ultrasound in C-28/I2 human chondrocyte cell line.
      ,
      • Parvizi J.
      • Parpura V.
      • Greenleaf J.F.
      • Bolander M.E.
      Calcium signaling is required for ultrasound-stimulated aggrecan synthesis by rat chondrocytes.
      . Integrins clustered with FAK to form a focal adhesion complex composed of Src, p130Cas and CrkII molecules that were successively phosphorylated
      • Whitney N.P.
      • Lamb A.C.
      • Louw T.M.
      • Subramanian A.
      Integrin-mediated mechanotransduction pathway of low-intensity continuous ultrasound in human chondrocytes.
      , promoting ECM synthesis
      • Hasanova G.I.
      • Noriega S.E.
      • Mamedov T.G.
      • Thakurta Guha S.
      • Turner J.A.
      • Subramanian A.
      The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds.
      ,
      • Xia P.
      • Ren S.
      • Lin Q.
      • Cheng K.
      • Shen S.
      • Gao M.
      • et al.
      Low-intensity pulsed ultrasound affects chondrocyte extracellular matrix production via an integrin-mediated p38 MAPK signaling pathway.
      ,
      • Cheng K.
      • Xia P.
      • Lin Q.
      • Shen S.
      • Gao M.
      • Ren S.
      • et al.
      Effects of low-intensity pulsed ultrasound on integrin-FAK-PI3K/Akt mechanochemical transduction in rabbit osteoarthritis chondrocytes.
      ,
      • Choi B.H.
      • Choi M.H.
      • Kwak M.G.
      • Min B.H.
      • Woo Z.H.
      • Park S.R.
      Mechanotransduction pathways of low-intensity ultrasound in C-28/I2 human chondrocyte cell line.
      ,
      • Whitney N.P.
      • Lamb A.C.
      • Louw T.M.
      • Subramanian A.
      Integrin-mediated mechanotransduction pathway of low-intensity continuous ultrasound in human chondrocytes.
      . The activated complex or ion channels initiated multiple intracellular cascades, mainly, the PI3K/Akt and the MAPK pathways. The PI3K/Akt pathway involves the phosphorylation of PI3K and Akt that can also induce the phosphorylation of mammalian target of rapamycin (mTOR). This pathway activation following ultrasound application enhanced the ECM production and reduced cell oxidation and apoptosis
      • Zuo D.
      • Tan B.
      • Jia G.
      • Wu D.
      • Yu L.
      • Jia L.
      A treatment combined prussian blue nanoparticles with low-intensity pulsed ultrasound alleviates cartilage damage in knee osteoarthritis by initiating PI3K/Akt/mTOR pathway.
      ,
      • Cheng K.
      • Xia P.
      • Lin Q.
      • Shen S.
      • Gao M.
      • Ren S.
      • et al.
      Effects of low-intensity pulsed ultrasound on integrin-FAK-PI3K/Akt mechanochemical transduction in rabbit osteoarthritis chondrocytes.
      . The phosphorylation of p38, ERK or JNK kinases increased after ultrasound application
      • Nishida T.
      • Kubota S.
      • Aoyama E.
      • Yamanaka N.
      • Lyons K.M.
      • Takigawa M.
      Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation.
      ,
      • Sekino J.
      • Nagao M.
      • Kato S.
      • Sakai M.
      • Abe K.
      • Nakayama E.
      • et al.
      Low-intensity pulsed ultrasound induces cartilage matrix synthesis and reduced MMP13 expression in chondrocytes.
      ,
      • Xia P.
      • Ren S.
      • Lin Q.
      • Cheng K.
      • Shen S.
      • Gao M.
      • et al.
      Low-intensity pulsed ultrasound affects chondrocyte extracellular matrix production via an integrin-mediated p38 MAPK signaling pathway.
      ,
      • Choi B.H.
      • Choi M.H.
      • Kwak M.G.
      • Min B.H.
      • Woo Z.H.
      • Park S.R.
      Mechanotransduction pathways of low-intensity ultrasound in C-28/I2 human chondrocyte cell line.
      ,
      • Kim E.J.
      • Kim G.Y.
      Effect of low intensity pulsed ultrasound in activating the mitogen-activated protein kinase signaling pathway and inhibition inflammation cytokine synthesis in chondrocytes.
      . The activation of the ERK pathway was implicated in chondrocytes differentiation
      • Sekino J.
      • Nagao M.
      • Kato S.
      • Sakai M.
      • Abe K.
      • Nakayama E.
      • et al.
      Low-intensity pulsed ultrasound induces cartilage matrix synthesis and reduced MMP13 expression in chondrocytes.
      and ECM synthesis
      • Nishida T.
      • Kubota S.
      • Aoyama E.
      • Yamanaka N.
      • Lyons K.M.
      • Takigawa M.
      Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation.
      ,
      • Sekino J.
      • Nagao M.
      • Kato S.
      • Sakai M.
      • Abe K.
      • Nakayama E.
      • et al.
      Low-intensity pulsed ultrasound induces cartilage matrix synthesis and reduced MMP13 expression in chondrocytes.
      , while the activation the p38 pathway was involved in diminishing the oxidative stress and MMPs expression, and enhancing the ECM synthesis
      • Nishida T.
      • Kubota S.
      • Aoyama E.
      • Yamanaka N.
      • Lyons K.M.
      • Takigawa M.
      Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation.
      ,
      • Xia P.
      • Ren S.
      • Lin Q.
      • Cheng K.
      • Shen S.
      • Gao M.
      • et al.
      Low-intensity pulsed ultrasound affects chondrocyte extracellular matrix production via an integrin-mediated p38 MAPK signaling pathway.
      ,
      • Kim E.J.
      • Kim G.Y.
      Effect of low intensity pulsed ultrasound in activating the mitogen-activated protein kinase signaling pathway and inhibition inflammation cytokine synthesis in chondrocytes.
      . These findings suggest that ultrasound activated chondrocytes' mechanoreceptors (e.g., integrins and ion channels), triggered the PI3K/Akt, ERK1/2, p38 and JNK pathways to promote CCN2, COL II, aggrecan and PGs productions in healthy chondrocytes, demonstrating the interplay between several mechanoreceptors and pathways to transduce ultrasound signals.
      Fig. 4
      Fig. 4Mechanotransduction pathways involved in healthy chondrocytes. Mechanical deformations induced by ultrasound activate the mechanoreceptors of chondrocytes: integrin, stretch-activated and Ca2+ ion channels. Once activated, they further activate the intracellular signaling pathways that induce gene transcription at nucleus level, increasing the expression of CCN2, collagen type II, aggrecan and proteoglycans. Created with BioRender.com.
      In OA chondrocytes (Fig. 5), the p-p38 and p-JNK cascades were increased in chondrocytes subjected to oxidative stress
      • Zuo D.
      • Tan B.
      • Jia G.
      • Wu D.
      • Yu L.
      • Jia L.
      A treatment combined prussian blue nanoparticles with low-intensity pulsed ultrasound alleviates cartilage damage in knee osteoarthritis by initiating PI3K/Akt/mTOR pathway.
      ,
      • Kim E.J.
      • Kim G.Y.
      Effect of low intensity pulsed ultrasound in activating the mitogen-activated protein kinase signaling pathway and inhibition inflammation cytokine synthesis in chondrocytes.
      and inflammation
      • Guan M.
      • Zhu Y.
      • Liao B.
      • Tan Q.
      • Qi H.
      • Zhang B.
      • et al.
      Low-intensity pulsed ultrasound inhibits VEGFA expression in chondrocytes and protects against cartilage degeneration in experimental osteoarthritis.
      , which increased VEGFA expression
      • Guan M.
      • Zhu Y.
      • Liao B.
      • Tan Q.
      • Qi H.
      • Zhang B.
      • et al.
      Low-intensity pulsed ultrasound inhibits VEGFA expression in chondrocytes and protects against cartilage degeneration in experimental osteoarthritis.
      , indicating a potential involvement in OA pathogenesis. After ultrasound application, the p38 pathway was suppressed, restoring the anabolic activities and inhibiting the VEGFA expression
      • Guan M.
      • Zhu Y.
      • Liao B.
      • Tan Q.
      • Qi H.
      • Zhang B.
      • et al.
      Low-intensity pulsed ultrasound inhibits VEGFA expression in chondrocytes and protects against cartilage degeneration in experimental osteoarthritis.
      ,
      • Xia P.
      • Ren S.
      • Lin Q.
      • Cheng K.
      • Shen S.
      • Gao M.
      • et al.
      Low-intensity pulsed ultrasound affects chondrocyte extracellular matrix production via an integrin-mediated p38 MAPK signaling pathway.
      . Regarding the JNK signaling, the effect of ultrasound on OA chondrocytes was unclear. While one study
      • Guan M.
      • Zhu Y.
      • Liao B.
      • Tan Q.
      • Qi H.
      • Zhang B.
      • et al.
      Low-intensity pulsed ultrasound inhibits VEGFA expression in chondrocytes and protects against cartilage degeneration in experimental osteoarthritis.
      did not report any statistically significant effect in the p-JNK expression after ultrasound application; another study
      • Zuo D.
      • Tan B.
      • Jia G.
      • Wu D.
      • Yu L.
      • Jia L.
      A treatment combined prussian blue nanoparticles with low-intensity pulsed ultrasound alleviates cartilage damage in knee osteoarthritis by initiating PI3K/Akt/mTOR pathway.
      found a decreased p-JNK expression (resulting in diminished inflammation and degrading proteins) when ultrasound was combined with PBNPs. Thus, it is not possible to ascertain if the effect was related to the ultrasound application or the combined therapy. Administration of IL-1β to chondrocytes activated the NFκB pathway responsible for cartilage matrix degradation. However, ultrasound suppressed this pathway, downregulating the degradation markers, and stimulating matrix production
      • Uddin S.M.Z.
      • Richbourgh B.
      • Ding Y.
      • Hettinghouse A.
      • Komatsu D.E.
      • Qin Y.X.
      • et al.
      Chondro-protective effects of low intensity pulsed ultrasound.
      . These findings indicate that ultrasound exerted protective effects by inhibiting the p38, JNK and NFκB pathways in OA chondrocytes to reduce their catabolic effects, such as cell apoptosis, oxidative stress, MMPs, VEGFA and inflammation expressions.
      Fig. 5
      Fig. 5Mechanotransduction pathways involved in OA chondrocytes. Cellular stress, induced either by inflammation (IL-1β) or oxidative stress (hydrogen peroxide or lipopolysaccharide), activates intracellular signaling pathways that lead to increased expression of reactive oxidative stress (ROS), MMPs, VEGFA, inflammation and cell apoptosis. When OA chondrocytes are exposed to ultrasound, these signaling pathways are inhibited. Created with BioRender.com.
      Studies investigating the intracellular pathways applied ultrasound at intensities ranging from 0.14 to 200 mW/cm2. Stimulating at 60 mW/cm2 improved the integrins expression
      • Sekino J.
      • Nagao M.
      • Kato S.
      • Sakai M.
      • Abe K.
      • Nakayama E.
      • et al.
      Low-intensity pulsed ultrasound induces cartilage matrix synthesis and reduced MMP13 expression in chondrocytes.
      compared to 30 mW/cm2. For the same power density, 0.14 mW/cm2, integrins expression increased with increasing number of sessions
      • Hasanova G.I.
      • Noriega S.E.
      • Mamedov T.G.
      • Thakurta Guha S.
      • Turner J.A.
      • Subramanian A.
      The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds.
      .
      Only few studies have investigated the effects of ultrasound on OA chondrocytes. Although most studies provide insightful information on therapeutic outcomes on healthy chondrocytes, these cells have not undergone degenerative changes that occur during OA. Even when using OA chondrocytes, some of them were obtained from OA-induced animals, which do not fully recreate the spontaneous OA development. These compromises the applicability of the studies' results to human OA cartilage. It would be beneficial that in vitro studies better reflected the natural human OA cartilage where ultrasound application is desired.
      Nearly all studies presented high risk of bias in the “Experimental conditions” domain due to lack of in vitro setup calibration and temperature control. The thickness of well bottom and the acoustic gel layer, culture media height/volume and distance between the transducer tip to the well bottom were poorly reported. The propagation of ultrasound waves is deeply affected by those variables by decreasing the pressure amplitude and intensity, compromising the therapeutic effect
      • Hensel K.
      • Mienkina M.P.
      • Schmitz G.
      Analysis of ultrasound fields in cell culture wells for in vitro ultrasound therapy experiments.
      ,
      • Leskinen J.J.
      • Hynynen K.
      Study of factors affecting the magnitude and nature of ultrasound exposure with in vitro set-ups.
      . Previous calibration should always be conducted to ensure the dosage received by the cells is not significantly altered and/or to allow dosage readjustments. Temperature rise is an important variable to consider during stimulation, since ultrasound application may increase the temperature of the cell culture system, particularly, for the setup using acoustic gel, which may be sufficient to induce biological effects in chondrocytes
      • Leskinen J.J.
      • Hynynen K.
      Study of factors affecting the magnitude and nature of ultrasound exposure with in vitro set-ups.
      • Padilla F.
      • Puts R.
      • Vico L.
      • Raum K.
      Stimulation of bone repair with ultrasound: a review of the possible mechanic effects.
      • Ito A.
      • Aoyama T.
      • Tajino J.
      • Nagai M.
      • Yamaguchi S.
      • Iijima H.
      • et al.
      Effects of the thermal environment on articular chondrocyte metabolism: a fundamental study to facilitate establishment of an effective thermotherapy for osteoarthritis.
      . In case of a significant temperature rise, appropriate controls should be established to account for the temperature effect in the chondrocytes' activity.
      Our systematic review has some limitations. We were not able to perform a meta-analysis of the studies due to the heterogeneity observed between studies (e.g., cells source, type or culture conditions used). Therefore, we calculated the SMDs for each study included in the data synthesis, but we did not report the pooled effect size, that could lead to erroneous and spurious conclusions. The SMDs were computed based on a sample size of two (minimum possible value to perform meta-analysis) because studies did not provide the number of independent experiments or replicates. Although the search strategy was adequate, two thirds of the included studies were only identified by the cross-referencing searches. To circumvent this limitation, we have performed an extensive manual search to include as many studies as possible. The studies' findings should be interpreted with caution. Most studies were judged with high risk of performance and detection bias, and presented pseudoreplication issues, which may compromise the analysis of statistical results, limiting the confidence of our conclusions.
      Recommendations for future research must be highlighted. Several studies
      • Nishikori T.
      • Ochi M.
      • Uchio Y.
      • Maniwa S.
      • Kataoka H.
      • Kawasaki K.
      • et al.
      Effects of low-intensity pulsed ultrasound on proliferation and chondroitin sulfate synthesis of cultured chondrocytes embedded in Atelocollagen gel.
      ,
      • Korstjens C.M.
      • Rijt R.H.H.
      • Albers G.H.R.
      • Semeins C.M.
      • Klein-Nulend J.
      Low-intensity pulsed ultrasound affects human articular chondrocytes in vitro.
      ,
      • Vaughan N.M.
      • Grainger J.
      • Bader D.L.
      • Knight M.M.
      The potential of pulsed low intensity ultrasound to stimulate chondrocytes matrix synthesis in agarose and monolayer cultures.
      ,
      • Zhang Z.J.
      • Huckle J.
      • Francomano C.A.
      • Spencer R.G.S.
      The effects of pulsed low-intensity ultrasound on chondrocyte viability, proliferation, gene expression and matrix production.
      ,
      • Zuo D.
      • Tan B.
      • Jia G.
      • Wu D.
      • Yu L.
      • Jia L.
      A treatment combined prussian blue nanoparticles with low-intensity pulsed ultrasound alleviates cartilage damage in knee osteoarthritis by initiating PI3K/Akt/mTOR pathway.
      • Ding W.
      • Du D.
      • Chen S.
      LIPUS promotes synthesis and secretion of extracellular matrix and reduces cell apoptosis in human osteoarthritis through upregulation of SOX9 expression.
      • Mukai S.
      • Ito H.
      • Nakagawa Y.
      • Akiyama H.
      • Miyamoto M.
      • Nakamura T.
      Transforming growth factor-β1 mediates the effects of low-intensity pulsed ultrasound in chondrocytes.
      ,
      • Guan M.
      • Zhu Y.
      • Liao B.
      • Tan Q.
      • Qi H.
      • Zhang B.
      • et al.
      Low-intensity pulsed ultrasound inhibits VEGFA expression in chondrocytes and protects against cartilage degeneration in experimental osteoarthritis.
      ,
      • Sekino J.
      • Nagao M.
      • Kato S.
      • Sakai M.
      • Abe K.
      • Nakayama E.
      • et al.
      Low-intensity pulsed ultrasound induces cartilage matrix synthesis and reduced MMP13 expression in chondrocytes.
      ,
      • Yuan L.J.
      • Niu C.C.
      • Lin S.S.
      • Yang C.Y.
      • Chan Y.S.
      • Chen W.J.
      • et al.
      Effects of low-intensity pulsed ultrasound and hyperbaric oxygen on human osteoarthritic chondrocytes.
      ,
      • Zhang Z.J.
      • Huckle J.
      • Francomano C.A.
      • Spencer R.G.S.
      The influence of pulsed low-intensity ultrasound on matrix production of chondrocytes at different stages of differentiation: an explant study.
      ,
      • Ito A.
      • Zhang X.
      • Yamaguchi S.
      • Aoyama T.
      • Akiyama H.
      • Kuroki H.
      Low-intensity pulsed ultrasound inhibits messenger RNA expression of matrix metalloproteinase-13 induced by interleukin-1 beta in intensity-dependent manner on chondrocytes.
      ,
      • Kim E.J.
      • Kim G.Y.
      Effect of low intensity pulsed ultrasound in activating the mitogen-activated protein kinase signaling pathway and inhibition inflammation cytokine synthesis in chondrocytes.
      stimulated chondrocytes with parameters specifically developed for bone healing
      • Palanisamy P.
      • Alam M.
      • Li S.
      • Chow S.K.H.
      • Zheng Y.P.
      Low-intensity pulsed ultrasound stimulation for bone fractures healing: a review.
      ,
      • Park K.
      • Hoffmeister B.
      • Han D.K.
      • Hasty K.
      Therapeutic ultrasound effects on interleukin-1β stimulated cartilage construct in vitro.
      ,
      • Guan M.
      • Zhu Y.
      • Liao B.
      • Tan Q.
      • Qi H.
      • Zhang B.
      • et al.
      Low-intensity pulsed ultrasound inhibits VEGFA expression in chondrocytes and protects against cartilage degeneration in experimental osteoarthritis.
      ,
      • Ito A.
      • Zhang X.
      • Yamaguchi S.
      • Aoyama T.
      • Akiyama H.
      • Kuroki H.
      Low-intensity pulsed ultrasound inhibits messenger RNA expression of matrix metalloproteinase-13 induced by interleukin-1 beta in intensity-dependent manner on chondrocytes.
      . Despite improving chondrocytes activity, these devices do not offer optimized protocols to specifically treat articular cartilage. Stimulating above the range for bone healing, up to 200 mW/cm2 or 5 MHz, enhanced the therapeutic effects on chondrocytes. These parameters range have already been tested at the clinical level
      • Draper D.O.
      • Klyve D.
      • Ortiz R.
      • Best T.M.
      Effect of low-intensity long-duration ultrasound on the symptomatic relief of knee osteoarthritis: a randomized, placebo-controlled double-blind study.
      ,
      • Karakaş A.
      • Dilek B.
      • Şahin M.A.
      • Ellidokuz H.
      • Şenocak Ö.
      The effectiveness of pulsed ultrasound treatment on pain, function, synovial sac thickness and femoral cartilage thickness in patients with knee osteoarthritis: a randomized, double-blind clinical, controlled study.
      . Efforts could be directed to develop new therapeutic devices and respective protocols for an optimized stimulation of articular cartilage. When planning in vitro studies, the stimulation setup should be calibrated and the temperature monitored as well as properly described, to allow reproducibility of the results. Setups presented liquid–air interfaces which caused waves reflection, influencing the ultrasound dosage
      • Tan L.
      • Ren Y.
      • van Kooten T.G.
      • Grijpma D.W.
      • Kuijer R.
      Low-intensity pulsed ultrasound (LIPUS) and pulsed electromagnetic field (PEMF) treatments affect degeneration of cultured articular cartilage explants.
      . Selecting materials with similar acoustic impedance, as water or culture medium, and/or ultrasound-absorbing materials, or developing a setup without air interfaces will minimize the stimulus interference
      • Vaughan N.M.
      • Grainger J.
      • Bader D.L.
      • Knight M.M.
      The potential of pulsed low intensity ultrasound to stimulate chondrocytes matrix synthesis in agarose and monolayer cultures.
      ,
      • Kopakkala-Tani M.
      • Leskinen J.J.
      • Karjalainen H.M.
      • Karjalainen T.
      • Hynynen K.
      • Töyräs J.
      • et al.
      Ultrasound stimulates proteoglycan synthesis in bovine primary chondrocytes.
      ,
      • Hensel K.
      • Mienkina M.P.
      • Schmitz G.
      Analysis of ultrasound fields in cell culture wells for in vitro ultrasound therapy experiments.
      . Upcoming research should give more emphasis to human OA chondrocytes to provide clear evidence on the ultrasound potential in human OA cartilage. Aiming to reduce the risk of bias and increase study's replication, future studies must clearly report the chondrocytes source and characteristics, culture and stimulation conditions, and implement two independent operators for semi-quantitative analysis or perform those analyzes blindly. Pseudoreplication issues should be recognized by the authors who must always report the number of independent replicates.
      In conclusion, this systematic review highlights the effects of using therapeutic ultrasound on chondrocytes cultures and elucidates the underlying mechanotransduction processes. However, further research should target the application of ultrasound in human OA chondrocytes to provide more robust knowledge on its potential use in human OA cartilage. The outcome tables and figures herein summarized provide detailed and useful information on stimulation protocols and setups, biological outcomes and mechanotransduction pathways implicated in the ultrasound-induced effects.

      Conclusions

      Ultrasound improved the ECM synthesis and downregulated its degradation, inflammation and apoptosis, triggering different mechanotransduction pathways on healthy and OA chondrocytes. Most studies were judged with high risk of performance, detection and pseudoreplication bias that underpin the need for well-defined experimental conditions. Future studies should monitor the temperature change, calibrate the stimulation setup and target human OA chondrocytes cultures. Our findings will help direct future pre- and clinical research, aiming to the clinical application of ultrasound on cartilage regeneration.

      Authors' contribution

      All authors were involved both in the idealization of the systematic review and contributed for the design of the manuscript. S.O. and R.A. were involved in the databases searches and data extraction. S.O. performed the data collection and organization in coordination with R.A., B.B.H., A.L. and O.C.. S.O. and R.A. appraised the risk of bias of the studies included in the systematic review. Conflicts were resolved by B.B.H., A.L. and O.C.. B.B.H., F.S.S. and J.EM. guided and provided advice during all steps of the development of the systematic review. All authors contributed to drafting and approving the final manuscript prior to submission to the peer-reviewed journal.

      Declaration of conflicting interests

      The authors declare that there is no conflict of interest.

      Acknowledgments

      This work was supported by Fundação para a Ciência e Tecnologia (FCT) national funds, under the national support to R&D units grant, through the reference projects UIDB/04436/2020 and UIDP/04436/2020; Stimcart -PTDC/EME-EME/4520/2021, BrainStimMap-PTDC/EME-EME/1681/2021 and supported by the PhD fellowship grant UI/BD/150951/2021. The funder was not involved in any aspect of the project, such as the study design and manuscript draft. The funder was not involved in the interpretation or publication of the study results.

      Appendix A. Supplementary data

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