Synergistic Effects of Therapeutic Ultrasound and Biomaterials in Osteoarthritis

Abstract

Osteoarthritis (OA) is a common degenerative joint disease characterized by progressive cartilage degradation, subchondral bone remodeling, and synovial inflammation. Current treatments cannot halt or reverse OA progression, necessitating the development of novel noninvasive therapies. Therapeutic ultrasound (US), particularly low-intensity pulsed US, has demonstrated efficacy in slowing OA progression. Therapeutic US generates significant thermal and nonthermal effects through noninvasive mechanical forces, exerting biological effects and regulating cell behavior. Therapeutic US has been explored for bone and cartilage repair and shows broad potential in tissue repair when combined with biomaterials. This review summarizes the enhanced or synergistic effects of US and biomaterials in OA. This study elucidated the molecular mechanisms underlying the effects of US on synovium, cartilage, subchondral bone, and mesenchymal stem cells. Notably, the combination of US with various biomaterials can modulate cellular behavior in OA through synergistic effects, including tissue regeneration, enhanced mechanical stimulation, drug delivery, and microenvironment regulation. For each cell type, we summarize the biological mechanisms underlying the therapeutic effects of US and biomaterials, demonstrating their potential to mitigate OA progression. Furthermore, this article explores the limitations and future research prospects of combining US and biomaterials as a therapeutic strategy. Overall, the integration of US and biomaterials holds significant promise as a novel treatment for OA, with potential applications in broader musculoskeletal tissue repair and regenerative medicine.

Impact Statement

Osteoarthritis (OA) is a complex degenerative disorder that remains challenging to manage. This review highlights the innovative therapeutic potential of combining ultrasound (US), particularly low-intensity pulsed US, with biomaterials for OA treatment. By leveraging synergistic effects such as enhanced tissue repair, targeted drug delivery, and microenvironment regulation, this approach offers a noninvasive and effective strategy to mitigate OA progression and paves the way for advancements in musculoskeletal regenerative medicine.

Keywords

therapeutic ultrasound biomaterials osteoarthritis tissue regeneration molecular mechanisms

Introduction

Osteoarthritis (OA) is a prevalent degenerative joint disorder associated with substantial socioeconomic burdens. It is characterized by progressive cartilage degradation, subchondral bone remodeling, and synovial inflammation, leading to structural joint damage, osteophyte formation, and synovitis.^1,2 Most patients remain untreated until OA progresses to moderate or advanced stages. At that point, common medications (such as nonsteroidal anti-inflammatory drug (NSAIDs) used for OA pain relief) can only ease symptoms but cannot stop the disease from getting worse, so many patients end up needing joint replacement surgery.¹ Therefore, there is a need for more effective, noninvasive treatments, particularly those using ultrasound (US) and biomaterials, to promote cartilage and bone repair and regeneration.

Therapeutic US represents a rapidly evolving field with broad clinical applications, including physical therapy, drug delivery, ablation, and regenerative medicine.^3–5 Since its initial approval from the Food and Drug Administration (FDA) in 1994 for bone fracture treatment, therapeutic US has been recognized as a noninvasive and safe approach for managing musculoskeletal disorders.⁶ However, the underlying mechanisms through which therapeutic US affects OA remain to be fully elucidated.

In recent years, biomaterials have been extensively applied in tissue repair, demonstrating remarkable potential for articular cartilage and subchondral bone regeneration.⁷ One of the most promising applications of US is its ability to modulate the physical properties of biomaterials, such as enhancing the mechanical performance of bioscaffolds and increasing cellular permeability for optimized drug delivery.⁸ The development of novel biomaterials has further expanded the applications of US, revealing diverse potential uses in regenerative medicine.⁹

This review systematically examines the synergistic therapeutic effects of therapeutic US combined with biomaterials in the treatment of OA. Emphasis is placed on elucidating the mechanisms by which US, particularly low-intensity pulsed US (LIPUS), influences various cell types involved in OA pathology. An in-depth analysis explores the pathways and mechanisms underlying the synergy between US and biomaterials. Finally, we critically evaluate current limitations of these combined therapies and propose future research directions to advance their clinical translation.

Overview of Therapeutic US

Definition

The use of US to treat diseased organs or tissues is referred to as therapeutic US.¹⁰ In medical applications, the frequency range for therapeutic US typically spans from 20 kHz to 3 MHz, whereas frequencies beyond this range are generally reserved for diagnostic purposes.³ Therapeutic US can be broadly categorized into LIPUS and high-intensity focused US (HIFU), depending on the energy level delivered.^11,12 HIFU has emerged as a promising modality for chronic pain management and has received approval for treating chronic neuropathic pain, such as in patients with bone metastases and those experiencing pain due to small joint OA.^13,14 LIPUS, a specialized form of US, typically operates at frequencies of 1–3 MHz with intensities ranging from 0.02 to 1 W/cm².^15,16

Biological effects of therapeutic US

Thermal effects

As US waves propagate through biological tissues, a portion of their energy is absorbed at a rate that is related to tissue density and acoustic impedance, converting some of this energy into heat and causing localized temperature elevation.¹⁷ This thermal effect promotes blood circulation, stimulates collagen production, and aids cell regeneration, thereby promoting tissue repair.^18–20 LIPUS operates in a pulsed mode, minimizing thermal effects while emphasizing significant nonthermal effects. A temperature increase of 4–8°C generally does not cause permanent damage to healthy biological tissues,²¹ yet it is sufficient to activate heat-sensitive enzymes such as metalloproteinases and modulate the local microenvironment.²² Under identical parameters, continuous US generates more pronounced thermal effects than pulsed US, making LIPUS more suitable for clinical applicability.²³

In contrast, HIFU achieves tissue ablation through higher energy delivery. When the target area temperature exceeds 65°C, it effectively acts on periosteal nerve endings to produce definitive analgesic effects.¹⁴ For knee OA treatment, HIFU relieves pain through selective denervation, requiring real-time temperature monitoring to maintain the target area at approximately 60°C.²⁴ The dissipation rate of US-induced thermal effects primarily depends on tissue characteristics and material properties. In well-vascularized tissues, the microcirculatory system effectively promotes heat diffusion.²⁵ However, heat dissipation is substantially reduced near biomaterials or metal/ceramic implants. Titanium alloy implants exhibit a surface temperature increase of 13°C after 30 s of US irradiation, markedly exceeding the 5°C safety threshold for bone tissue.²⁶ Additionally, model systems containing magnetic beads exhibit approximately 10% higher temperature rise compared with pure agarose models.²⁷ The heat shock response (HSR) serves as a critical protective mechanism against environmental stress.²⁸ LIPUS can induce heat shock protein 70 (HSP70) expression in gingival tissues, promoting bone regeneration.²⁹ However, since LIPUS causes only limited temperature increase, its role in chronic inflammation regulation may not depend on HSR.³⁰ Conversely, HIFU’s high-temperature effects may denature HSPs, impairing their function.³¹

Cavitation effects

Cavitation refers to the formation, oscillation, and collapse of microbubbles (MBs) when US waves propagate through a liquid medium.³² This phenomenon manifests in two distinct forms: transient and stable cavitation. Transient cavitation, used in tumor treatment and lithotripsy, involves rapid bubble expansion and collapse, releasing energy to damage tissue.^33,34 Stable cavitation, conversely, involves sustained bubble oscillation under continuous US exposure, which enhances fluid dynamics, increases membrane permeability, and facilitates drug delivery.^35,36 Both mechanisms contribute significantly to biological effects in target tissues, with transient cavitation poses risks of cellular damage and requires careful avoidance in gas-filled organs.³⁷

Mechanical effects

Mechanical effects refer to the impact of US waves on tissues through changes in acoustic pressure and microvibrations. As US propagates through a medium, it generates mechanical forces in the direction of wave propagation, which can induce fluid movement³⁸ or create stress within solid materials.²³ In cavitation, the stable oscillation of bubbles under continuous US exposure also produces mechanical effects.³⁹

Interaction Mechanisms Between Therapeutic US and Biomaterials

Biomaterials offer distinct advantages in tissue engineering and regenerative medicine due to their excellent biocompatibility and tunable physicochemical properties.^9,40 These materials have shown significant therapeutic effects in tissue repair, bone regeneration, anti-inflammatory treatment, and immune modulation.^7,41,42 Nevertheless, single-component biomaterials frequently prove inadequate for addressing the complex regulatory demands of pathological microenvironments. As a noninvasive physical modality, therapeutic US enables precise modulation of biomaterial properties through mechanical forces, cavitation effects, and thermal effects, thereby enhancing their functional performance.

Tissue repair and regeneration

Therapeutic US applies cyclic mechanical stress to simultaneously activate cellular mechanotransduction pathways and modulate biomaterials properties, including stiffness and topological structure.⁴³ This interaction establishes a synergistic regulatory network of “mechanical stimulation-material response-cellular behavior.” In piezoelectric biomaterial, US induces localized electrical currents through the direct piezoelectric effect, demonstrating promising applications in nerve regeneration and OA treatment.^44,45 For hydrogel-based materials, US-induced shear forces dynamically alter their three-dimensional (3D) network structures.⁴⁶ Notably, fibrin hydrogels exhibit optimal response at 3.7 MHz, whereas combination with cartilage-mimicking phase-change materials increases this frequency to 4.1 MHz, significantly enhancing cartilage matrix synthesis efficiency.⁴⁷ In fibrous scaffold, LIPUS-generated propagates through the porous structure to cells, enhancing primary myoblasts differentiation and myofiber survival during muscle tissue repair.^48,49

Mechanical enhancement and drug delivery

Therapeutic US modulates the physical properties of biomaterials (e.g., MB size and hydrogel porosity) through cavitation effects and mechanical shear forces, thereby enhancing the delivery efficiency of drugs or regenerative factors.^50,51 Studies have demonstrated that diclofenac sodium (a commonly prescribed NSAID for OA pain management)-loaded polyester microcapsules, when combined with hydrogels, can establish a synergistic delivery system under US exposure, facilitating controlled drug release.⁵² Cavitation-mediated effects significantly improve cell membrane permeability in US-sensitive hydrogel carriers, facilitating targeted drug delivery.⁵³ In cardiac repair applications, LIPUS triggers dual effects: triggering phase transition in nanoscale oxygen carriers while creating microporous hydrogels structures. This combined action enhances oxygen diffusion and improves transplanted cells viability.⁵⁴

Microenvironment modulation

Biomaterials enhance therapeutic efficacy by regulating pathological microenvironments (pH/reactive oxygen species (ROS)/hypoxia) in combination with US-induced cavitation and thermal effects.⁵⁵ Specifically, pH-sensitive nanocomplexes generate cavitation-mediated microjets and shockwaves that enhance tumor vascular permeability, substantially increasing nanodrug penetration depth.⁵⁶ US-mediated mechanical force facilitates nanocarrier transmembrane transport and intracellular redox homeostasis through ROS activation, inducing charge reversal of carriers for efficient gene release.⁵⁷ Recent findings reveal that US-activated nanozymes can generate oxygen to alleviate joint cavity hypoxia, whereas cavitation-enhanced sonodynamic effects promote ROS production, effectively suppressing synovial inflammation and angiogenesis.⁵⁸ Notably, hyaluronic acid (HA) hydrogels subjected to 30 W US irradiation reach 53.7°C, leading to gold cluster dissociation and subsequent drug release.⁵⁹

Mechanisms of Therapeutic US in OA

Current studies indicate multiple pathological factors in OA, including abnormal extracellular matrix (ECM) metabolism, articular cartilage destruction, synovial inflammation, and imbalanced bone remodeling.⁶⁰ Therapeutic US presents a promising treatment approach for OA, demonstrating potential to promote cartilage regeneration, mitigate synovial inflammation, reduce subchondral bone sclerosis, enhance bone repair, and alleviate pain.^61–64 However, methodological variations in US parameters and experimental conditions (e.g., cell types, exposure duration, and culture methods) across studies hinder direct comparison of results and optimization of LIPUS treatment protocol. Representative studies are summarized in Table 1 and discussed subsequently.

Table 1.

Representative Studies on Therapeutic US for OA

Author	Main function	In vitro study and US parameters	In vivo study and US parameters	Main outcomes	References
Uddin et al. (2016)	Chondroprotection/cartilage repair	Human cartilage explants and C28/I2 chondrocytes 1 MHz, 30 mW/cm², 200 μs pulses at 100 Hz	N/A	LIPUS enhanced cartilage repair by promoting proteoglycan synthesis, chondrocyte migration and proliferation, and MSC chondrogenesis, while inhibiting IL-1β/NF-κB signaling to reduce MMP13 and ADAMTS5 expression	⁶⁵
Ding et al. (2020)	Chondroprotection/cartilage repair	Primary human OA chondrocytes 1.5 MHz, 20% duty cycle, 20 min/day × 7 days (0, 30, 60, 90 mW/cm²)	N/A	LIPUS at 30, 60, and 90 mW/cm² significantly inhibited chondrocyte apoptosis and upregulated both mRNA and protein levels of COL2A1 and ACAN compared with 0 mW/cm²	⁶⁶
Naito et al. (2010)	Chondroprotection/cartilage Repair	Nasal septal chondrocytes 30 mW/cm², 20 min/day for 7 days	Rats with knee OA model 30 mW/cm², 20 min/day for 28 days	Compared with the non-LIPUS and sham groups, the LIPUS group showed increased CPII levels, improved Mankin scores, and enhanced type II collagen expression in OA cartilage	⁶⁷
Xia et al. (2015)	Chondroprotection/cartilage repair	N/A	Rabbits underwent anterior cruciate ligament transection (ACLT) 3 MHz, 20% duty cycle, 40 mW/cm², 20 min/day, 6 days/week for 6 weeks	The early US group showed milder cartilage damage, higher type II collagen (lower MMP13), elevated integrin β1/p-FAK, and reduced p-ERK1/2/p-p38 versus early OA controls	⁶⁸
Sang et al. (2021)	Chondroprotection/cartilage repair	N/A	OA model induced by ACLT 3 MHz, 20% duty cycle, 40 mW/cm², 20 min/day for 8 weeks	LIPUS enhanced chondrocyte proliferation and differentiation through FAK signaling activation while reducing inflammatory cytokines IL-6, IL-8, and TNF-α in the synovial fluid of OA mice	⁶⁹
Nishida et al. (2017)	Chondroprotection/cartilage repair	HCS-2/8 cells, primary rat epiphyseal cartilage or articular cartilage cells 3.0 MHz LIPUS, 20 min, 30/45/60 mW/cm²	N/A	LIPUS increased chondrogenic marker expression and cartilage-regenerative CCN2 protein production in cultured chondrocytes	⁷⁰
Pan et al. (2024)	Chondroprotection/cartilage repair	Primary rat chondrocytes 1.5 MHz LIPUS, 1000 Hz, 30/45/60 mW/cm², 10/20/30 min	Rats with destabilization of medial meniscus (DMM) surgery. LIPUS, 30 mW/cm², 1.5 MHz, 1000 Hz, 20 min/day, 5 days/week, 6 weeks)	LIPUS suppressed YAP through autophagy recovery and YAP-RIPK1 inhibition, reducing OA progression and cartilage degradation in vivo	⁷¹
Hsieh et al. (2018)	Antisynovitis/antifibrotic	N/A	A PTOA model was established by ACLT and meniscectomy in rats 1.0 MHz LIPUS, 0.1 W/cm² for 4 weeks	Early LIPUS intervention mitigates cartilage degeneration through synovial anti-inflammatory effects, MMP13 suppression, and type II collagen upregulation	⁷²
Sato et al. (2014)	Antisynovitis/antifibrotic	Rabbit knee synovial membrane cell line, HIG-82 3 MHz, 30 mW/cm², 20% duty cycle, 20 min	N/A	LIPUS may regulate synovial cell metabolism and cell apoptosis via integrin/FAK/MAPK pathway particularly	⁷³
Liao et al. (2021)	Antisynovitis/antifibrotic	N/A	Mice with DMM surgery LIPUS (30 mW/cm², 20 min/day, 2 weeks)	LIPUS treatment for 2 weeks significantly reduced synovial fibrosis, hyperplasia, and proliferation in the DMM model by suppressing Wnt/β-catenin signaling in FLS	⁷⁴
Feltham et al. (2021)	Antisynovitis/antifibrotic	N/A	Rat knee intra-articular fracture model 1.5 MHz, 30 mW/cm², 1 kHz, 200 ms	Two lines of evidence support LIPUS’s anti-inflammatory effect postinjury: reduced synovial macrophages and lower IL-1β in joint fluid, thereby limiting cartilage degradation	⁷⁵
Sasaki et al. (2024)	Antisynovitis/antifibrotic	N/A	Rats arthritis with transdermal diclofenac administration 1.5 MHz, 60 mW/cm², 20% DC, 10 min	Phonophoresis reduced CD68+/CD11c+ cells (vs. LIPUS/diclofenac) and IL-1β/COX-2 (vs. arthritis), with no CD206+ changes or intergroup differences in cytokines	⁷⁶
Zhang et al. (2020)	Antisynovitis/antifibrotic	RAW 264.7 cells 1.5 MHz LIPUS, 20% duty cycle, 30 mW/cm²	DMM mouse model LIPUS for 20 min/day, 6 days/week	LIPUS attenuated synovitis (DMM/air pouch models), inhibited IL-1β maturation, and enhanced autophagy via SQSTM1-PKM in macrophages	⁷⁷
Li et al. (2019)	Improve subchondral bone remodeling	N/A	A rat OA model was established by ACLT 3 MHz US, 20% duty cycle, 30 mW/cm², 20 min/day, 5 days/week × 6 weeks	Low-intensity US could improve cartilage degeneration and subchondral sclerosis during OA progression	⁷⁸
Yılmaz et al. (2017)	Improve subchondral bone remodeling	N/A	Cartilage degeneration was provided using monoiodoacetate (MIA) 3 MHz, 40 mW/cm², 20 min/day for 15 days	The LIPUS group showed significantly higher osteoblastic activity than MIA-treated controls	⁷⁹
Yamaguchi et al. (2016)	Improve subchondral bone remodeling	N/A	Osteochondral defect rat model 1.5 MHz, 30 mW/cm², 1 kHz, 20 min/day, 5 days/week for 4/8 weeks	LIPUS irradiation significantly increased BV/TV vs. controls in osteochondral defect rat models. Combination MSC injection treatment promoted both cartilage repair and BV/TV improvement	⁸⁰
Yi et al. (2020)	Improve subchondral bone remodeling	Condylar subchondral bones come from TMJOA models	TMJOA models 1 MHz LIPUS, 30 mW/cm², 200 μs, 20 min/day for 3 and 6 weeks	LIPUS promoted the quality of subchondral bone by suppressing osteoclast activity and TGF-β1/Smad3 pathway in rabbit TMJOA versus controls	⁸¹
Fujita et al. (2019)	Improve subchondral bone remodeling	N/A	TMJOA was induced by 5-day bilateral TMJ mechanical overloading in rats. OA model 1.5 MHz, 30 mW/cm², 20% duty cycle, 20 min/day × 5 days	LIPUS treatment attenuates cartilage degeneration, downregulates subchondral trabecular bone resorption, and protects the early progression of TMJOA	⁸²
Chen et al. (2023)	Promote MSC growth and differentiation	MSC and Human chondrocytes (C28/I2). 20 min/day LIPUS	Rat knee articular cartilage defect model 1 MHz, 20% DC, 0.33 Hz, 30–250 mW/cm² for 20 min/day	LIPUS enhances MSC chondrogenesis via TNF signaling, achieving faster and superior cartilage repair versus MSCs or LIPUS alone	⁸³
Sahu et al. (2020)	Promote MSC growth and differentiation	Human bone marrow-derived MSCs 5/2/8 MHz with 14 kPa pressure (<30 mW/cm²) for 5 min	N/A	Long-term 3D-cultured MSCs under cLIUS showed upregulated collagen II/SOX9 and enhanced cartilage formation	⁸⁴
Xia et al. (2017)	Promote MSC growth and differentiation	BMSCs 20/30/40/50 mW/cm² for 20 min/day for 10 days	N/A	LIPUS promotes TGF-β1-induced chondrogenesis of BMSCs through the integrin-mTOR signaling pathway	⁸⁵
Xia et al. (2021)	Promote MSC growth and differentiation	MSCs derived from bone marrow 3 MHz, 20% duty cycle, 20/30/40/50 mW/cm²	The MSC suspension was injected into the right knee joint after ACLT operation for 4 weeks The LIPUS parameters were the same as the in vitro study: 20 min/day for 40 days	LIPUS upregulated SDF-1/CXCR4 to promote MSC migration in vitro and enhanced MSC-mediated cartilage repair in vivo, which was autophagy-dependent	⁸⁶
Hua et al. (2022)	Promote MSC growth and differentiation	iMSC is differentiated from iPSC cell lines 1.5 MHz, 100 Hz, 20–60 mW/cm², 20–80% duty cycle	5-mm calvarial bone defects LIPUS: 30 min/qod	LIPUS at an intensity of 40 mW/cm² and a duty cycle of 50% can promote the osteogenic potential of iMSCs	⁸⁷
Lai et al. (2010)	Promote MSC growth and differentiation	MSCs were isolated from human bone marrow 1 MHz LIPUS, 20% duty cycle, 200 mW/cm²	N/A	LIPUS alone can increase osteogenic differentiation of hMSCs and LIPUS enhances TD-mediated chondrogenic differentiation of hMSCs	⁸⁸

3D, three dimensional; ACAN, aggrecan; BV, bone volume; FLS, fibroblast-like synoviocyte; LIPUS, low-intensity pulsed ultrasound; IL-1β, interleukin-1 beta; MSC, mesenchymal stem cell; OA, osteoarthritis; RIPK1, receptor-interacting protein kinase 1; TMJ, temporomandibular joint; TV, tissue volume; US, ultrasound; YAP, Yes-associated protein.

Effects on chondrocytes

The degeneration of articular cartilage is considered a central feature of OA pathogenesis, with chondrocytes maintaining cartilage homeostasis by regulating ECM metabolism.⁸⁹ As mechanosensitive cells, chondrocytes respond to external mechanical signals.⁶¹

Extensive preclinical studies have confirmed the protective effects of LIPUS on cartilage, including preventing chondrocyte degeneration and promoting matrix synthesis^61,90 (Fig. 1). LIPUS promotes chondrocyte proliferation, increases glycosaminoglycan (GAG) content, and downregulates matrix metalloproteinase (MMP)-1 expression, thus inhibiting cartilage degradation.^91,92 Additionally, LIPUS mitigates interleukin-1 beta (IL-1β)-induced cartilage damage by boosting ECM proteins, including type II collagen (Col II) and aggrecan (ACAN), while facilitating chondrocyte proliferation, migration, and differentiation.⁶⁵ Ding et al.⁶⁶ found that LIPUS activates SRY-Box transcription factor 9 (SOX9), a transcription factor that enhances ECM synthesis and secretion, while reducing apoptosis in OA chondrocytes. In vivo studies using rat OA models have confirmed these findings, showing that LIPUS stimulates ECM synthesis of Col II and ACAN.⁶⁷ These results highlight the therapeutic potential of LIPUS in modulating ECM metabolism and protecting cartilage in OA.

FIG. 1.

LIPUS regulates chondrocyte function through multiple pathways. Specifically, LIPUS promotes the expression of SOX9, Col II, and ACAN in OA chondrocytes by activating mechanosensitive integrin/FAK pathways and modulating the MEK/ERK and PI3K/AKT signaling cascades. Additionally, LIPUS may hinder chondrocyte degradation in OA by inhibiting p38 phosphorylation. The activation of TRPV4 ion channels further facilitates calcium influx and the subsequent activation of the MEK/ERK pathway. LIPUS also enhances the ERK pathway through the generation of ROS. Furthermore, LIPUS regulates chondrocyte catabolism and inflammation by inhibiting IL-1β and the downstream binding of the NF-κB pathway. It has also been shown to mitigate autophagy impairment induced by IL-1β, inhibit the interaction between YAP and PIPK1, and affect downstream NF-κB signaling, thereby exerting its biological regulatory functions. ACAN, aggrecan; ERK, extracellular signal-regulated kinase; IL-1β, interleukin-1 beta; LIPUS, low-intensity pulsed ultrasound; OA, osteoarthritis; TRPV4, transient receptor potential vanilloid 4.

The integrin/focal adhesion kinase (FAK)/mitogen-activated protein kinase (MAPK) signaling pathway is critical in mediating LIPUS-induced cartilage repair. Xia et al.⁶⁸ showed in a rabbit OA model that LIPUS increases integrin β1 expression, inducing FAK phosphorylation and subsequent extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation, which promotes Col II synthesis. Further studies revealed that LIPUS upregulated Col II and integrin β1 expression in both normal and OA chondrocytes while specifically suppressing p38 phosphorylation in OA chondrocytes, highlighting LIPUS’s unique regulatory effect under pathological conditions.⁹³ This inhibitory effect was further confirmed in another study, where LIPUS slowed vascular endothelial growth factor A-induced cartilage degradation and synovitis by inhibiting p38 activity.⁹⁴ LIPUS also enhanced chondrocyte proliferation and differentiation, alleviating synovial inflammation in OA mice via FAK signaling activation.^69,95 Moreover, LIPUS influences the integrin-FAK-phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway, promoting ECM synthesis of ACAN and Col II in OA chondrocytes.⁹⁶ In this context, integrins, as mechanosensitive receptors on the cell surface, perceive external mechanical signals and convert them into biochemical signals,⁹⁷ a process underlying LIPUS-mediated therapeutic effects.

LIPUS elevated ROS production and activated ERK1/2 phosphorylation, leading to upregulation of cartilage-regenerative genes.⁹⁸ Research has shown that LIPUS enhances calcium influx into chondrocytes via the transient receptor potential vanilloid 4 (TRPV4) channel, subsequently activating p38 and ERK1/2 phosphorylation to enhance cellular communication network factor 2 (CCN2) expression, a key cartilage regeneration factor.⁷⁰ Similarly, LIPUS stimulated CCN2 expression through the ERK1/2 pathway in human medial meniscus cells, aiding meniscus repair.^99,100 These findings suggest that LIPUS exerts distinct pathway-specific effects in different joint disease models.

LIPUS regulates multiple signaling pathways beyond MAPK to regulate chondrocyte growth and differentiation. It suppresses IL-1β-induced cartilage degradation by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-p65 and inhibitor of kappa B alpha (IκBα) phosphorylation, while enhancing chondrocyte migration, proliferation, and differentiation.⁶⁵ LIPUS also restores autophagy, a process essential for maintaining intracellular homeostasis, and delays OA progression by inhibiting the interaction between Yes-associated protein (YAP) and receptor-interacting protein kinase 1, reducing YAP expression and downstream NF-κB signaling.⁷¹

In summary, the chondroprotective effects of LIPUS are mediated through multiple signaling pathways, including MAPK, NF-κB, and integrin-associated mechanotransduction, which regulate chondrocyte proliferation, differentiation, autophagy, and the balance between anabolic and catabolic processes.

Synovial tissue and synoviocytes

The synovium maintains joint homeostasis through complex immune and inflammatory interactions with cartilage.¹⁰¹ Synoviocytes, particularly fibroblast-like synoviocytes (FLSs) and macrophages, play a central role in synovial inflammation, proliferation, and remodeling.¹⁰²

Increasing evidence suggests that US effectively suppresses synovial inflammation in both animal models and cellular studies of synovitis¹⁶ (Fig. 2). In collagen-induced arthritis rats, US treatment significantly alleviated synovial hyperplasia after 4–6 weeks.¹⁰³ Early LIPUS intervention decreases synovial inflammation and MMP13 expression while enhancing Col II synthesis, thereby slowing cartilage degeneration.^72,104 LIPUS exerts its anti-inflammatory and protective effects by activating ERK1/2 and p38 phosphorylation through the integrin/FAK/MAPK pathway.⁷³ Additionally, LIPUS modulates synovial function by upregulating hyaluronan synthases (HAS2 and HAS3) and inhibiting hyaluronidase (HYAL2) activity, leading to the accumulation of high-molecular-weight hyaluronan for joint lubrication.¹⁰⁵ The abnormal activation and functional changes of FLS are considered key factors in the onset and progression of OA. Studies indicated that LIPUS inhibits pathological FLS activation in OA by suppressing Wnt/β-catenin signaling activity, thereby preventing synovial fibrosis and hyperplasia.⁷⁴

FIG. 2.

LIPUS combats synovitis and fibrosis through multiple pathways. LIPUS regulates the apoptosis and survival of synovial cells via the integrin/FAK/MAPK pathway. LIPUS inhibits synovial fibrosis by suppressing the Wnt/β-catenin signaling pathway in FLS. Furthermore, LIPUS alleviates synovial inflammation by enhancing autophagy levels in macrophages and inhibiting the maturation of IL-1β. The stimulation of LIPUS also activates the mechanosensitive ion channels PIEZO1 and TRPV1, which suppress NF-κB nuclear translocation, subsequently reducing the release of proinflammatory cytokines. FLS, fibroblast-like synoviocyte.

Macrophages are key immune cells within the synovium, controlling joint inflammation and tissue homeostasis. Studies have shown that pulsed US at 3.0 W/cm² for 5 min significantly reduces inflammatory factor expression in lipopolysaccharide (LPS)-induced macrophages.¹⁰⁶ In post-traumatic OA rats, LIPUS reduced CD68+ macrophages in the synovium and lowered IL-1β levels in joint fluid, demonstrating its anti-inflammatory effects.⁷⁵ When combined with diclofenac, LIPUS synergistically alleviated joint inflammation and pain, potentially through M1 macrophages suppression.⁷⁶ LIPUS also increased the number of Lubricin-positive cells in OA models, suggesting it mitigates cartilage degradation by reducing early OA inflammation.¹⁰⁷ LPS-induced macrophages showed p65 nuclear translocation, which LIPUS inhibited by activating mechanosensitive ion channels PIEZO1 and TRPV1, reducing proinflammatory cytokine release through the p38 MAPK pathway. Importantly, blocking PIEZO1 and TRPV1 significantly diminished LIPUS’s anti-inflammatory effects, highlighting their central role in LIPUS-mediated inhibition of NF-κB signaling.¹⁰⁸ Similarly, LIPUS attenuated LPS-induced inflammation in U937 cells by preventing IκBα degradation and p65 nuclear translocation.¹⁰⁹

Notably, LIPUS promoted autophagy, facilitating the formation of sequestosome 1 - pyruvate kinase M (SQSTM1)-pyruvate kinase muscle isozyme (PKM) complexes and reducing PKM2 levels in LPS-adenosine triphosphate (ATP)-treated macrophages. This process suppressed mature IL-1β production, ultimately alleviated synovial inflammation and gait abnormalities in OA animal models.⁷⁷ These findings collectively demonstrate LIPUS’s multifaceted anti-inflammatory actions through macrophage modulation.

Subchondral bone

As OA progresses, subchondral bone loss worsens, disrupting the balance between osteoblasts and osteoclasts, which contributes significantly to OA onset and progression.¹¹⁰

LIPUS exerts protective effects on subchondral bone remodeling in OA. One study examined the effects of LIPUS on joint cartilage and subchondral bone changes during OA progression under normal and functional disuse conditions. The results showed that LIPUS not only alleviated cartilage degeneration but also improved subchondral bone sclerosis during OA progression, with more pronounced effects observed under normal joint use conditions.⁷⁸ In monoiodoacetate (a glycolysis inhibitor that induces chondrocyte apoptosis)-induced rats, LIPUS enhanced osteoblast activity and bone mineral density, facilitating concurrent cartilage and subchondral bone regeneration.⁷⁹ Moreover, combining LIPUS with mesenchymal stem cell (MSC) therapy significantly improved cartilage repair and enhanced the bone volume/tissue volume ratio.⁸⁰

Osteoblasts and osteoclasts are known to be sensitive to mechanical stimulation.¹¹¹ Early LIPUS intervention attenuated knee OA progression by preserving tissue integrity, improving subchondral bone microstructure, and reducing both pain perception and diminishing sensory innervation. Moreover, daily LIPUS treatment inhibited osteoclastogenesis, potentially through neuromodulatory mechanisms.⁶³ In temporomandibular joint (TMJ) OA models, LIPUS improved subchondral bone quality by dual inhibition of osteoclast activity and the transforming growth factor (TGF)-β1/Smad3 signaling.⁸¹ Further studies showed that LIPUS inhibits IL-6 expression and the TGF-β1/Smad3 pathway in subchondral bone, aiding TMJOA treatment.¹¹² Additionally, LIPUS counteracts mechanical overload-induced TMJOA by decreasing osteoclast numbers, restoring proteoglycan expression, and preventing cartilage degradation.⁸²

Collectively, these findings suggest that LIPUS modulates osteoblast–osteoclast interactions, playing a key role in maintaining bone homeostasis and remodeling subchondral bone.

MSCs

MSCs are multipotent cells capable of differentiating into various cell types, including chondrocytes and osteoblasts. US stimulation, through its mechanical, cavitation, and thermal effects, significantly enhances MSC proliferation, differentiation, and migration.¹¹³

US parameters can direct MSC differentiation toward specific cell types. It promotes chondrogenic differentiation by enhancing TGF-β signaling, demonstrated by elevated proteoglycan production (3-fold increase) and intensified alcian blue staining.¹¹⁴ LIPUS also suppresses the TNF signaling pathway, promoting MSC chondrogenesis and optimizing the anti-inflammatory microenvironment for chondrocytes. In vivo studies confirm these findings, showing superior cartilage repair in rat knee defects when combining LIPUS with MSC therapy versus controls.⁸³ Continuous LIPUS induces increased SOX9 expression in MSCs, a key transcription factor for chondrogenesis, through ERK1/2 phosphorylation. Prolonged continuous low-intensity US stimulation also increases collagen II and chondroitin sulfate in MSCs cultured in hydrogels.⁸⁴ As a crucial degradative process for maintaining intracellular homeostasis, autophagy is modulated by LIPUS and contributes to MSC chondrogenic differentiation. Specifically, LIPUS exerts dual effects through the integrin-mammalian target of rapamycin (mTOR) signaling pathway: it upregulates integrin and p-mTOR expression to suppress autophagy and promote bone marrow mesenchymal stem cell (BMSC) chondrogenesis,¹¹⁵ while simultaneously mediating TGF-β1-induced chondrogenesis in BMSCs, an effect that can be reversed by specific inhibitors.⁸⁵ In the anterior cruciate ligament transection-induced OA model, LIPUS combined with MSC therapy significantly enhanced cell migration capacity and promoted cartilage regeneration through autophagy pathway activation.⁸⁶ These findings suggest that LIPUS-mediated autophagy regulation exhibits stage-specific characteristics, likely dependent on the distinct physiological states of cells.

US also positively influences MSC osteogenic differentiation. Studies have shown that LIPUS stimulates MSC osteogenesis via the Wnt/β-catenin signaling pathway, involving hypoxia-inducible factor 1-alpha (HIF-1α) and ras homolog family member A (RhoA) activation.^87,116 Moreover, LIPUS enhances osteogenic differentiation by modulating specific miRNAs, such as miR-31-5p and miR-675-5p.^116,117 When MSCs are treated with dexamethasone/TGF-β1 (TD) or BMP-2, LIPUS not only promoted osteogenic differentiation but also amplified the chondrogenic effects induced by TD.⁸⁸ These findings highlight the therapeutic potential of LIPUS in OA and other bone-related diseases by promoting MSC proliferation and differentiation.

Synergistic Effects of Therapeutic US and Biomaterials

Biomaterials, including natural, synthetic, inorganic, and metallic types, offer distinct therapeutic benefits (Fig. 3). The synergistic application of therapeutic US and biomaterials is vital for tissue repair, drug delivery, and microenvironmental modulation. US accelerates tissue repair by stimulating cellular proliferation, whereas biomaterials provide a stable microenvironment to enhance healing. The combination approach provides new possibilities for the treatment of OA, with this review focusing on these key mechanisms (Fig. 4).

FIG. 3.

Various types of biomaterials can synergistically collaborate with the mechanical forces of US to exert biological effects that regulate cell behavior. US, ultrasound.

FIG. 4.

Biomaterials, particularly with US, have witnessed significant advancements in tissue engineering and regenerative medicine and are now widely employed for joint repair. US can modulate the physical properties of biomaterials, promote tissue repair and regeneration, enhance drug delivery, and regulate the local microenvironment. The synergistic effect of US and biomaterials promotes cartilage repair, reduces synovial inflammation, regulates subchondral bone remodeling and bone homeostasis, and ultimately promotes OA tissue repair. OA, osteoarthritis; US, ultrasound.

Effects on chondrocytes

Biomaterials such as collagen and HA, known for their biocompatibility and ability to mimic the natural ECM, are widely used in cartilage tissue engineering (Table 2). The synergistic interaction between US and biomaterials promotes cartilage repair primarily through mechanical stress. US-generated acoustic radiation forces and shear stresses directly stimulate chondrocytes while inducing microstrains in biomaterials, thereby mimicking the mechanical microenvironment of natural cartilage. Furthermore, cavitation effects enhance mass transport within the materials. Numerous experimental studies have confirmed this collaborative repair mechanism. For instance, Guo et al.¹³⁰ demonstrated that LIPUS treatment significantly enhanced repair outcomes in rabbit articular cartilage defects using an allogeneic chondrocyte-calcium alginate gel composite, yielding smoother cartilage tissue, improved tissue integration, and elevated collagen II expression. Similarly, another study indicated that low-intensity US enhanced the viability of human articular chondrocytes cultured in alginate while increasing the expression of Col II and proteoglycan.¹³¹ In contrast, some studies reported limited effects of LIPUS on cell viability under specific conditions. Specifically, LIPUS stimulated GAG synthesis in monolayer or agarose-cultured chondrocytes by day 2, but this effect dissipated by day 5, indicating that the potential for pulsed low-intensity US-stimulated matrix synthesis may be limited in cartilage tissue engineering.¹³²

Table 2.

US Combined with Biomaterials for Cartilage Repair

Author	Biomaterials	Constituent	In vitro study and US parameters	In vivo study and US parameters	Main outcomes	References
Guo et al. (2013)	Natural	Calcium alginate gel	Primary chondrocytes isolated from New Zealand knee joint cartilage 60 mW/cm², 1 MHz, 20 min/day for 3 days	Articular cartilage defect in New Zealand model 60 mW/cm², 1 MHz, 20 min/day for 8 and 12 days	The LIPUS-optimized allogeneic chondrocyte-calcium alginate gel composite can effectively repair articular cartilage defects	¹¹⁸
Choi et al. (2006)	Natural	Alginate bead	Human articular chondrocytes 0, 100, 200, and 300 mW/cm², 10 min/day for 2, 7, or 15 days.	N/A	The survival rate of chondrocytes increased, along with higher expression of Col II and proteoglycans	¹¹⁹
Vaughan et al. (2010)	Natural	Agarose constructs	Adult bovine articular chondrocytes 30 or 100 mW/cm² for 20 min/day	N/A	US induces a 20% increase in sGAG content at day 2 of culture in agarose, this response was lost by day 5	¹²⁰
Noriega et al. (2013)	Natural	3D chitosan matrices	Bovine chondrocytes 5.0 MHz, 51 s, 4 applications/day	N/A	US stimulation treatment affected the expression of rho-A, ROCK-I, and mDia1 and modulated the cytoskeleton organization	¹²¹
Babaei et al. (2022)	Synthetic	3D-printed polyurethane scaffolds dip-coated with gellan gum, hyaluronic acid, and glucosamine	Human chondrocyte cell line C28/I2 100, 200, and 300 mW/cm² for 20 min/day	N/A	LIPUS can stimulate cell proliferation and vitality, promote the expression of ACAN and Col II genes, and enhance chondrogenic phenotype	¹²²
Hasanova et al. (2011)	Natural	3D chitosan matrices	Bovine chondrocytes 0.14 mW/cm², 5.0 MHz, 51 s, and applied 1, 2, 4, and 8 times/day	N/A	LIUS can enhance cell proliferation, as well as the expression of proteoglycans and Col II, while downregulating the expression of MMP-3	¹²³
Noriega et al. (2007)	Natural	3D chitosan scaffolds	Healthy human chondrocytes 1.5 MHz for 161 s, 5.0 MHz for 51 s, and 8.5 MHz for 24 s, 2 times/day for 10 days	N/A	Continuous US for predetermined time intervals possessed higher cellular viability and higher levels of Col II and ACAN expression when compared with controls	¹²⁴
Takeuchi et al. (2008)	Natural	Collagen sponge	Pig articular chondrocytes 30 mW/cm², 1.5 MHz, 20 min/day for 3, 7, 10, and 14 days	N/A	LIPUS promotes chondrocyte proliferation and Col IX production via the integrin/PI3K/Akt signaling pathway	¹²⁵
Hsu et al. (2006)	Synthetic	Polyester composites	Human chondrocytes 67 mW/cm², 1 MHz for 10 min/day	N/A	US enhanced chondrocyte proliferation and GAG production, with the strongest effect observed at 28 days	¹²⁶
Vinikoor et al. (2023)	Synthetic	Piezoelectric hydrogel, made of short electrospun poly-L-lactic acid nanofibers embedded inside a collagen matrix	Adipose-derived stem cells 0.33 watt/cm², 40 kHz, and 20 min/day for 14 days	Rabbit osteochondral defect model 0.33 watt/cm², 40 kHz, 20 min/day, 5 days/week for 1 or 2 months	Piezoelectric hydrogel activated by the external US can induce chondrogenesis in vitro and treat severe osteochondral defects in vivo	¹²⁷
Chen et al. (2020)	Synthetic	Liposomes, alginate beads	Human OA and normal chondrocytes 0.5 W/cm², 1.0 MHz, 20 min/day for 7 days	Male spontaneous OA Dunkin–Hartley guinea pigs 3 times/week (once every other day) for 8 weeks	Liposome encapsulation collaborated with LIPUS can reduce the effective dose and administration frequency of rapamycin, promote the production of proteoglycans and Col II in chondrocytes, and have significant anti-OA effects in vivo	¹²⁸
Zuo et al. (2021)	Synthetic	Nanoparticles	Primary cells of knee joint cartilage 30, 45, and 60 mW/cm², 1.5 MHz for 20 min/day	New Zealand rabbits with knee OA model 60 mW/cm², 1.5 MHz, 20 min/day and 5 days/week for 6 weeks	Combined PBNPs/LIPUS treatment could alleviate ROS and apoptosis of chondrocytes by activating the PI3K/Akt/mTOR pathway. It may also reduce inflammatory cytokines and inhibit ECM degradation by reducing MMPs expression	¹²⁹

Col II, type II collagen; GAG, glycosaminoglycan; LIPUS, low-intensity pulsed ultrasound; MMP, matrix metalloproteinase; OA, osteoarthritis.

Recent investigations have increasingly focused on US’s effects on chondrocytes within 3D scaffolds. Low-intensity diffuse US stimulation was found to modulate cytoskeletal organization in chondrocytes seeded in 3D chitosan scaffolds.¹³³ LIPUS treatment on chondrocytes seeded in 3D-printed polyurethane scaffolds coated with gellan gum, HA, and glucosamine promoted chondrocyte proliferation, viability, and expression of cartilage phenotype-related genes.¹³⁴ Similarly, low-intensity US stimulation of bovine chondrocytes in 3D chitosan matrices enhanced cell proliferation and the expression of proteoglycan and Col II.¹³⁵ Continuous US stimulation in 3D chitosan scaffolds significantly enhanced chondrocyte viability and matrix synthesis, particularly increasing Col II and proteoglycan expression.¹³⁶ Mechanistically, LIPUS promotes chondrocyte proliferation and Col IX production in 3D scaffolds via the integrin/PI3K/Akt pathway.¹³⁷ Furthermore, Col II-modified polyester composite scaffolds combined with pulsed US enhanced chondrogenic potential of cultured chondrocytes.¹³⁸ Functional biomaterials, such as piezoelectric hydrogels, have revealed that US-induced localized electrical charges can stimulate stem cell migration, endogenous growth factor production, and chondrocyte marker expression. When injected into a rabbit osteochondral defect model, piezoelectric hydrogels, under US stimulation, facilitated cartilage healing.¹³⁹

The cavitation effect of US enhances cell membrane permeability, making it particularly effective for anti-inflammatory treatments. Studies show that combining LIPUS with liposome-encapsulated rapamycin in chondrocytes within alginate beads significantly increased proteoglycan and Col II production while suppressing IL-6 expression. Liposomes prolonged rapamycin’s efficacy, reduced adverse effects, and allowed LIPUS to lower the required drug dose and dosing frequency in OA treatment.¹⁴⁰ Furthermore, LIPUS improved the ROS scavenging ability of Prussian blue nanoparticles, reducing ROS levels, apoptosis, and MMP expression through activation of the PI3K/Akt/mTOR pathway, thereby protecting articular cartilage.¹⁴¹ These findings highlight US’s dual capacity to modulate the cellular microenvironment while offering significant therapeutic benefits. In summary, US combined with biomaterials promotes chondrocyte proliferation, matrix synthesis, enhances drug delivery, and protects cartilage, demonstrating its potential in OA treatment.

Effects on synovial tissue and cells

The combined effects of US and biomaterials on synovial tissue have been primarily reflected in anti-inflammatory repair and drug delivery mechanisms. US enhances drug release and cellular uptake through cavitation and mechanical effects, enabling precise control of local drug concentration while promoting intracellular delivery via cell membrane permeabilization.¹⁴² US with MBs improved skin permeability, enhancing diclofenac sodium gel delivery, which reduced synovial neovascularization and inflammation in arthritic regions.¹⁴³ US-driven sonodynamic therapy effectively killed activated synovitis cells while amplifying the therapeutic efficacy of nanozyme recombinant human serum albumin conjugated with SPX (Rh/SPX-HSA), further suppressing synovial inflammation.⁵⁸ Additionally, US-triggered MB destruction facilitated drug release from nanocarriers in the synovial cavity, reducing joint swelling, bone erosion, and inflammation in collagen-induced arthritis models.¹⁴⁴

Macrophages, as key immune cells in OA joints, drive cartilage degradation and accelerate OA progression through M1/M2 polarization imbalance and proinflammatory factor secretion.¹⁴⁵ US combined with biomaterials promoted macrophages polarization, cytokine secretion, and immune microenvironment improvement. US-stimulated release of biomimetic peptide nanofibers from hydrogels activated mitochondrial glycolysis, attenuated ROS production, and enhanced M2 macrophage polarization.¹⁴⁶ Piezoelectric biomaterials such as BaTiO3/collagen membranes, when activated by LIPUS, generated synergistic electrical and mechanical stimuli that enhanced Ca²⁺ influx via PIEZO1 channels, promoting bone regeneration.¹⁴⁷ Additionally, US-responsive IL-4-encapsulated microdroplets (MDs) not only suppressed inflammatory phenotypes in macrophages and enhanced M2 polarization but also promoted osteogenic differentiation in hBMSCs.¹⁴⁸

In summary, integrating US with biomaterials facilitates macrophage polarization, accelerates tissue regeneration, improves drug delivery efficiency, and modulates the microenvironment, effectively alleviating synovial inflammation.

Effects on subchondral bone

The pathological changes in subchondral bone in OA are closely associated with disrupted bone homeostasis. US combined with biomaterials demonstrates significant effects in regulating bone homeostasis and promoting bone regeneration. US induces hydrogel deformation, providing mechanical stimulation that promotes osteoblast differentiation.¹⁴⁹ Studies have shown that osteoblasts encapsulated in hydrogels with optimal stiffness exhibit upregulation of mineralization markers such as calcium, cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2) under US stimulation, thereby enhancing mineralized tissue formation. In vivo experiments using nucleotide-binding oligomerization domain (NOD) scid gamma mice further confirmed that BMSC-laden optimized hydrogels with 4-week US treatment significantly promoted calvarial defect repair through synergistic cell delivery and mechanostimulus-induced osteogenesis.¹⁵⁰ Furthermore, US-responsive biomimetic hydrogel promotes BMSC osteogenic differentiation by inducing M2 macrophage secretion of BMP-2 and insulin-like growth factor I. In murine femoral defect models, these hydrogels significantly enhanced BMP-2 and type I collagen expression, demonstrating optimal bone healing efficacy.¹⁴⁶ Additional studies showed that LIPUS-generated mechanical stimulation further increased COX-2 and PGE2 expression in osteoblasts, elevating osteogenic markers including alkaline phosphatase and osteocalcin.¹⁵¹ 3D culture systems demonstrated superior osteogenic potential, with US-treated tricalcium phosphate scaffolds showing enhanced osteoblast adhesion and elevated phosphorylation of ERK1/2 and p38 phosphorylation compared with 2D cultures.¹⁵²

Piezoelectric biomaterials, capable of transducing mechanical stimuli into bioelectrical signals, work synergistically with LIPUS to promote osteoblast proliferation and differentiation.¹⁵³ For example, BaTiO3-coated scaffolds under intermittent LIPUS stimulation generated continuous electrical signals that activated mitochondrial function and cytoskeletal reorganization, improving osteoblast function. This combination of mechanical and electrical stimulation upregulated osteogenesis-related genes, particularly BMP-2, and reduced apoptosis, promoting osteoblast activity.¹⁵⁴

The combination of drug delivery and microenvironment modulation demonstrates significant potential in regulating bone homeostasis. US-responsive MDs, such as nuclear factor of activated T-cells cytoplasmic 1-polylactic acid (NFATc1-PLA)-exosome scaffolds, enhanced osteogenic differentiation of hBMSCs while modulating cytokine expression.¹⁵⁵ MDs-IL4 promoted macrophage polarization and further enhanced osteogenic differentiation.¹⁴⁸ Remarkably, these MDs exhibit temporally controlled structural transformations following US stimulation, with MDs-NFATc1 treatment significantly reducing osteoclast numbers, indicating a strong effect in inhibiting osteoclastogenesis.¹⁵⁶ Further studies have demonstrated that US-controlled “explosive” hydrogels loaded with resveratrol nanobubbles release drugs under low-intensity focused US, enhancing osteogenesis and maintaining bone microenvironmental homeostasis.¹⁵⁷ This innovative approach combining precise drug delivery with US-mediated modulation represents a promising therapeutic paradigm for regulating bone homeostasis.

Effects on MSCs

The combination of various hydrogel materials and US has shown notable effects in promoting chondrogenic differentiation of MSCs. In studies where rabbit MSCs were cultured in fibrin-HA or alginate hydrogels and treated with LIPUS in a chondrogenic medium, fibrin-HA hydrogels showed greater efficacy in promoting chondrogenesis, as indicated by increased production of sulfated GAGs and collagen, likely due to their superior mechanical properties. LIPUS further enhanced MSC chondrogenic differentiation in these hydrogels.¹¹⁸

Similarly, scaffolds combined with LIPUS have promoted hMSC proliferation and chondrogenic differentiation. For instance, hMSCs seeded on a 3D-printed polyethylene glycol-diacrylate hydrogel scaffold showed increased proliferation and significantly higher levels of chondrogenic markers, including GAGs and Col II, following LIPUS treatment.¹¹⁹ Further studies revealed that when lipid-coated, perfluorobutane-filled MBs were introduced, LIPUS treatment of hMSCs on this 3D scaffold led to a 40% increase in proliferation (compared with 18% with LIPUS alone). After 3-week chondrogenic differentiation, GAG production increased by 17% (vs. 5% increase with LIPUS alone), whereas Col II production rose by 78% (vs. 44% increase with LIPUS alone).¹²⁰ Similarly, in vivo studies using nonporous polyglycolic acid (PGA) scaffolds implanted in mice also showed increased chondrogenic biomarkers following LIPUS treatment.^121,122 Furthermore, the combination of LIPUS and β-tricalcium phosphate (β-TCP) scaffolds synergistically promotes MSC proliferation and osteogenic differentiation. In experiments where BMSCs/β-TCP composites were subcutaneously implanted in rats, the LIPUS-treated group demonstrated significantly elevated alkaline phosphatase activity, osteocalcin levels, and improved angiogenesis and bone formation.¹²³

US combined with MBs has been shown to induce localized shear forces and controlled mechanical stress within cells, amplifying the mechanical effects of LIPUS. Nanoliposomes have been shown to act as nanomechanical force generators on cell membranes, promoting osteogenesis and bone formation in BMSCs through mechanisms involving TRPM7 ion channels and intracellular calcium oscillations.¹²⁴ Furthermore, 3D-printed scaffolds with high porosity, when combined with LIPUS, significantly promoted MSC proliferation, alkaline phosphatase activity, calcium deposition, and protein content.¹²⁵ Collectively, these findings highlight the potential of LIPUS combined with hydrogel scaffolds to enhance both chondrogenic and osteogenic differentiation of MSCs in vitro and in vivo.

Discussion

Numerous in vitro studies have shown that US can modulate ECM metabolism in OA joints and influence the microenvironment of synovium, cartilage, and subchondral bone.^126,127 US also enhances the proliferation and differentiation of MSCs derived from various tissues, including bone marrow, adipose tissue, and the umbilical cord.¹¹³ However, variations in experimental parameters, such as well thickness, gel layer, and medium volume, may introduce bias by affecting wave transmission and cellular responses.¹⁵⁸ Furthermore, temperature increases in the culture system may influence biological effects.^18,61 Detailed analyses have revealed that at an US intensity of 30 mW/cm², systems using coupling gel to connect the transducer to the culture plate exhibited a temperature increase of 3°C, whereas immersion of the transducer in water resulted in only a 0.2°C temperature change. This difference stems from the poor heat transfer performance of air medium leading to heat accumulation.^128,158 Therefore, standardized US configuration calibration and temperature control measures are strongly recommended, particularly for nonimmersion systems.

The therapeutic effects of US vary between preclinical and clinical studies. US primarily shows positive effects in OA animal models, but these models are mostly limited to a single disease stage. There is a need to assess animal models at different disease progression stages to better mimic human OA development. Particularly lacking are comparative studies between young and aged models, whereas OA mainly occurs in the elderly population. Future studies should systematically compare the therapeutic effects of LIPUS in animal models of different ages, which is critical for clinical translation. The choice of appropriate animal models is critical, with species such as pigs, horses, and sheep offering closer anatomical resemblance to humans in terms of cartilage thickness and joint structure.¹²⁹ Despite promising results, US has not yet been incorporated into guidelines for knee OA management.^159,160 This underscores the necessity for large-scale, multicenter clinical trials with stringent control over variables such as US intensity, frequency, and patient demographics to enhance the robustness of findings.

US demonstrates significant tissue penetration capability, but its therapeutic effects may vary significantly across different tissue types due to the involvement of multiple parameters, including frequency, intensity, duty cycle, and duration of intervention.¹⁶¹ Compared with the mechanical forces required for bone regeneration, the mechanical stimulation needed for soft tissue repair is relatively weaker. In tissue engineering applications, various natural and synthetic scaffold materials have been developed to enhance US-mediated mechanical stimulation. For instance, MBs significantly amplify US cavitation effects to achieve controlled drug release, whereas hydrogels and scaffold materials provide cells with excellent structural support and an ideal microenvironment, thereby optimizing the mechanobiological effects of US. However, research on US parameters tailored to different material properties remains relatively scarce.

Effective OA treatment requires addressing joint cartilage, synovial inflammation, and subchondral bone remodeling. While most research has focused on bone tissue engineering, soft tissue repair also holds significant potential, including tendons, skeletal muscle, ligaments, and cartilage.¹⁶² Developing novel US-responsive biomaterials requires a thorough understanding of US mechanisms, such as mechanotransduction,¹⁶³ cavitation effects, and cellular signaling cascades that determine biological responses. Further research is needed to explore the synergistic mechanisms between US and biomaterials.

In clinical translation, US, especially LIPUS, offers multiple advantages including localized application, noninvasiveness, ease of operation, and significant therapeutic benefits. As research progresses, LIPUS can target unmet clinical needs in the orthopedic field and is expected to significantly enhance OA management for numerous patients worldwide. Most importantly, LIPUS has shown unique synergistic therapeutic potential, but its efficacy and safety still require verification through more large-scale clinical trials.

Conclusion

Future research should focus on developing tunable US parameters and biomaterials for the treatment of OA across various pathological stages. This optimization strategy aims to enhance synergistic therapeutic mechanisms while expanding potential clinical applications. A deeper understanding of the mechanisms by which US operates, as well as the design requirements for biomaterials, will facilitate the provision of more personalized and precise therapeutic options for complex diseases.

Authors’ Contributions

W.H.: Writing original draft and literature search. X.H., C.P., and X.S.: Editing and revisions. T.X.: Conceptualization and revisions of the article. All authors have read and approved the final version of the article.

Footnotes

Funding Information

This study was supported by National Natural Science Foundation of China (grant nos. 82072556 and 82272610). The authors thank Figdraw for the assistance in creating schematic diagram.

Disclosure Statement

The authors have declared that no competing interest exists.

Declaration of Generative AI and AI-Assisted Technologies in the Writing Process

The authors declare that they did not use AI-assisted technologies to create this article.

Data Availability

The raw data supporting the conclusions of this article will be made available by the authors without reservation and can be obtained from the corresponding author.

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