Exosome-Based Therapeutics for Musculoskeletal Disorders: Advances in Engineering, Targeting, and Biomaterial Integration

Abstract

Musculoskeletal disorders represent an escalating global challenge that adversely affects both the quality of life and healthcare systems. Despite the availability of conventional therapeutic approaches, these methods are constrained by inadequate targeting, limited tissue regeneration capabilities, and potential long-term safety concerns. In recent years, exosomes have emerged as promising agents for precise intervention and functional regeneration, owing to their properties of active targeting, cargo delivery capability, modifiability, and biocompatibility, particularly when used in conjunction with engineering and biomaterial delivery strategies. While the therapeutic potential of exosomes in the management of musculoskeletal diseases is increasingly acknowledged, the current literature lacks a comprehensive integration of three critical dimensions: exosome engineering strategies, advanced biomaterial delivery systems, and their prospective therapeutic applications across various diseases. Therefore, this study concentrates on engineering methodologies aimed at augmenting the therapeutic efficacy of exosomes, encompassing the pretreatment of blast cells, the modification of exosomes, and their incorporation with biomaterials. Furthermore, we systematically introduce delivery systems utilizing hydrogels, scaffolds, microneedles, and fiber membranes, which enhance exosome delivery by facilitating spatial positioning control and achieving sustained release effects. Building on this foundation, we conduct an in-depth examination of the mechanisms and applications of exosomes in the treatment of musculoskeletal disorders. Additionally, this review provides the analysis of biogenesis, isolation, extraction, and preservation strategies of exosomes while also identifying the key factors impeding their clinical application. Based on this synthesis, we propose that exosomes represent a transformative paradigm for targeted, minimally invasive, and tissue-specific interventions in musculoskeletal medicine.

As the structural foundation of the human body, the skeletal system plays a vital role not only in maintaining morphology and providing mechanical support but also in orchestrating essential physiological functions such as hematopoiesis, organ protection, and mechano-sensing. , Its functionality arises from a complex composite architecture consisting of mineralized matrix, collagen fibers, and embedded bone cells, which enables both mechanical integrity and biochemical responsiveness. ,

However, a wide spectrum of skeletal disorders, including traumatic bone defects, osteoarthritis (OA), rheumatoid arthritis (RA), and spinal cord injuries (SCI)significantly compromises mobility and quality of life, especially in aging populations. − These conditions are further complicated by limited vascularization and poor regenerative capacity of hard tissues, posing substantial challenges for effective treatment. , Current pharmacological and surgical interventions often suffer from nonspecific targeting, limited long-term efficacy, and high costs. − Artificial grafts and nanocarriers, including liposomes and inorganic nanoparticles, offer some advances but remain limited by issues such as cytotoxicity, low biodegradability, and insufficient control over release kinetics. ,

Amid these limitations, exosomesmembrane-bound extracellular vesicles (30–150 nm)have emerged as a powerful platform for therapeutic delivery. Exosomes facilitate intercellular communication by transporting functional nucleic acids, proteins, lipids, and metabolites. Their inherent biocompatibility, low immunogenicity, and capacity for selective tissue targeting make them highly attractive for orthopedic applications. In preclinical studies, exosomes derived from mesenchymal stem cells (MSCs) and other cell types have demonstrated robust regenerative potential in bone, cartilage, tendon, and spinal tissues. However, the clinical application of exosomes is impeded by several systemic challenges. First, batch heterogeneity, stemming from variations in cell sources, results in unpredictable therapeutic efficacy, significantly obstructing standardized production. Second, conventional separation methods, such as ultracentrifugation, are constrained by low throughput and high costs, rendering them inadequate for meeting clinical dosage requirements. Furthermore, exosomes are susceptible to aggregation, degradation, and functional decline during prolonged storage, thereby restricting their commercial distribution. Collectively, these factors contribute to the absence of robust manufacturing processes and quality control standards, culminating in an unclear regulatory framework.

To tackle this intricate and multifaceted challenge, a range of engineering strategies have been devised. These strategies encompass the pretreatment of exosome-derived cells, the regulation of culture conditions, the internal cargo loading, and external surface modification of exosomes, all of which have significantly enhanced batch uniformity and therapeutic targeting. Concurrently, the integration of various biological materials and the development of hydrogels, scaffolds, and other delivery systems have further optimized delivery efficiency, extended the activity cycle, and facilitated large-scale production. − This dual-track innovation, characterized by both engineering and materialization, not only offers a tangible solution for enhancing storage stability but also establishes a technical framework that facilitates regulatory approval through standardized manufacturing processes, thereby expediting the clinical application of exosome therapy. At the molecular level, advancements in gene editing and membrane modification technologies have equipped exosomes with precise lesion recognition capabilities and programmable biological activities, overcoming the targeting ambiguities and functional constraints inherent in natural vesicles. In terms of delivery, the use of biomaterials creates spatiotemporal and dynamic regulatory networks. These networks enable on-demand drug release and prolonged retention driven by the pathological microenvironment, thereby extending the local treatment window. Within the system dimension, the regulatory capacity of engineered exosomes on signaling pathways, coupled with the remodeling function of the physical microenvironment provided by biomaterials, generates an orthogonal synergy. This synergy effectively addresses multiple challenges in tissue regeneration, such as immune imbalance, loss of innervation, and barriers to vascularization. This dual-track evolution of “molecule-carrier” systems has facilitated the transition of three clinical paradigms: the therapeutic objective has advanced from symptom suppression to the reconstruction of tissue structure; the precision of interventions has progressed from the organ scale to the dynamic adaptation of the cellular microenvironment; and the pathway of transformation has evolved from laboratory exploration to large-scale production and regulatory oversight. The newly developed system of regenerative medicine addresses significant clinical challenges, including osteoarthritis, traumatic bone defects, and spinal cord injuries, by transforming these conditions from a state of irreversible degradation to a state of functional regeneration. This advancement represents a pivotal milestone in the field of precision musculoskeletal treatment. ,

While exosome therapy has demonstrated considerable potential in the musculoskeletal domain, the current body of literature reveals significant gaps. Notably, there is a deficiency in engineering strategies and a systematic framework for biomaterial delivery systems. Furthermore, the principles of disease adaptation remain inadequately defined, impeding the systematic development of exosome engineering strategies and biomaterial carriers tailored to the diverse pathological characteristics of orthopedic diseases. In response to these challenges, this thesis innovatively establishes a trinity framework of “engineering-material delivery-disease orientation”. This is achieved by analyzing the molecular basis of exosome biogenesis and therapeutic potential, integrating engineering strategies with delivery platforms to construct coherent interaction mechanisms, and examining the latest preclinical research on prevalent orthopedic conditions such as osteoarthritis, traumatic bone defects, rheumatoid arthritis, and spinal cord injury. Finally, the disease-specific treatment matrix was established, and the decision tree for “engineered exosome-biological integration” was developed based on the characteristics of the pathological microenvironment. This framework addresses a theoretical gap in system integration within the field and offers a pathology-driven design blueprint to advance the translation of laboratory-derived exosomes into clinical-grade applications for precision regenerative medicine in musculoskeletal disorders.

Overview of Exosomes

Exosomes, small EVs (30–150 nm), are crucial in intercellular communication and increasingly significant in diagnostics, therapeutics, and regenerative medicine. , Once considered mere cellular debris, exosomes are now acknowledged for their capability to transport bioactive molecules between cells, rendering them essential tools in biomedical research and clinical applications. This part offers a comprehensive exploration of exosomes, from their discovery and biogenesis to their diverse roles in physiology, pathology, and innovative medical applications.

History: From Discovery to Functional Insights

The journey of exosomes began in the mid-20th century with a broader study of EVs. However, it was not until the 1980s that exosomes were specifically identified, following observations that maturing reticulocytes released small vesicles, later termed “exosomes”. , Foundational studies by Johnstone and Stahl established that exosomes originate within multivesicular bodies (MVBs) and are released into the extracellular space when MVBs fuse with the plasma membrane. ,

As research advanced into the 2000s, the significance of exosomes in various physiological processes became evident. They were found to be integral to immune responses, neuronal communication, and tissue repair. , Simultaneously, their roles in pathological conditions, particularly in cancer, were uncovered, where exosomes modulate the microenvironment and facilitate tumor cell growth and metastasis. ,

Over the last two decades, technological innovations have led to remarkable developments in exosome research. Enhanced isolation and characterization techniques, including ultracentrifugation, nanoparticle tracking analysis (NTA), and high-resolution imaging, have enabled researchers to explore exosomes in unprecedented detail. Not only have these technologies advanced our knowledge of exosome biology, but they have also facilitated their use as biomarkers and natural transporters for drug delivery, gene therapy, and RNA-based treatments. −

Biogenesis: A Complex and Regulated Process

The biogenesis of exosomes is a highly regulated and multistep complex process that plays a crucial role in exosomal biosynthesis, intercellular communication, and the maintenance of cellular homeostasis (Figure ). Initially, the cytoplasmic membrane invaginates to form early sorting endosomes (ESEs). Subsequently, ESEs progressively mature into late sorting endosomes (LSEs) through the concerted actions of the trans-Golgi network and the endoplasmic reticulum (ER). The LSE membrane buds inward to generate MVBs, which enclose intraluminal vesicles (ILVs). Ultimately, the fate of MVBs dictates exosome release. If MVBs fuse with lysosomes, ILVs are degraded. Conversely, upon fusion with the plasma membrane, ILVs are secreted into the extracellular space as exosomes, thereby facilitating intercellular communication. ,

1.

Biogenesis and structure of exosomes.

Exosome biogenesis relies on key molecular players like Rab proteins, Syntenin-1, TSG101, ALIX, ESCRT proteins, and tetraspanins, which guide their formation, sorting, and release. − Current studies are uncovering the complexities of these interactions, emphasizing their importance in normal physiology and disease.

Extraction: Isolation and Preservation Techniques

Isolation Technology of Exsomes

To advance the application of exosome research in clinical settings, it is imperative to address the challenges of scalability, standardization, and the preservation of functional activity in isolation techniques. − While ultracentrifugation is esteemed as the gold standard due to its ability to produce high-purity exosomes through density differentiation via high-speed centrifugal force, its limited throughput and the extensive processing time, which can extend up to 24 h, present substantial obstacles to the production of clinical-grade exosomes. Density gradient centrifugation, which utilizes sucrose, iodixanol, or other density media to create a continuous gradient for enriching exosomes at points of equal density through ultracentrifugation, is predominantly confined to basic research. This limitation is attributed to its exceedingly low sample processing capacity and the high cost of the media involved. To address the scalability issue, tangential flow filtration technology employs the transmembrane pressure differential to facilitate the lateral flow and filtration of the sample solution. This method selectively intercepts exosomes based on membrane pore size, making it suitable for processing large sample volumes and demonstrating industrial applicability. Nevertheless, its limitations in achieving high purity often necessitate supplementary purification techniques. Particle size exclusion chromatography, which relies on the size disparity between exosomes and impurity molecules, allows exosomes to elute first, as they are unable to penetrate the pores of the medium. This method has become the preferred choice for functional verification due to its gentle separation characteristics, which best preserve the native structure of the vesicles. However, the restricted column loading capacity of ≤1.5 mL and the high cost of chromatographic media pose significant challenges to the direct clinical application of size exclusion chromatography.

Emerging technologies characterized by high specificity are catalyzing a transformational shift in separation paradigms. Immunoaffinity capture, which is predicated on the principle of antigen–antibody specific binding, employs antibodies targeting exosome surface marker proteins to facilitate targeted enrichment via solid-phase carriers. This approach underpins the development of precision medicine. Nonetheless, the substantial cost associated with antibodies presents a significant impediment to widespread adoption. , In contrast, microfluidic chips, which amalgamate microscale fluid manipulation with multiphysics separation mechanisms, offer a promising alternative. By incorporating acoustic screening, two-dimensional electrophoresis, and online detection modules, these chips can achieve single-vesicle resolution in samples, thereby demonstrating considerable potential for advancing precision medicine. The chemical precipitation method employs polymers, such as poly­(ethylene glycol), to disrupt the hydration layer on the surface of exosomes, thereby facilitating vesicle condensation and separation through low-speed centrifugation. This method is advantageous for diagnostic purposes due to its rapid execution. However, its high rate of cocontamination raises concerns regarding its reliability. Consequently, the selection of appropriate technology must consider factors such as yield, purity, scalability, cost, and ease of use while adhering to the principle of scenario adaptation. Specifically, basic research prioritizes methods with high purity, rapid diagnostics benefit from straightforward processes, therapeutic development depends on scalable platforms, and microfluidic technology holds promise for precision intervention scenarios (Table ).

1. Comparison of Various Isolation and Extraction Techniques for Exosomes.

technique	purity	throughput	cost	scalability	function preservation	primary research use	clinical applicability
ultracentrifugation (UC)	high	low	moderate-high	low	moderate	small-scale studies	limited
density gradient centrifugation	very high	very low	high	very low	good	subpopulation analysis	niche research
tangential flow filtration (TFF)	moderate	high	moderate	high	moderate	preconcentration step	large-volume processing
size exclusion chromatography (SEC)	high	low	high	low	excellent	functional validation	purification in cascades
polymer precipitation	low	moderate	low	moderate	poor	rapid screening	diagnostic prototypes
immunoaffinity capture	exceptional	low	very high	low	good	biomarker-specific studies	targeted therapy development
microfluidic systems	high/tunable	low	high	low	good	single-vesicle analysis	point-of-care diagnostics

Commercialized Exosome Isolation Platforms

With the ongoing advancements in exosome isolation technology, its industrial application is experiencing consistent growth. Initial commercial kits, such as ExoQuick, utilized polymer precipitation to facilitate rapid separation, thereby significantly decreasing the reliance on ultracentrifugation. In contrast, Qiagen’s membrane affinity purification technology enhances standardization through superior reproducibility, establishing a foundation for the Good Manufacturing Practice (GMP) system. Following this, the MagCapture platform achieved clinical-grade throughput by employing phosphatidylserine magnetic bead capture technology, emerging as the first system capable of meeting the processing demands of up to a thousand cases per day.

Through the application of tangential flow filtration technology, the Exodisc device effectively combines centrifugation and nanofiltration modules, thereby enhancing the efficiency of urine treatment. Additionally, the innovative ultrathin silicon nitride membrane technology facilitates continuous production with minimal pressure drop, which directly contributes to reducing the structural costs associated with CAR-exosomes and other therapeutic products. Furthermore, the ExoSearch chip establishes a “capture-detection” closed-loop analysis paradigm. Meanwhile, the iKnife factory employs artificial intelligence to dynamically optimize process parameters, setting a new industry standard by minimizing production volatility. ,

Preservation Methods for Exosomes

Preserving exosomes is vital for their research and practical use, as it directly impacts their stability, functionality, and reliability of experimental outcomes. One of the primary considerations in exosome preservation is storage conditions, which can significantly affect their size, number, contents, and biological activities. Research indicates that storing biofluids and isolated exosomes at −80 °C is optimal for preserving their integrity over long durations. Short-term storage at 4 °C is also possible, but it is not recommended for long durations due to potential alterations in exosome properties. Lyophilization, or freeze-drying, is a promising technique for stabilizing exosome products, making them easier to transport. In addition, the preservation of exosomes is a multifaceted challenge that requires careful consideration of storage conditions, isolation techniques, and maintenance of their molecular integrity. Advances in these areas will enhance the reliability of exosomal studies and facilitate their application in diagnostics and therapeutics.

Transformation: From Functions to Applications in Biomedicine

Applications of Exosomes in Biomedicine

The unique biophysical properties of exosomes underpin their roles in health and disease. They exhibit a characteristic size and morphology, with a “saucer-like” appearance under electron microscopy and a buoyant density in sucrose gradients between 1.13 and 1.19 g/mL. The stability and functionality of their lipid and protein composition, rich in cholesterol, sphingomyelin, phosphatidylserine (PS), and proteins like tetraspanins (CD9, CD63, and CD81), enable their role in long-range intercellular communication, highlighting their involvement in diverse biological processes and potential clinical applications. , In immune regulation, exosomes derived from immune cells can either stimulate or suppress immune responses, while in tissue homeostasis, they promote repair by delivering GFs and signaling molecules. , In pathogenesis, exosomes are important factors in the progression of diseases, particularly cancer, influencing tumor growth, spread, and drug resistance. ,

Exosomes hold significant promise for both diagnosis and therapy. Their molecular cargo, indicative of the originating cells’ physiological state, makes them ideal noninvasive disease biomarkers. Exosomes extracted from bodily fluids such as blood, urine, and saliva offer crucial insights into disease conditions, facilitating liquid biopsies and early detection. Exosomes are being investigated as natural delivery vehicles in therapy because they can encapsulate and protect therapeutic agents. , Their innate targeting capabilities offer a promising approach for delivering drugs, genes, and RNA therapies to specific cells or tissues, enhancing the treatment efficacy. In regenerative medicine, exosomes derived from stem cells can improve tissue repair and regeneration, providing promising treatment options for injuries and degenerative diseases. −

Exosomes have emerged as multifaceted entities with the potential to transform biomedicine. They are valuable for both fundamental research and clinical applications due to their importance in intercellular communication, diagnostics, and therapeutics. As our knowledge of these nanovesicles increases, the potential for exosomes to revolutionize healthcare continues to grow, heralding a new era of precision medicine and innovative treatment strategies.

Challenges in the Clinical Application of Exosomes

As natural nanoscale delivery vectors, the clinical translation of exosomes remains constrained by fundamental challenges inherent in their biological characteristics. A primary obstacle is cargo heterogeneity, which impedes the consistency of therapeutic outcomes. Even when exosomes are derived from the same cell, there is significant variability in the composition of bioactive molecules, such as proteins, nucleic acids, and lipids, that they carry. This heterogeneity arises not only from dynamic changes in the cellular microenvironment but also from the loss of subpopulations during the isolation and purification processes. Such variability can lead to fluctuations in therapeutic efficacy and significantly complicates the evaluation of treatment effectiveness. Furthermore, the immunogenicity and off-target effects associated with modified exosomes remain inadequately explored. , The genetic engineering or chemical modification of exosomes to improve targeting capabilities may introduce novel epitopes, potentially eliciting immune clearance. Additionally, the nonspecific binding of targeted ligands, such as peptide-modified exosomes that may cross-react with cell surface receptors of normal tissues, could elevate the risk of unintended accumulation in organs such as the liver and kidneys, thereby compromising the safety of the therapeutic intervention.

The advancement of large-scale production technology for functional exosomes remains insufficient to meet clinical demands, posing a significant barrier to their transition from laboratory research to clinical application. Consequently, the development of efficient large-scale production methods for functional exosomes is imperative for their clinical utilization. Therapeutic interventions often necessitate milligram to gram quantities of exosomes, yet current extraction techniques struggle to achieve an optimal balance between yield and quality. Furthermore, the stability of exosomes during long-term storage presents an additional challenge for large-scale application. Cryopreservation may compromise membrane integrity, whereas storage at ambient temperatures can result in the degradation of exosomal contents. ,

In the future, the challenges associated with the clinical application of exosomes may be progressively addressed through advancements in technology. Precision separation techniques have the potential to resolve issues related to cargo heterogeneity, while modifications to reduce immunogenicity could enhance safety profiles. Furthermore, breakthroughs in large-scale production and storage technologies are anticipated to facilitate standardized preparation. As these obstacles are overcome, exosomes are poised to assume a more significant role in clinical practice.

Strategies of Exosomes in the Management of Orthopedic Diseases

Exosomes, as an emerging therapeutic modality in regenerative medicine, demonstrate significant promise in tissue repair, attributed to their low immunogenicity, efficient intercellular communication capabilities, and diverse biological effects. Nonetheless, the treatment of orthopedic diseases poses distinct challenges due to the intricate microenvironment and restricted regenerative capacity of these tissues. Nanoscale exosomes exhibit potential in facilitating the repair of complex degenerative alterations in bone and cartilage through various approaches, including their native form, modified configurations, or integration with biomaterials ,

Native Exosomes Load Cargo and Promote Tissue Repair

Exosomes originating from cells like mesenchymal stem cells (MSCs), , chondrocytes, and osteoblasts, play a vital role in this research. They carry diverse biological molecules like proteins, lipids, and nucleic acids and act as carriers of molecular signals that stimulate essential regenerative processes.

Native Exosomes Regulate Cartilage Repair

In treating cartilage damage, exosomes have shown significant potential as therapeutic agents. Exosomes from chondrocytes and MSCs are essential for regulating the chondrogenic environment. These exosomes can enhance the proliferation of chondrocytes while simultaneously reducing inflammation, a dual action necessary for maintaining and regenerating cartilage tissue,. In this context, exosomes are primarily beneficial due to their capacity to carry anti-inflammatory cytokines and microRNAs (miRNAs). These molecules help create a more favorable environment for tissue healing by mitigating the chronic inflammation that frequently accompanies orthopedic injuries and degenerative diseases. Exosomes can transport miR-140-5p, crucial for cartilage regeneration and inhibiting OA progression by modulating inflammation and enhancing chondrocyte viability. , The increasing amount of evidence highlights the potential of exosome-based treatments in regenerative medicine, especially for conditions involving cartilage damage. By leveraging the natural abilities of exosomes to transfer targeted therapies while suppressing inflammatory responses, innovative treatment approaches are being developed that could significantly improve outcomes for patients with degenerative joint diseases. Qian et al. demonstrated that miR-26b-5p, derived from M2-Exos, modulates macrophage phenotype by targeting the TLR3 signaling pathway. Additionally, it inhibits chondrocyte hypertrophy by targeting COL10A1. Subsequent experiments revealed that intra-articular administration of a miR-26b-5p agonist attenuated synovial inflammation and cartilage degradation in osteoarthritic mice.

Native Exosomes Repair Bone Defects

Native exosomes possess the capability to accurately target the bone microenvironment by utilizing their intrinsic cargo of protein signaling molecules, including bone morphogenetic proteins (BMPs)-related proteins and integrins, as well as regulatory nucleic acids, such as bone-promoting microRNAs. These components influence the fate and behavior of critical cellular entities, including mesenchymal stem cells, osteoblasts, and osteoclasts, thereby facilitating bone regeneration and remodeling at both the molecular and cellular levels. Specifically, exosomes derived from bone marrow mesenchymal stem cells (BMSC-Exos) have been shown to effectively deliver BMP-2, contributing to the repair of bone tissue. −

Zhang et al. verified that BMSC-Exos effectively activate critical osteogenic signaling pathways through the incorporation of BMP-2. As a highly effective osteogenic inducer, BMP-2 interacts with its specific cell membrane receptor, initiating the phosphorylation of Smad1/5/8. The phosphorylated Smad proteins subsequently form a complex with Co-Smad4 and translocate into the nucleus, where they synergistically activate the principal osteogenic transcription factor RUNX2. This activation leads to the upregulation of osteocalcin (OCN), osteopontin (OPN), and other target genes. Consequently, this process directly promotes the differentiation of mesenchymal stem cells into osteoblasts and facilitates the mineralization of the bone matrix, thereby effectuating bone repair. Yu et al. employed ultracentrifugation to isolate BMSC-derived exosomes (BMSC-Exos) and subsequently delivered miR-136-5p to the site of injury. The miR-136-5p specifically targeted LRP4, resulting in the suppression of LRP4 protein expression and the consequent activation of the Wnt/β-catenin signaling pathway. This activation led to an increase in the nuclear translocation of β-catenin and an elevated expression of the downstream transcription factor Runx2. Additionally, there was a more than 2-fold increase in the activity of alkaline phosphatase (ALP), an indicator of osteogenic differentiation, as well as in the synthesis of bone matrix protein. In a related study, Wang et al. demonstrated that augmenting the expression of lncRNA H19 within BMSC-derived exosomes could modulate bone repair via the miR-467/HoxA10 axis. The long noncoding RNA (lncRNA) H19 functions as a competitive endogenous RNA by binding to miR-467, thereby alleviating its suppression of the target gene HoxA10. This interaction activates the HoxA10 transcription factor, leading to upregulation of essential osteogenic genes, including RUNX2. In a murine fracture model, exosomes exhibiting elevated levels of lncRNA H19 effectively ameliorated the disruption of osteogenic differentiation. In vitro assays demonstrated a 42% increase in alkaline phosphatase (ALP) activity and a 35% enhancement in calcium nodule formation. Concurrently, the mRNA and protein levels of osteogenic marker genes RUNX2, osteopontin (OPN), and osteocalcin (OCN) were significantly upregulated.

Furthermore, native exosomes have the capability to deliver specific signaling molecules that precisely modulate the recruitment, polarization, and functional state of immune cells, including macrophages and T cells. This modulation transforms a pro-inflammatory injury microenvironment into a pro-regenerative repair environment, thereby establishing favorable conditions for bone repair. Wang et al. demonstrated that M2-derived exosomes influence macrophage polarization by activating the PI3K/AKT signaling pathway, which enhances the bone immune microenvironment and facilitates the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), ultimately expediting bone healing. M2-derived exosomes, containing active components such as mRNA, can be internalized by M1 macrophages, leading to the activation of the PI3K/AKT pathway and inducing the conversion of pro-inflammatory M1 macrophages to the anti-inflammatory M2 phenotype. M2 macrophages subsequently secrete anti-inflammatory cytokines, such as interleukin-10 (IL-10), and osteogenic factors, including bone morphogenetic protein-2 (BMP-2) and oncostatin M (OSM), which collectively inhibit the inflammatory environment and promote the osteogenic differentiation of BMSCs.

Exosomes Enhance Tissue Repair by Promoting Angiogenesis

Angiogenesis refers to the process of forming new blood vessels from the existing blood vessel network, which plays an important role in tissue repair as it supplies essential nutrients and oxygen to healing tissues. Exosomes contribute to this process by carrying angiogenic factors that promote vascularization, thus facilitating the repair of not only bone but also other orthopedic tissues that require a robust blood supply for effective regeneration. , Behera et al. elucidated that exosomes containing long noncoding RNA H19 (lncRNA-H19) facilitate bone regeneration by modulating the interplay between angiogenesis and osteogenesis. Specifically, lncRNA-H19 competitively interacts with miR-106a, alleviating its transcriptional suppression of target gene angiopoietin-1 (Angpt1). Subsequently, activated Angpt1 associates with the Tie2 receptor, inducing its tyrosine phosphorylation, which subsequently enhances the phosphorylation of endothelial nitric oxide synthase (eNOS) at serine residues. This cascade results in increased nitric oxide (NO) production, ultimately promoting endothelial cell angiogenesis and osteogenic differentiation of bone marrow stromal cells (BMSCs). In comparison to the control group, there was a 2.1-fold increase in the length of new blood vessels, a 1.8-fold increase in branch points, a 68% enhancement in the activity of bone repair-related phosphatase, and a 1.5-fold increase in calcium nodule formation. Chen et al. employed miR-142-3p encapsulated within regulatory T cell (Treg)-derived exosomes (TREMD-Exos) to target the TGFBR1/SMAD2 signaling pathway, thereby synergistically enhancing angiogenesis and osteogenic differentiation. The miR-142-3p enriched in TREMD-Exos inhibited the expression of TGFBR1 and the phosphorylation of SMAD2 through direct binding to TGFBR1. In bone marrow-derived mesenchymal stem cells (BMSCs), this pathway inhibition resulted in a 2.5- to 3.0-fold increase in the expression of osteogenic markers such as Runx2, ALP, and OCN, an 80% enhancement in ALP activity, and augmented calcium nodule formation. In human umbilical vein endothelial cells (HUVECs), overexpression of miR-142-3p led to a 60% increase in cell proliferation, a 1.7-fold enhancement in migratory capacity, and a 2.1-fold increase in vascular branching points.

Special Functions Given by Engineering

The field has advanced into creating modified exosomes, offering an innovative method to boost the therapeutic efficacy of these vesicles (Figure ).

2.

Processing strategies of engineered exosomes and forms of integration with materials.

Pretreatment of Blast Cells: Source-Regulated Secretory Behavior

By pretreating progenitor cells responsible for exosome secretion, we can optimize the functional attributes of the resultant exosomes in a source-specific manner. This optimization is primarily achieved through the modulation of the biological state or molecular expression profile of the progenitor cells. Genetic modification of these cells via gene transfection technology enables the continuous secretion of exosomes with predetermined targeting capabilities. Specifically, fusing a target ligand with an exosome membrane protein gene allows the progenitor cells to secrete exosomes carrying the target molecule. This method facilitates the precise regulation of ligand density, thereby significantly enhancing the recognition capabilities of surface markers on bone and joint tissues. Furthermore, the prior incorporation of specific functional biomolecules into progenitor cells can substantially augment the therapeutic efficacy of subsequently secreted exosomes. Ma et al. employed a track-etch-membrane nanoelectroporation system (TM-nanoEP) to facilitate the delivery of a plasmid mixture encoding BMP-2 and VEGF-A into human adipose-derived mesenchymal stem cells (hAdMSCs). Subsequent to transfection, the cells’ intrinsic transcriptional machinery synthesized the mRNA, which was subsequently encapsulated into exosomes. Upon application of an external voltage stimulus, the transfection efficiency achieved was 75%, while cell viability was maintained above 75%. Furthermore, the yield was approximately 10-fold greater than that of untreated cells. Notably, the transcriptional generation of BMP-2 and VEGF-A mRNA was approximately 1000-fold higher compared to untreated cells.

Moreover, the characteristics of exosomes can be substantially altered through precise regulation of the culture environment and conditions during the production by cells. For instance, modifications in medium composition, such as the type and concentration of serum, the addition of specific growth factors or small molecule compounds, and adjustments to the physical parameters of the cell culture, including oxygen tension, fluid shear stress, or matrix stiffness, can have significant effects. Han et al. demonstrated that three-dimensional (3D) culture significantly enhanced the efficacy of mesenchymal stem cell-derived exosomes (3D-EXOs) in spinal cord repair. Exosomes derived from 3D culture were enriched with neuroprotective proteins and immunomodulatory microRNAs, maintaining cell stemness as evidenced by a 3.89-fold increase in SOX2 expression compared to a two-dimensional (2D) culture. Additionally, the average particle size of 60 nm was more favorable for cellular uptake. Various culture strategies, encompassing two-dimensional monolayer culture, three-dimensional sphere culture, conventional culture, hypoxia culture, and starvation treatment, can effectively influence cells to secrete exosome populations that exhibit variations in yield, particle size distribution, surface marker expression, and the composition of critical components, including nucleic acid molecules, proteins, and lipids. − Li et al. demonstrated that miR-21-5p was significantly upregulated following the hypoxic preconditioning of exosomes, facilitating their uptake by target cells. By targeting SPRY1 to activate the PI3K/AKT signaling pathway, this upregulation promotes angiogenesis, particularly the formation of type H vessels, and is coupled with osteogenesis in human umbilical vein endothelial cells (HUVECs). In vitro experiments revealed that these exosomes significantly enhanced the proliferation, migration, and tube formation capabilities of endothelial cells. Furthermore, in vivo experiments indicated that the exosomes increased new bone formation, bone mineral density, and the number of type H vessels, ultimately contributing to the effective repair of osteoporotic fractures.

The capacity to directionally modify the biological characteristics of exosomes by altering their “birth environment” offers a crucial strategic foundation for acquiring exosomes with specific functions or targets. This significantly enhances their potential applications in disease diagnosis, treatment, and regenerative medicine.

Internal Modification: Efficient Loading of Therapeutic Cargo

Loading exosomes with therapeutic molecules further expands their utility. These engineered exosomes, by encapsulating drugs, GFs, or other bioactive substances, can directly deliver their cargo to recipient cells, utilizing their inherent role in cell communication to improve tissue repair and regeneration. ,

To efficiently load therapeutic molecules into the inner cavity of exosomes, active loading techniques are essential. The choice of the loading strategy varies according to the nature of the therapeutic cargo. For nucleic acid analogues such as siRNA or mRNA, electroporation is commonly employed. This technique transiently disrupts the exosome membrane using specific electrical parameters, facilitating the efficient import of these molecules. For instance, therapeutic nucleic acids, including miRNA, siRNA, lncRNA, and mRNA designed through gene editing, can be incorporated into exosomes. These molecules, which play crucial roles in regulating osteogenesis or chondrogenesis, can directly activate repair pathways in recipient cells while minimizing the structural damage associated with exogenous drug delivery. In contrast, proteins and other macromolecular therapeutics often require methods such as repeated freeze–thaw cycles or the application of membrane permeabilizers to temporarily increase the permeability of the exosome membrane, thereby enabling the entry of these larger molecules. Hydrophobic drug molecules exhibit physical and chemical properties akin to those of lipid membranes, allowing them to be spontaneously incorporated into the lipid bilayer of exosomes via a straightforward incubation and diffusion process. This method effectively preserves the drugs’ inherent biological activity. The primary objective of these internal modification techniques is to overcome the intrinsic drug-loading limitations of exosomes and optimize their capacity to transport therapeutic agents. , Lin et al. successfully encapsulated the VEGF165 plasmid into exosomes via electroporation. These engineered exosomes containing the VEGF165 plasmid were effectively internalized by rat bone marrow mesenchymal stem cells (rBMSCs). Subsequent to transfection, there was a 3.5-fold increase in VEGF165 mRNA expression and a 2.8-fold enhancement in protein secretion. In the animal study, the experimental group exhibited a 2.5-fold increase in the density of CD31+ blood vessels compared to the control group, 4 weeks postoperation. Furthermore, the colocalization of VEGF with osteogenic markers, specifically COL1 and OCN, substantiated the coupling relationship between angiogenesis and osteogenesis.

Furthermore, the dependence on energy–substance interactions can significantly improve the delivery efficiency. In a study conducted by Kim et al., a multifunctional composite carrier, termed NV-IONP, was developed by integrating iron oxide nanoparticles (IONP) with exosome-like nanovesicles derived from mesenchymal stem cells (NV). The superparamagnetic properties of the IONP encapsulated within the NV-IONP facilitated the generation of a directional magnetic force when subjected to an external magnetic field. This mechanism effectively mitigated the entrapment of nanovesicles in the lungs and liver following systemic injection, thereby enhancing the accumulation of nanovesicles at the site of spinal cord injury by a factor of 10 compared to the control group.

External Modification: Precise Targeting and Functional Enhancement

Surface engineering of exosome membranes can endow them with the capability to actively recognize and target specific tissues or cells, thereby enhancing their functional efficacy. , One approach involves gene editing at the level of the maternal cell, enabling the secreted exosomes to express specific targeting peptides on their native membrane proteins. Alternatively, metabolic engineering can be employed, wherein specialized glycosylation precursors are introduced during maternal cell culture. These precursors are incorporated into the glycan structures of exosome membrane proteins, modifying their interactions with receptors and potentially increasing their accumulation at sites of inflammation. Furthermore, the application of efficient bioorthogonal click chemistry allows for the direct covalent attachment of recognition elements with high affinity and specificity, such as aptamers or antibody fragments, to the surface of purified exosomes. This technique facilitates precise and adaptable modification of exosomal surface ligands. , The incorporation of environmentally responsive groups enhances the ability of exosomes to sense pathological microenvironments, which is achieved through the use of pH-sensitive chemical bonds.

You et al. modified exosomes with dextran sulfate (DS) due to its specific affinity for the macrophage receptor (SR-A), which is abundantly expressed in the inflamed joints of individuals with rheumatoid arthritis (RA). The covalent attachment of DS to the surface of adipose-derived stem cell (ADSC)-derived exosomes (DS-Exos) was achieved through metabolic glucose engineering in conjunction with copper-free click chemistry. This modification improved the targeting capability of the exosomes and increased their accumulation in arthritic joints in vivo by a factor of 1.52. Lee et al. used an integrated strategy to construct bone-targeting EMs. Azides were displayed on the surface of MSCs through feeding MSCs with tetraacetylated N-azidoacetyl-d-mannosamine (Ac4ManNAz). Subsequently, azide-displayed EMs (N3-EMs) were fabricated through extrusion and loaded with a smoothened agonist (SAG) to build N3-EM-SAG. SAG can activate hedgehog signaling to enhance osteogenesis. Finally, dibenzocyclooctyne (DBCO)-functionalized bone-targeting ligands and alendronate (ALD) were used to modify the surface of N3-EM-SAG by the bioorthogonal click reaction between DBCO and azides to form ALD-EM-SAG. Systemic administration experiments in CD-1 nude mice showed that ALD-EM-SAG exhibited strong targeting ability to various bone tissues.

The primary objective of these external modifications is to address the limitations associated with the inherent tropism of exosomes, thereby significantly enhancing the efficiency and specificity of their delivery of therapeutic cargo to the targeted lesion site or cell.

Biomaterials-Mediated Delivery and Enhanced Function

Biomaterials employed as carriers for exosome delivery are selected based on distinct loading and release mechanisms. These biomaterials can be broadly categorized into natural polymers, synthetic polymers, inorganic materials, and stimuli-responsive materials. And these biomaterials are engineered into specific physical forms to create delivery systems, with their structural design critically impacting the loading, protection, release kinetics, and interaction of exosomes with target tissues.

Integration of Different Biomaterials with Exosomes

Natural Polymers

Natural polymeric materials, sourced from living organisms, exhibit notable advantages, such as excellent biocompatibility, biodegradability, and low toxicity. Exemplary materials within this category include gelatin, chitosan, sodium alginate, and hyaluronic acid. These materials are typically characterized by high porosity, hydrophilic surfaces, low compressive modulus, and enzyme-sensitive degradation with degradation periods spanning from days to weeks. Such properties facilitate a high-capacity physical embedding environment for exosomes and govern the sustained release behavior through mechanisms such as hydrogen bonding or electrostatic adsorption, thereby significantly mitigating the burst release phenomenon. Lin et al. used natural polymers HA and Gel to create injectable hydrogels via Schiff base reaction, which precisely controls exosome release. SEM revealed an interconnected porous structure with pore density increasing with HA-CHO concentration. The optimal pore size for exosome encapsulation and release was found in 4% HA-CHO or 16% Gel-ADH hydrogels. Rheological tests indicated limited modulus variation with HA-CHO concentration but significant shear thinning properties. The hydrogel transitions between “Sol” and “Gel” states at high and low strain, respectively, allowing stable exosome encapsulation and responsive diffusion based on mechanical changes.

Nevertheless, the inherent low mechanical strength of natural materials poses a risk of structural failure and atypical exosome release when applied to weight-bearing bone defects. Concurrently, the rapid enzymatic degradation occurring within an inflammatory microenvironment may result in premature exposure and subsequent proteolytic degradation of exosomes, thereby constraining their effectiveness in orthopedic applications involving weight-bearing and inflammatory conditions.

Synthetic Polymers

Synthetic polymers, such as polyethylene glycol, polylactic acid, polyacetate copolymers, and polycaprolactone, exhibit a stable molecular architecture and consistent properties, alongside superior mechanical characteristics when compared to natural materials. The remarkable characteristics of these materials encompass the precise engineering of the network aperture and a broad spectrum of adjustable degradation cycles, facilitating more versatile functional modifications. In their study, Gao et al. utilized porous polylactic acid-glycolic acid copolymer (PLGA) microspheres modified with polydopamine (PDA) as carriers for exosomes. The PDA coating enables efficient and stable exosome loading due to its superior adhesion properties. Concurrently, the porous PLGA microspheres are injectable and suitable for filling irregular bone defects via minimally invasive surgical techniques. Furthermore, the porous architecture offers ample adsorption space for exosomes, and the affinity coating with PDA eliminates the need for a freeze-drying step, thereby better preserving the biological activity of the exosomes.

In the context of treating orthopedic diseases with exosomes, synthetic materials frequently exhibit limitations, including suboptimal biocompatibility, inadequate degradation congruence, and a propensity to induce localized inflammatory responses.

Inorganic Biomaterials

The function of inorganic biomaterials is to serve as rigid scaffolds for bone conduction. Notable examples of such materials include hydroxyapatite and titanium alloys. , These materials are characterized by high compressive strength, a specific micropore scale, a relatively low specific surface area, and a tendency toward brittleness. These inherent properties result in low efficiency and a significant burst release of exosomes when loaded solely through physical adsorption. Consequently, surface functionalization has emerged as a crucial strategy. This includes the coordination and binding of the calcium apatite layer in hydroxyapatite, the formation of a nanowhisker structure to increase the specific surface area of titanium alloys via alkaline heat treatment, or the application of other active material coatings to introduce multiple forces. Zhai et al. employed laser melting 3D printing to create a porous cylindrical titanium alloy scaffold with a high porosity structure, which promotes cell migration and material loading. Following autoclaving, the scaffold was immersed in a poly-l-lysine solution overnight, resulting in a positively charged coating on its surface. This modification enhanced the adsorption capacity for negatively charged exosomes through electrostatic interactions. Gao et al. employed low-temperature deposition 3D printing to fabricate porous hydroxyapatite (HA) scaffolds. These scaffolds, when coated with polylysine, are capable of adsorbing exosomes through electrostatic interactions, achieving a loading efficiency exceeding 85%. Under physiological conditions, the functionalized scaffolds can release exosomes over a period exceeding 14 days, with an initial release of approximately 40% within the first 24 h, followed by a sustained release that preserves biological activity.

Nonetheless, the inherent brittleness of these materials may result in particle shedding when applied to dynamic load-bearing bone regions. This shedding could lead to the unintended migration of encapsulated exosomes to nontarget tissues, presenting a significant risk in orthopedic environments subjected to dynamic loading.

Intelligent Responsive Materials

The fundamental principle of intelligent responsive materials lies in their ability to interpret pathological signals, thereby facilitating precise spatiotemporal release. Notable examples of such materials include thermosensitive substances, enzyme-responsive gels, and photothermal materials. These materials are engineered to respond to specific stimulus, such as temperature fluctuations, particular enzyme concentrations, or near-infrared light, to initiate exosome release as needed. , For instance, a hydrogel formulated with poloxamer 407 (17.9% w/w) and poloxamer 188 (5% w/w) can be administered as a liquid at room temperature (25 °C) and undergo rapid gelation at physiological body temperature (37 °C) upon injection into the joint cavity. This phase transition results in the formation of a solid network that not only provides structural support but also effectively prevents the clearance of synovial fluid through gelation. In vivo fluorescence imaging demonstrated that the retention signal intensity of Exo-Gel was five times greater than that of free exosomes, with the therapeutic concentration remaining effective for up to 28 days.

This innovative design encounters two primary challenges: first, the intricate synthesis process frequently results in a substantial reduction of exosome activity; second, the in vivo pathological signal gradient, including the concentration of specific enzymes, may be inadequate to achieve a predetermined response threshold. These limitations significantly hinder its practical effectiveness in the treatment of orthopedic conditions necessitating precise delivery, such as bone infections or bone tumors.

Intrinsic Properties of the Material Significantly Influencing the Therapeutic Efficacy of Exosomes

The intrinsic physical characteristics of biomaterials, notably porosity, stiffness, and degradation rate, serve as critical design parameters that significantly influence their loading efficiency, release kinetics, and overall biological performance at the target site when they are employed as exosome carriers.

Porosity

Porosity plays a critical role in modulating the diffusion pathways of exosomes and the interface of cell contact by influencing the microstructural characteristics of the material. While a structure characterized by high porosity and large pore size facilitates the efficient loading of exosomes, as well as the infiltration and migration of cells, it may also induce an initial burst release effect, leading to the premature depletion of active molecules. Conversely, structures with dense or small pore sizes considerably restrict the diffusion rate, resulting in a slow and sustained release pattern. This is particularly advantageous for tissue repair processes that necessitate prolonged regulation, such as nerve or osteochondral regeneration. An optimal range of porosity not only enhances the spatiotemporal distribution of exosomes but also establishes a conducive physical microenvironment for effective paracrine signal transmission between exosomes and target cells. ,

In the domain of exosome therapy for orthopedic disorders, porosity is a fundamental determinant influencing osseointegration and angiogenesis. An optimal bone repair scaffold necessitates a highly interconnected and suitably sized porous architecture. This structure not only facilitates the infiltration of host osteoblasts, vascular endothelial cells, and MSCs but also provides an extensive surface area and space for the accommodation of exosomes. Nevertheless, the design of pores requires meticulous calibration: excessively large or open pores may result in the premature loss of exosomes during the initial phase of implantation, potentially compromising their capacity to sustain an effective concentration within the bone defect. Conversely, appropriately small or nanoscale pores can retard the release of exosomes, aligning with their long-term role in promoting osteogenic differentiation, angiogenesis, and immune modulation.

Stiffness

The stiffness of materials, characterized by their elastic modulus, indirectly influences the efficacy of exosomes primarily through mechanotransduction signals. Cells exhibit a high sensitivity to substrate stiffness, and materials that closely replicate the physiological stiffness of the target tissue can more effectively facilitate cellular adhesion, spreading, proliferation, and differentiation. Additionally, such materials can modulate the exosome secretion profile of both the loaded cells and infiltrating host cells.

In the field of orthopedics, the stiffness of materials plays a crucial role in replicating the mechanical microenvironment of the target bone tissue. Given that bone possesses inherently high stiffness, materials employed for bone repair in load-bearing regions typically necessitate a high elastic modulus. This is essential to providing initial mechanical support and facilitating physiological stress stimulation. The alignment of material stiffness with that of bone significantly influences the behavior of loaded or infiltrated cells through mechanotransduction signals. Specifically, a stiffer substrate is more favorable for the differentiation of mesenchymal stem cells (MSCs) into osteoblasts and enhances the secretion of osteogenesis-related exosomes while also maintaining the balance between osteoclastic and osteogenic activities. Conversely, in the context of cartilage repair, such as in the treatment of osteoarthritis or tendon/ligament regeneration, materials with relatively lower stiffness and viscoelastic properties, which more closely approximate the modulus of cartilage or soft tissue, are required. These materials are critical for mimicking the mechanical behavior of native tissue and promoting the maintenance of the chondrocyte phenotype or the differentiation of tendon stem cells, thereby preventing tendinopathy. ,

Degradation Rate

The degradation rate is a pivotal factor influencing both the carrier lifetime and the timing of exosome release. This degradation process is characterized by the dynamic evolution of the pore structure, specific surface area, and local microenvironment. These alterations not only continuously modulate the diffusion and release of exosomes but also impact the stability of exosomes and the efficiency of cellular interactions. The kinetics of material degradation are intricately linked to exosome release behavior: materials undergoing bulk dissolution or hydrolysis typically exhibit rapid degradation, leading to a short-term, substantial release of exosomes, whereas slow degradation facilitates a prolonged and sustained release profile. An optimal degradation rate must be precisely aligned with the physiological processes of tissue repair or therapy. Concurrently, the degradation rate must achieve a delicate balance between maintaining structural support for continuous delivery and providing sufficient space for new tissue growth; premature or excessively slow degradation can compromise therapeutic efficacy.

For biomaterials intended for bone repair, it is crucial that the degradation rate is precisely synchronized with the rate of new bone formation. If degradation occurs too rapidly, it may result in the premature collapse of the scaffold, thereby compromising mechanical support and disrupting the continuous supply of exosomes, which are essential for the maturation and mineralization of the callus. Conversely, excessively slow degradation can impede new bone ingrowth and potentially trigger a foreign body reaction. Ideally, the biomaterial should maintain structural integrity during the initial phase to ensure the stable release of repair exosomes, followed by gradual degradation to accommodate the formation of new bone tissue during the intermediate and later stages. In the context of intra-articular delivery for osteoarthritis treatment, the degradation rate of injectable hydrogels must align with the therapeutic window of exosomes to effectively inhibit synovial inflammation, protect cartilage, or facilitate subchondral bone repair. It is imperative to avoid rapid degradation that could lead to the swift clearance of exosomes by synovial fluid, as well as to prevent residual materials from impairing joint function. ,

In conclusion, porosity, stiffness, and degradation rate do not function independently; rather, they interact synergistically to influence the loading, release, and stability of exosomes within biomaterial carriers as well as their interaction efficiency with the cellular and tissue microenvironment. A comprehensive understanding and precise regulation of these fundamental physical parameters are crucial strategies for achieving spatiotemporally controlled delivery of exosomes, thereby maximizing their therapeutic potential, particularly in the domains of regenerative medicine, immune regulation, and precision medicine.

Construction of Biomaterial Delivery Systems to Deliver Exosomes

Hydrogel Systems

Hydrogel systems, characterized by a highly aqueous three-dimensional network structure, offer excellent biocompatibility and a gentle encapsulation environment for exosomes. These systems may be composed of naturally derived materials such as gelatin and CS or synthetic polymers like polyethylene glycol. By adjustment of the cross-linking density, the pore size and swelling behavior of the network can be modulated, thereby controlling the diffusive or reactive release of exosomes. Despite these advantages, the weak mechanical strength of hydrogel systems limits their direct application in weight-bearing bone defect sites, rendering them more suitable for soft tissue filling or the repair of nonweight-bearing bone cavities. ,

Wu et al. focused on optimizing the fundamental physical characteristics, including the pore structure and mechanical properties, of alginate hydrogel to enhance the release profile and functional behavior of exosomes (Treg-Exo) and SDF-1α. The objective was to achieve controlled release and minimize peripheral loss. Scanning electron microscopy (SEM) analysis revealed that the hydrogel exhibited an interconnected porous architecture, which facilitated the storage of exosomes and SDF-1α and ensured their uniform distribution. Furthermore, the release kinetics were modulated through the diffusion within the pore network, enabling a sustained release of both agents. Additionally, the hydrogel demonstrated an appropriate storage modulus (G′) and loss modulus (G″), rendering its mechanical stiffness suitable for both injection and tissue repair applications. This optimal stiffness ensures that the hydrogel retains its structural integrity postinjection, preventing rapid degradation and thereby reducing the diffusion loss of exosomes at the fracture site, while promoting local retention. Concurrently, its mechanical properties are compatible with the fracture microenvironment, providing a stable platform for interactions between exosomes and target cells, effectively facilitating the repair of fracture damage. Chen et al. incorporated exosomes derived from hUVECs (hUVEC-Exos) into alginate/GelMA interpenetrating polymer network (IPN) hydrogels to establish composite materials. These composite materials promote M2 macrophage polarization and enhance the regeneration of bone and blood vessels.

Scaffold Systems

The scaffold system typically comprises synthetic polymers, such as polylactic acid-glycolic acid copolymer and polycaprolactone, as well as inorganic materials such as hydroxyapatite and titanium alloys. These scaffolds are engineered into a three-dimensional configuration with specific pore structures and geometries using techniques such as 3D printing, thermally induced phase separation, or sintering. , The high porosity and interconnected pores of the scaffold are instrumental in facilitating cellular proliferation and nutrient exchange. The mechanical properties of these scaffolds can be extensively tailored through the selection of materials and structural design to accommodate various bone defect requirements. Polycaprolactone (PCL) scaffolds offer degradation support ranging from weeks to years; however, their inherent hydrophobicity and the acidic nature of their degradation products may adversely affect exosome activity. Conversely, scaffolds composed of hydroxyapatite and titanium alloys exhibit superior osteoconductivity and mechanical strength, yet they necessitate surface functionalization strategies to address challenges related to low exosome loading efficiency and burst release. , The primary challenge associated with scaffold forms lies in attaining a uniform distribution and regulated release of exosomes at the defect site while maintaining structural stability. This is particularly crucial to preventing uncontrolled delivery resulting from scaffold deformation or fragmentation in dynamic loading environments.

Kyung Kim et al. employed freeze-drying to create silk fibroin (SF) scaffolds, which were then coated with exosomes from human adipose mesenchymal stem cells (AMSCs). Silk fibroin (SF), derived from Bombyx mori, exhibits excellent biocompatibility, injectable mechanical propertiescharacterized by an appropriate storage modulus (G′) and loss modulus (G″)and controllable degradability. The porous architecture of the scaffolds not only facilitates the sustained release of exosomes but also mitigates enzymatic degradation and preserves the integrity of exosomes through its barrier function. Ten weeks postimplantation, the bone volume fraction (BV/TV) in the Exo-SF group was observed to have doubled compared to the control group, with the newly formed bone structure displaying increased maturity. Furthermore, the β-sheet structure of SF imparts significant mechanical strength via hydrogen bonding, and methanol cross-linking can prevent postoperative inflammation and immune rejection.

Microneedle Systems

Microneedle (MN) systems, particularly soluble microneedle arrays composed of materials such as gelatin, hyaluronic acid, or polylactic-co-glycolic acid, offer a minimally invasive and effective route for transdermal or transmucosal delivery. The sharp tip of the microneedle can penetrate the skin or tissue barrier, allowing for the rapid release of exosome-loaded needle bodies into the local microenvironment as the needle material dissolves. This delivery method presents distinct advantages for treating conditions such as osteoarthritis, where intra-articular injection poses challenges, or bone infection lesions that necessitate high local drug concentrations, thereby facilitating the rapid accumulation of exosomes at the target site. The drug loading and release kinetics of microneedles are primarily influenced by the dissolution rate of the needle material and the drug loading strategy. Nonetheless, the limited drug loading capacity and relatively short duration of sustained release remain significant challenges in their application for large bone defects.

Li et al. successfully encapsulated Exo and polydopamine nanoparticles (PDA NPs) within the tip of a microneedle patch. This innovative approach effectively circumvented the discomfort associated with gastrointestinal digestion and the administration of oral drugs via injection, thus enhancing patient medication adherence. The PDA NPs, characterized by their reducing functional groups such as catechol and imine, were strategically encapsulated within an outer layer of HAMA shell. The biomaterial configuration enabled spatiotemporally controlled release of PDA NPs, which effectively attenuated ROS-mediated inflammation. Besides, the study’s findings demonstrated that PDA@Exo not only promoted osteogenesis but also regulated cartilage metabolism and modulated macrophage polarization.

Fiber Membrane Systems

In fiber membrane systems, both natural polymers, such as gelatin and chitosan, and synthetic polymers, such as polycaprolactone and polylactic acid-co-glycolic acid, are frequently processed using electrospinning technology to create nano-to-micrometer fiber networks. These networks exhibit a remarkably high specific surface area and a biomimetic extracellular matrix morphology, which enhances cell adhesion and proliferation. Exosomes can be incorporated directly into the spinning solution or affixed to the fiber surface via physical adsorption or covalent binding. The release behavior of exosomes is modulated by the fiber diameter, orientation, packing density, and material degradation rate. Fibrous membranes, characterized by their flexibility, are commonly employed to encase bone grafts, fill irregular bone defects, and serve as barrier membranes to guide bone regeneration. A significant challenge lies in balancing the superior load-release properties afforded by the high specific surface area with the structural integrity required for the membrane to maintain its shape and position within complex three-dimensional bone defects.

Zha et al. constructed a coaxial electrospinning nanofiber membrane with polylactic acid (PLA) as a hydrophobic core and chitosan (CS) as a hydrophilic shell by coaxial electrospinning technology. PLA can provide mechanical support, and CS can provide rich modification sites. As a biomimetic extracellular matrix, the fiber membrane not only has a high loading capacity and controlled release characteristics but also can stably bind exosomes to avoid their diffusion, thereby improving local retention efficiency. At the same time, its nanofiber structure can provide a large cell contact area, which is conducive to nutrient transport and cell adhesion and proliferation. Moreover, the mechanical matching and wettability optimization between CS and PLA enhance the biocompatibility, which can cooperate with the high transfection efficiency of exosomes to continuously release the VEGF gene, thereby effectively promoting vascularization and osteogenesis.

In summary, the engineering strategy of exosomes and their delivery systems should be meticulously aligned with the pathological characteristics and anatomical requirements of the target orthopedic conditions. It is essential to evaluate the advantages and disadvantages of various systems concerning drug loading capacity, release controllability, mechanical properties, biocompatibility, and clinical application convenience in order to optimize the therapeutic efficacy of exosomes. Furthermore, the profound synergy between engineered exosomes and biomaterial-based delivery systems offers innovative solutions to surmount the delivery challenges specific to orthopedic diseases through multilevel functional integration and spatiotemporal coupling mechanisms.

Application of Exosomes in Orthopedic Diseases

Orthopedic diseases present distinct challenges to therapeutic strategies owing to their intricate microenvironment and restricted capacity for tissue regeneration. Nanoscale exosomes, particularly when modified or utilized as biomaterials, have demonstrated potential in augmenting repair mechanisms related to complex degenerative conditions impacting bone and cartilage. It is important to acknowledge that the strategies for applying and designing exosomes exhibit significant specificity depending on the disease context.

In the case of traumatic bone injury, the primary objective is to expedite the repair and regeneration of bone defects. This involves strategies that focus on selecting or engineering exosome sources enriched with potent pro-angiogenic and osteogenic differentiation factors and precisely targeting these exosomes to the injury site using optimized delivery systems to enhance new bone formation and vascularization. Conversely, in osteoarthritis, the emphasis shifts toward cartilage protection, inflammation inhibition, and degeneration delay. The design approach frequently involves utilizing exosomes derived from specific cells, which are either naturally enriched or engineered to contain anti-inflammatory factors and molecules related to cartilage matrix synthesis. This aims to modulate the inflammatory microenvironment within the joint cavity and promote chondrocyte anabolism. In the context of rheumatoid arthritis, a systemic autoimmune disorder, the central strategy revolves around immune regulation and inhibition of synovial hyperplasia. The design of exosomes typically prioritizes their immunomodulatory properties, which may originate from regulatory immune cells or be engineered to express immunosuppressive molecules at high levels. The objective is to target hyperactive immune cells and synovial fibroblasts, inhibit their abnormal activation and the release of proinflammatory factors, and restore immune homeostasis. In the context of spinal cord injury, the challenge is the complex environment associated with nerve regeneration disorders. The primary strategy involves crossing the blood-spinal cord barrier and inhibiting glial scar formation. The design approach often involves selecting exosomes with neurotrophic properties, axonal growth promotion, and antiapoptotic capabilities. These exosomes are frequently combined with biological material scaffolds or subjected to physical and chemical modifications to enhance their penetration and retention time in the injured core area, with the aim of creating a microenvironment conducive to nerve axon regeneration and functional recovery.

Consequently, the selection of exosome sources, content engineering, and optimization of delivery methods must be meticulously aligned with the pathological mechanisms and therapeutic end points of specific diseases (Table ).

2. Comparative Design Framework for Exosome-Based Therapies in Skeletal Disorders.

disease type	core pathological features	engineering strategies	delivery systems	key case studies & innovations	references
traumatic bone defect	vascular injury	•targeting: integrin peptides for bone homing	porous scaffolds (HA/Ti alloy)	metabolic engineering + click chemistry-modified bone-targeting peptides → 150% ↑ bone accumulation	,
	inflammatory imbalance	•functional enhancement: co-loading VEGF + BMP-2 for angiogenic-osteogenic coupling	self-healing hydrogels
	large-scale defects	•immunomodulation: M2-Exos to suppress inflammation
osteoarthritis (OA)	cartilage degradation	•targeting: CAP peptides for cartilage penetration	thermosensitive hydrogels	CRISPR/Cas9 delivery of FGF18 gene + chondrocyte targeting → synergistic gene editing and lubrication	,
	chronic synovitis	•anticatabolism: siRNA silencing MMP13	microneedle arrays
	penetration barriers	•metabolic regulation: ECM synthesis promotion
rheumatoid arthritis (RA)	autoimmune attack	•immune tolerance: IL-10/TGF-β delivery for M2 polarization	systemic administration	MMP-responsive PEG-cP exosomes → 152% ↑ accumulation in inflamed joints	,
	synovial hyperplasia	•smart response: MMP-activatable PEG deshielding	local fibrin hydrogels
	Th17/Treg imbalance	•combo therapy: immunosuppressant loading
spinal cord injury (SCI)	neuroinflammation	•anti-inflammatory repair: M2-Exos for microglial polarization	conductive hydrogels	conductive hydrogel integrated M2-Exos → activates Ca2+-PTEN/AKT/mTOR axis for neural regeneration	,
	glial scar	•gene regulation: PTEN-silencing siRNA for axonal growth	intranasal delivery
	axonal regeneration failure	•barrier repair: endothelial targeting

Traumatic Bone Defect

Selection Principle and Design Idea of Exosomes

Traumatic bone defects are frequently associated with localized vascular damage, disruption of the inflammatory microenvironment, and discontinuity of the bone matrix. However, bone with large defects is unable to heal spontaneously because it exceeds the intrinsic regeneration capacity of itself, , generally requiring surgical intervention to fill the defect site with natural or synthetic bone grafts to restore bone integrity. To better promote in situ bone regeneration, exosomes are increasingly utilized in bone regeneration, along with seed cells and biomaterials, , over cell-based therapies due to their noncytotoxic nature, low immunogenicity, nontumorigenicity, high circulation stability, and excellent biocompatibility − (Table ).

3. Application of Exosomes and Engineered Exosomes in Traumatic Bone Defect.

treated disease	year	exosome source	modification	signaling pathway/molecule	in vitro	in vivo	animal model	ref
traumatic bone defect	2024	MSCs	NGF stimulates MSCs to produce exosomes	MAPK and PI3K/Akt pathway	N-Exos promoted cellular function and neurotrophic	N-Exos-stimulated neural cells enhanced osteogenesis	rats with femur critical-size defects
					effects of neural cells
		NSCs	Exos serve as carriers for metabolic modulators	PLIN5	NSC-Exo supported cell survival, bone formation, blood vessel growth, and balanced lipid metabolism within cells	endogenous PLIN5 accelerated callus growth and promoted angiogenesis-osteogenesis coupling	the distraction osteogenesis model
	2023	Schwann cells	SC-Exos and BMSCs were incorporated into a GelMA and SilMA hybrid hydrogel bioink	let-7c-5p/TGF-β	SC-Exos enhanced the osteogenic differentiation of BMSCs, with the effect increasing as SC-Exos concentration rose	the SC-Exos@G/S group demonstrated the most effective bone formation, nearly filling the defect area with new bone tissue	critical-sized rat calvarial full-thickness defect
		hAdMSCs	Exos containing VEGF-A and BMP-2 mRNAs were encapsulated in a custom injectable PEGSA hydrogel	TGF-β/VEGF	The t-sEVs within the PEGS-A hydrogel cage exhibited the greatest mineral deposition and enhanced angiogenesis	the PEGS-A/sEVs hydrogel group exhibited extensive new trabecular or lamellar bone, with the highest concentration of vessels and OCN-positive areas	rats with femur critical-size defects
		BMSCs	the exos were loaded into 3D hydroxyapatite (HA) scaffolds	PTEN/AKT pathway	3D/H-Exos promote proliferation, migration, and tube formation of hUVECs	3D/HA-Exos increased newly formed bone tissues and blood, and the trainer fibers formed were more mature	critical-sized rat calvarial full-thickness defect
	2022	SHED	hypoxic preconditioned H-Exo was applied to injectable porous PLGA microspheres coated with bioinspired polydopamine	VEFG	H-Exo promoting cell homing, osteogenesis and angiogenesis	the PMS–PDA + H-Exo group exhibited a collagen-rich ECM and substantial, highly vascularized new bone tissue in the defect area	critical-sized rat calvarial full-thickness defect
		MSC	Exos combined with PEI was developed as a novel gene vector (EP/D NPs)	BMP2	EP/D NPs promoted angiogenesis and osteogenic differentiation using the RTP801 promoter, while reducing PEI-induced inflammation	EP/D NPs enhance bone healing by stimulating new bone and blood vessel growth while preventing inflammation and fibrous sac formation	rabbit femoral condyle defect models
	2021	BMSC	Creating a lyophilized BMSC-OI-exo delivery system on a hierarchical MBG scaffold	let-7a-5p, let-7c-5p, miR-328a-5p and miR-31a-5p	BMSC-OI-exo exhibited optimal osteoinductivity and could significantly promote cell migration at low concentration	the scaffold-loaded exosomes initially released quickly and then transitioned to a steady slow-release, resulting in significant new bone formation	critical-sized rat calvarial full-thickness defect
				BMP/Smad pathway
		AMSC	Exos were coated on SF scaffolds		exosome-coated SF scaffolds promoted the growth and osteogenic differentiation of hBMSCs	Exo-SF-BMSC scaffolds enhance the production of collagenous tissues and regeneration of bone-like tissue	critical-sized rat calvarial full-thickness defect
		ATDC5	VEGF plasmid-loaded exosomes were incorporated into 3D-printed scaffolds modified with the exosomal anchor peptide CP05	VEGF	Exosomes enhanced osteogenesis ability	numerous new bone tissues, blood vessels, and mature collagen fibers were observed	rat radial bone defect model
		MSC	MSCs are employed to functionalize three-dimensional printed titanium alloy scaffolds	PI3K/Akt and MAPK	the EXO-D10 and EXO-D15 groups showed increased levels of osteo-specific markers (Runx2, ALP, and OPN), along with higher ALP activity and calcium deposition	the presence of collagen, osteoblasts, and Haversian-like structures indicates that exosome-treated scaffolds enhance bone regeneration	rat radial bone defect model
	2020	ATDC5	The complex of exosome-VEGF165 plasmid was incorporated into the dopamine-modified silk fibroin-polycaprolactone membrane	VEGF	the exosome-VEGF165 plasmid complex significantly boosted VEGF expression and directly influenced osteogenic differentiation	the experimental group showed significantly more new bone growth, better structural integrity, and more CD3-labeled capillaries	critical-sized rat calvarial full-thickness defect

The effective repair of bone defects necessitates not only the rapid filling of the bone void but also the reconstruction of the vascular network to ensure an adequate supply of nutrients. Furthermore, modulation of the inflammatory response is essential to foster a pro-regenerative phenotype, thereby facilitating functional bone regeneration. The selection of cargo transported by exosomes must be meticulously tailored to meet the specific demands of the pathological environment. Cytokines with anti-inflammatory properties can mitigate the inhibitory effects of excessive inflammation on the osteogenic process, while bone-promoting factors and functional microRNAs can activate osteogenic differentiation pathways and regulate the differentiation trajectory of mesenchymal stem cells (MSCs) to enhance osteogenic potential.

To optimize the targeting and efficacy of exosomes in relation to the specific characteristics of the pathological microenvironment of traumatic defects, further modifications are necessary. For instance, surface modification with integrin peptides can enhance the homing capability of exosomes to bone defect sites, while chemical modifications can improve their stability in inflammatory environments. Throughout the modification process, it is crucial to adhere to core principles that preserve the structural integrity of the exosome membrane and maintain the functionality of bioactive molecules, thereby preventing any loss of biological activity due to excessive modification. Furthermore, the design of biomaterials and delivery systems should aim to achieve synergistic effects. Biomaterials should exhibit excellent biocompatibility and osteoconductivity, exemplified by composite materials such as hydroxyapatite and collagen, which mimic the structure of the natural bone matrix and function as scaffolds for bone regeneration (Figure ).

3.

Mechanisms of engineered exosomes employed in treating traumatic bone defects, including pretreated precursor cells, surface modifications, hydrogels, and scaffolds. BMP2, bone morphogenetic protein-2; Lipo, liposome; NGF, nerve growth factor; Sep, Sephin1; HA, hyaluronic acid; HA-ADH, ADH-grafted HA; HA-CHO, aldehyde-modified HA; and ERS, endoplasmic reticulum stress.

Loading Cargo to Promote Bone Regeneration and Inhibit Inflammation

In order to promote cell regeneration and tissue repair, BMSC-derived exosomes (BMSC-Exos) enhance osteogenesis in various fracture models, with miRNA and lncRNA within the exosomes playing a crucial role. − Zhang et al. conducted a study in which bone marrow mesenchymal stem cells (BMMSCs) carrying bone morphogenetic protein 2 (BMP-2) were locally injected into a rat bone defect model. The treatment group exhibited a significant enhancement in bone regeneration 20 weeks postoperation. Quantitative analysis using microcomputed tomography (micro-CT) revealed a 2.5-fold increase in bone volume fraction (BV/TV) and a 3.2-fold increase in imaging scores relative to the control group. Histological examination further confirmed that the newly formed woven bone successfully bridged the fracture site. Huang et al. demonstrated that the overexpression of exosomal miR-19b led to a 40% increase in mineralized nodule area and a 1.5-fold enhancement in alkaline phosphatase (ALP) activity. In an in vivo mouse model of femoral fracture, the local administration of exosomes derived from bone marrow stromal cells (BMSCs) carrying miR-19b resulted in a 35% augmentation in callus area and a 50% increase in the proportion of mineralized callus at 4 weeks, as verified by X-ray staining. Besides miRNA, lncRNA also showed some osteogenic potential. Wang et al. reported that exosomal lncRNA-H19 reversed the inhibition of osteogenesis via the miR-467/HoxA10 axis. In their animal model study, the group receiving exosome treatment exhibited a 34.2% reduction in callus width at 3 weeks postfracture, a 28.7% enhancement in bone mineral density (BMD) at 6 weeks, and a 40.5% improvement in maximum load capacity during the three-point bending test. Moreover, exosomes are able to regulate other cells that can help bone regeneration, such as adipose-derived stem cells (ADSCs), endothelial progenitor cells (EPCs), and neural stem cells (NSCs). , Also, many other cells also derive exosomes that indeed have the ability to regulate bone regeneration.

In addition, for the requirement of vascular repair in traumatic bone defects, exosomes should carry nucleic acids that regulate angiogenesis. Chen et al. administered TREMD-Exos encapsulated miR-142–3p at the fracture site, which were effectively internalized by both bone marrow-derived mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cells (HUVECs). Microcomputed tomography (micro-CT) analysis revealed an approximate 40% enhancement in callus volume (BV/TV) and a significant increase in bone mineral density (BMD). Furthermore, CD31 immunohistochemistry and laser speckle contrast imaging (LSCI) demonstrated a 1.9-fold augmentation in vascular density within the fracture region. The lncRNA-H19 also can promote angiogenesis as well as osteogenesis via the Angpt1/Tie2-NO signaling pathway. Behera et al. conducted an experiment in which they administered MSC-derived exosomes carrying lncRNA-H19 into mice with bone defects, resulting in a notable enhancement of bone microstructure in CBS heterozygous mice. Microcomputed tomography (micro-CT) analysis revealed a 35% increase in bone mineral density (BMD), a 42% increase in bone volume fraction (BV/TV), and a 30% increase in trabecular bone number (Tb.N). Additionally, vascularization was significantly enhanced, as evidenced by a 2.3-fold increase in hemoglobin content within Matrigel plugs and the restoration of blood perfusion in the lower limbs.

Modified Exosomes Increased Therapeutic Efficacy

Enhancements in the extraction method and pretreatment of blast cells have substantially increased the production and targeted delivery efficiency of functional exosomes, which means increasing their “quantity”. Zhong et al. employed the continuous extrusion technique for the preparation of exosomes, which not only effectively regulated the size of the exosomes to enhance their uniformity but also significantly increased the yield. The exosome analogues produced via this method exhibit properties and functions comparable to those of exosomes extracted by using traditional methods. However, the yield is 10-fold greater, demonstrating a clear advantage over conventional extraction techniques. Furthermore, enhancing both the abundance and efficacy of therapeutic agents encapsulated within individual exosomes constitutes an additional strategy to augment the “quantity” of exosomes. Specifically, Liang employed the layer-by-layer self-assembly technique to modify the surface of BMSC-derived exosomes using positively charged polyethylenimine, thereby creating a layered structure. This approach facilitated the electrostatic binding of negatively charged nucleic acids to the BMSC-derived exosomes, significantly improving the efficiency of exosome-mediated delivery of large nucleic acids. Wu et al. investigated the effects of treating BMSCs with 50 μg/mL of Fe₃O₄ in conjunction with a 100 mT static magnetic field to augment exosome production and therapeutic efficacy. Their findings indicated that the exosome yield in the combined treatment group was 40% greater than that of the control group. Additionally, the enrichment of miR-1260a in the combined treatment group was 4-fold higher compared to the control. Mechanistically, miR-1260a targets HDAC7 and COL4A2, thereby promoting bone regeneration and angiogenesis.

Once adequate therapeutic doses are achieved, the subsequent crucial challenge involves ensuring efficient and specific enrichment of these exosomes within the target lesion area. Following systemic administration, exosomes frequently encounter obstacles such as nonspecific distribution, rapid clearance, and inadequate penetration within the complex pathological microenvironment. These issues lead to a significantly lower effective dose at the bone defect site than anticipated. Consequently, the active engineering of exosomes to target the microenvironment or specific cells associated with bone defects has become an essential strategy to enhance their therapeutic efficacy. Chen et al. investigated the modification of functional components in secreted exosomes (3D-Exos) through the three-dimensional culturing of human adipose-derived stem cells (hADSCs) to form tissue-like spheres. This process induced transcriptomic reprogramming, activating skeletal developmental pathways such as Ras/Wnt and downregulating cell-cycle genes. As a result, 3D-Exo exhibited a distinct miRNA profile, characterized by the significant upregulation of 12 miRNAs associated with bone formation, including miR-3648, and the downregulation of inhibitory miRNAs such as let-7i-5p and miR-29b-3p. This reprogramming enhanced the affinity of exosomes for bone tissue. In vivo distribution experiments demonstrated that the accumulation of 3D-Exo in bone tissues, including limb bones and the spine, was markedly higher than in the liver and other organs. Furthermore, the peak fluorescence intensity of 3D-Exo in skull defects was 4.5 times greater than that of exosomes derived from traditional two-dimensional cultures (2D-Exo).

Engineered exosome technology is dedicated not only to enhancing “quantity” but also to fundamentally improving “quality”. This qualitative enhancement encompasses two critical dimensions. First, it substantially mitigates the potential toxicity risk associated with exosomes, thereby enhancing the safety of clinical applications. In a study by Li et al., liposome-mediated BMP2 gene transfection was applied to human mesenchymal stem cells (hMSCs), allowing the transfected cells themselves to bear the liposome-induced cytotoxicity as the “factory”. Consequently, the secreted exosomes (MSC-BMP2-Exo) exhibited no biological toxicity, as evidenced by a cell survival rate exceeding 95% in the CCK-8 assay. Furthermore, BMP2 gene transfection resulted in a 3.5-fold increase in BMP2 mRNA expression in hMSCs and significantly facilitated bone regeneration by activating the BMP2/Smad signaling pathway.

Another critical facet of “quality improvement” involves endowing exosomes with the intrinsic capability to navigate and address complex pathological environments. This enhancement enables them to precisely target multiple interconnected tissues or pathological pathways simultaneously, thereby eliciting a synergistic reparative effect. This advancement is particularly crucial in the intricate context of traumatic bone defects, which involve multiple tissue injuries and regeneration disorders. The pivotal “quality” leap lies in employing engineering strategies to design multifunctional exosomes capable of facilitating vascular-bone coupling repair or neural-vascular-bone coupling regeneration. Such advancements aim to achieve more comprehensive and efficient tissue regeneration. In a study by Lian et al., adipose-derived mesenchymal stem cells (ADMSCs) were pretreated with nerve growth factors (NGF) for 48 h to induce the secretion of functional exosomes (N-Exos). NGF significantly reprogrammed the exosomal miRNA expression profile of ADMSCs by activating the TrkA receptor and initiating the intracellular MAPK/PI3K-Akt signaling pathway. This resulted in the upregulation of 12 key miRNAs, including miR-195-5p, let-7a-5p, and miR-29b-3p, by more than 2-fold. Upon delivery of these enriched miRNAs to target cells via N-Exos, a robust multitissue synergistic repair capability was observed. At the neural level, N-Exos significantly enhanced Schwann cell migration and stimulated a 1.8- to 2.3-fold increase in the secretion of neurotrophins BDNF and NT-3. Concurrently, N-Exos markedly enhanced the axonal outgrowth of dorsal root ganglion (DRG) neurons, which was associated with a 4.1-fold upregulation in the expression of the neuropeptide calcitonin gene-related peptide (CGRP). In terms of vascular development, the density of CD31⁺ vessels was 4.5 times greater than that observed in the control group. Regarding bone regeneration, the conditioned medium from N-Exos-treated neuronal cells significantly promoted the osteogenic differentiation of bone marrow stromal cells (BMSCs). This was evidenced by a 5.7-fold increase in the expression of key osteogenic genes, namely RUNX2 and osteocalcin (OCN), a 3.2-fold augmentation in the in vitro mineralization area, and an increase in the new bone volume fraction (BV/TV) to 38.7%. Wang et al. employed ex vivo periodic tensile stress (TS) to pretreat bone marrow endothelial cells (BMECs) and subsequently harvested their exosomes for injection to address bone defects. TS facilitates “vascular-osteogenic coupling” by reprogramming standard BMECs into H-type endothelial cells (THECs) through the activation of the YAP/TAZ-Notch signaling pathway. THECs exhibit an elevated level of expression of VEGFR2/PDGF-B and secrete increased levels of FGF-1 and TGF-β, thereby demonstrating enhanced angiogenic and osteogenic induction capabilities. Five weeks postinjection, the vascularized bone volume proportion reached 28%, a significant increase compared to the control group’s 9%.

Material Integration Expands the Clinical Application of Exosomes

Higher Targeting Ability and Controlled Release Behavior

Using exosomes directly to treat bone defects faces challenges like transient release and excessively high concentrations in circulation. Many studies have attempted to integrate exosomes with other biomaterials into composites to achieve targeting delivery and controlled release of exosomes. ,−

Building on the inherent properties of hyaluronic acid (HA) such as excellent biocompatibility, ease of modification, and hydrogel formation, Chen et al. further modified HA into HA-ADH and OHA-Q to enhance its antibacterial and hemostatic capabilities. The incorporation of stromal-cell-derived factor 1-alpha (SDF-1α) and exosomes from M2 macrophages with the modified HA hydrogel demonstrated significant efficacy in repairing traumatic bone defects. The hydrogel’s porous architecture facilitates efficient substance exchange and exosome release, while the positively charged modified quaternary ammonium group effectively inhibits bacterial proliferation. Moreover, this quaternary ammonium group enhances platelet aggregation, and the hydrogel’s high swelling rate allows for rapid absorption of blood, thereby promoting hemostasis. Subsequent experiments revealed 40% and 60% increases in bone volume fraction (BV/TV) at 14 and 21 days, respectively, compared to the control group, alongside a reduction in fracture gap and enhanced angiogenesis. Regarding antibacterial efficacy, the hydrogel at a 4% concentration achieved a clearance rate exceeding 95% against methicillin-resistant Staphylococcus aureus (MRSA) and over 99% against Staphylococcus aureus and Escherichia coli. In terms of hemostatic performance, blood loss within 60 s was significantly reduced compared to the control group. Wang et al. engineered a hydrogel composed of dental pulp stem cell-derived exosomes (CY@D-Exos) encapsulated with the natural derivative of adenosine, cordycepin, utilizing intermittent ultrasonic shock. These exosomes concurrently restore the viability of aged BMSCs and endothelial cells, thereby significantly enhancing bone regeneration and angiogenesis in these aged subjects. The CY@D-Exos were further incorporated into the GelMA hydrogel matrix. This incorporation leveraged the hydrogel’s sustained release properties, enabling a prolonged and concentrated release of exosomes at the target area, thereby extending their therapeutic action and increasing their local concentration. Xu et al. anchored exosomes to a self-healing hydrogel composed of guanidine-hyaluronic acid (HA-G) and silica-rich nanoclay, which is characterized by its self-healing and injectable properties, enabling it to conform to irregularly shaped defects and facilitate bone tissue healing. To ensure the sustained release of EMs, the HA component was functionalized as HA-G-DP by incorporating pyridyldithiol (DP) groups. Additionally, the EMs were modified with thiol groups to enable binding to the hyaluronic polymer chain via degradable disulfide bonds. These EMs integrated with functional hydrogel significantly enhanced bone regeneration at the defect.

Synergistic Repair of Vascular and Muscular Tissues

Li et al. isolated derivatives containing miR-21-5p from exosomes of ADSCs that had been preconditioned under hypoxic conditions. Following encapsulation in GelMA, the exosome derivative was demonstrated to specifically target SPRY1 in hUVECs, thereby activating the PI3K/AKT signaling pathway. This activation facilitated localized type H angiogenesis and bone regeneration in vivo. Ma et al. utilized electroporation to deliver a mixture of BMP-2 and VEGF-A expression plasmids into AMSCs and then collected the exosomes containing BMP-2 mRNAs and VEGF-A mRNAs. Subsequently, they encapsulated the exosomes within an injectable hydrogel cage composed of PEGylated poly­(glycerol sebacate) acrylate (PEGS-A). This exosome-functionalized hydrogel could rapidly solidify at body temperature and allow for controlled exosome release. Notably, the PEGS-A hydrogel was synthesized through a thiol-Michael addition click reaction loading with exosomes. This exosome-functionalized hydrogel effectively reduced the risk of membrane disruption and therapeutic RNA degradation and then facilitated angiogenic-osteogenic coupled bone regeneration. Pan et al. designed injectable hydrogel microspheres based on hyperbranched polyethylene glycol diacrylate and sulfhydryl-modified HA, with RGD grafted onto the surface to enhance cell adhesion. BMSC-Exos overexpressing miR-29a were incorporated into these hydrogel microspheres to promote osteogenesis and angiogenesis. Lin et al. utilized SF and PCL to fabricate a nanofiber film via electrospinning technology. This electrospun film was further functionalized with polydopamine (PDA) to enhance its adhesion properties. Simultaneously, they employed electroporation to introduce VEGF165 plasmids into exosomes derived from the chondroprogenitor cell line (ATDC5), which were subsequently loaded onto the electrospun film. This composite film was capable of directly inducing osteogenic differentiation of mouse osteogenic precursor cells (MC3T3-E1) and promoting angiogenesis and osteogenesis. Gao et al. employed a hypoxic preconditioning approach on stem cells from human exfoliated deciduous teeth (SHEDs) to enhance the secretion of exosomes and osteogenic and angiogenic capabilities. Concurrently, they designed injectable porous poly­(lactic-co-glycolic acid) (PLGA) microspheres with the PDA coating. These microspheres are capable of effectively adsorbing and controllably releasing exosomes. The local application of these microspheres in calvarial defects of rats significantly enhanced vascularized bone regeneration.

Jin et al. designed a polycaprolactone (PCL)-based asymmetric bilayer scaffold to treat muscle injuries associated with bone defects. The oriented fiber layer guides myofibroblast orientation and myogenic differentiation, while the random fiber layer promotes BMSC differentiation. The scaffold was positively charged via surface chemical modification with PEI, enabling it to bind BMSC-Exos to exert angiogenic and immunomodulatory effects.

Composition Simulation and Mechanical Support

Several studies have combined exosomes with bioceramics, metals, and ECM to form composite materials. ,− , Due to the similarity in chemical composition between bioceramics and the inorganic matrix of bone, bioceramics exhibit exceptional osteoconductivity and bioactivity. They are capable of forming chemical bonds with host bone tissue, thereby providing a conducive interface for osteoblast attachment and proliferation, which facilitates new bone ingrowth. Exosomes sourced from ADSCs and BMSCs under standard culture conditions and under OI conditions were compared. The most effective osteogenic exosomes (BMSC-OI-Exos) were identified and then embedded into the micropores of mesoporous bioactive glass (MBG) through lyophilization, creating an exosome-functionalized scaffold. The porous structure of MBG can keep exosomes biologically active and aid in their continuous release. The scaffold demonstrated remarkable bone repair capabilities through the regulation of Bmpr2/Acvr2b competitive receptor activation via the Smad pathway by exosomal miRNAs like let-7a-5p, let-7c-5p, miR-328a-5p, and miR-31a-5p.

Metal materials, characterized by their high elastic modulus and mechanical strength, offer immediate and stable mechanical support for bone defects, particularly in load-bearing regions. They effectively resist movement-induced loads and prevent collapse of the defect area. Furthermore, their inherent corrosion resistance and long-term biocompatibility contribute to structural stability and, when subjected to surface modification, enhance osseointegration capabilities. Kang et al. used Mg²⁺ and gallic acid (GA) to synthesize Mg-GA metal–organic framework (MOF). Subsequently, PLGA/Mg-GA MOF composite scaffolds were prepared by electrospinning using a polymer blend of PLGA and Mg-GA MOF, and then, human ADSC-derived exosomes were incorporated onto the scaffolds. These modified scaffolds are capable of slowly releasing exosomes, Mg²⁺ and GA, thereby concurrently promoting osteogenesis, angiogenesis, and exhibiting anti-inflammatory effects.

Exosomes, functioning as natural agents of intercellular communication, have shown considerable promise in the repair of bones. However, their use in clinical settings is limited by issues such as temporary release and increased systemic levels. By incorporating exosomes with both natural and synthetic polymers along with other biomaterials such as bioceramics, metals, and ECM components, researchers can achieve controlled and sustained exosome release, targeted delivery, and modulation of the local microenvironment, thereby enhancing therapeutic outcomes. Natural polymers, such as HA and SF, offer excellent biocompatibility and biodegradability, facilitating the effective delivery of exosomes while promoting bone healing. Similarly, synthetic polymers such as GelMA and PLGA provide greater control over mechanical properties and degradation rates, so the exosomes can be released in a regulated manner and the stability of the drug they carry is stabilized. When combined with bioceramics and metals, such as hydroxyapatite or titanium alloy, these composite materials not only support bone regeneration but also enhance angiogenesis and immune modulation, which are key factors in the success of bone repair.

Osteoarthritis

Selection Principle and Design Idea of Exosomes

OA, the prevalent type of arthritis, is marked by joint pain and functional impairment. The pathological environment of OA is characterized by a dynamic imbalance affecting the entire joint organ. Chondrocytes exhibit a reduced number, functional decline, diminished synthesis capacity, and accelerated senescence and apoptosis. This results in significant degradation of the extracellular matrix, particularly collagen II and proteoglycans, such as aggrecan, along with abnormally elevated activities of matrix metalloproteinases and aggrecanases. Concurrently, chronic low-grade inflammation pervades the joint cavity, with synovial tissue becoming activated to release proinflammatory factors. Damaged chondrocytes also contribute to the inflammatory cycle, perpetuating a deleterious process termed “inflammatory senescence”. The subchondral bone undergoes abnormal remodeling, evidenced by sclerosis of the bone plate, destruction of the trabecular structure, microfractures, and cystic degeneration. This is accompanied by aberrant blood vessels and nerves infiltrating from the bone end into the degenerative cartilage area, exacerbating cartilage destruction.

Exosomes can directly influence damaged chondrocytes by sending signals that encourage their growth and survival, prevent cell death, boost the production of essential matrix components, and effectively suppress the activity of enzymes that degrade the matrix, such as MMPs and ADAMTS, in an attempt to counteract catabolic processes. In terms of inflammation control, exosomes serve as key regulators, capable of reducing the production of pro-inflammatory factors by synovium cells, macrophages, and chondrocytes, while enhancing the secretion of anti-inflammatory factors, thus disrupting the cycle of chronic inflammation. By targeting the abnormal growth of blood vessels and nerves from the subchondral bone to the cartilage layer, exosomes can inhibit the vascular endothelial growth factor pathway by delivering specific antiangiogenic molecules, decrease pathological angiogenesis, and potentially regulate the expression of factors related to nerve growth to alleviate pain and abnormal nerve function , (Figure ).

4.

Illustrating the pathological alterations associated with osteoarthritis and the therapeutic application of exosomes in its treatment. ,,,, ROS, reactive oxygen species; MSC, mesenchymal stem cell; IL-1β, interleukin-1beta; NF-κB, nuclear factor kappa-B; STAT, signal transducers and activators of transcription; ASO, antisense oligonucleotides; MMP, metalloproteinase; CAP, chondrocyte affinity peptide; DSPE-PEG, polyethylene glycol-grafted 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; PDA, polydopamine; FGF, fibroblast growth factor; and COI II, type II collagen.

Regulating the Inflammatory Environment and Promoting Cartilage Repair

Exosomes exhibit therapeutic effects on OA through protecting cartilage and alleviating inflammation. MSC-Exos have the capacity to attenuate inflammation and facilitate osteochondral regeneration by modulating the balance between M1 and M2 macrophage phenotypes and decreasing the levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) at the lesion site, thereby reducing the site of injury. MSC^IPFP-Exos, isolated from infrapatellar fat pad MSCs, preserve articular cartilage integrity via dual modulation of apoptosis inhibition and ECM metabolic equilibrium, as evidenced in recent mechanistic investigations. The mechanism may involve miR-100-5p within MSC^IPFP-Exos, which significantly enhances chondrocyte autophagy levels by partially inhibiting the mammalian target of rapamycin (mTOR). Guan et al.’s innovative method of incorporating aldehyde-modified chondroitin sulfate (OCS) and BMSC-Exos into GelMA hydrogels. The amine groups in the GelMA matrix form dynamic Schiff base bonds with the aldehyde moieties on OCS, creating a reversible covalent cross-linking network. This hydrogel’s porous architecture enables controlled release of BMSC-Exos over extended periods. Mechanistically, GMOCS-Exos exert therapeutic effects by modulating M2 macrophage polarization to suppress inflammatory responses, enhancing anabolic metabolism in injured chondrocytes, and inhibiting aberrant bone repair following growth plate injuries. Additionally, the hydrogel system promotes cartilage regeneration through direct ECM replenishment, providing both structural support and bioactive signaling for tissue remodeling. The study by Jiang et al. demonstrated that exosomes derived from human umbilical cord Wharton’s jelly MSCs (hWJ-MSCs) possess pleiotropic regenerative effects. These exosomes significantly enhance migration and proliferation of BMSCs and chondrocytes while simultaneously inducing M2 macrophage polarization. Mechanistically, the acellular cartilage ECM scaffolds loaded with exosomes derived from hWJ-MSCs achieve therapeutic outcomes by suppressing pro-inflammatory responses via macrophage phenotype modulation and promoting osteochondral regeneration. Lu et al. engineered an ROS-responsive bilayer hydrogel system with dual functionalities. The upper layer consists of a poly­(vinyl alcohol) (PVA)-based hydrogel incorporating dendritic cells (DCs) conjugated via biodegradable linkers. This layer undergoes controlled degradation in the presence of elevated reactive oxygen species (ROS) to release DCs so that downregulating inflammatory response and creating an immunosuppressive microenvironment conducive to chondrogenesis The lower layer consists of HA hydrogel encapsulating BMSC-Exos, which can mimic the cartilage ECM and promote cartilage regeneration.

Improving Matrix Degradation and Maintaining a Metabolic Environment

Inhibiting the degradation of the extracellular matrix and preserving a conducive metabolic environment for chondrocytes are effective strategies for safeguarding the integrity of the cartilage matrix and mitigating or preventing its erosion and thinning. Zhang et al. revealed that exosomal CD73 present in MSC-Exos activates adenosine, subsequently influencing the AKT and EPK signaling pathways to promote the regeneration and matrix synthesis of chondrocytes. Zhang and colleagues observed that MSCs treat TMJ OA (TMJ-OA) through relieving pain and inflammation in the early stage. The therapeutic effects of MSCs are partly attributed to the ECM remodeling via chondrocyte proliferation and anabolic activation. At the molecular level, MSC-derived adenosine initiates the parallel AKT/ERK/AMPK signaling pathway, which reciprocally rescues IL-1β-impaired s-glycosaminoglycan­(s-GAG) synthesis, and suppresses IL-1β-driven nitric oxide/MMP13 overproduction, establishing a feedback loop for cartilage homeostasis. Chen et al. uncovered that MSC-Exos can effectively ameliorate mitochondrial dysfunction in degenerating chondrocytes. Based on this, they incorporated cartilage ECM and MSC-Exos into a GelMA solution to create a bioink. This bioink was then used with stereolithography and 3D printing to fabricate composite scaffolds with radial channels. These scaffolds effectively restore cartilage mitochondrial dysfunction, enhance chondrocyte migration, and promote M2 polarization of synovial macrophages, which can synergistically promote osteochondral repair in vivo. Wu et al. demonstrated that exosomes derived from TNF-α-primed IPFP-MSCs exhibit enhanced therapeutic efficacy. Mechanistically, TNF-α preconditioning augmented exosome secretion while endowing the exosomes with protective properties against ECM degradation.

Cartilage Targeting Increased to Improve the Effective Concentration

The rapid clearance of nonspecific exosomes from the joint cavity is facilitated by synovial fluid dynamics and the activity of synovial phagocytes. Furthermore, the avascular and alymphatic nature of cartilage, coupled with its dense extracellular matrix, acts as a formidable barrier to exosome penetration. Consequently, the effective accumulation of natural exosomes in cartilage lesions is challenging, necessitating the development of engineered strategies to enhance the targeting capabilities.

Yan et al. chose Sortase A to conjugate exosomes with chondrocyte affinity peptide (CAP) and subsequently anchored cholesterol-modified antisense oligonucleotide (ASO) targeting MMP13 to the exosome membrane through membrane insertion. Zhang et al. designed positively charged short-length cartilage peptide carriers and anchored them to the exosome surface via simple lipid insertion. Compared to unmodified exosomes, the cationic exosomes were able to penetrate the full thickness of the cartilage, achieving effective drug delivery. In addition to surface charge modification, using tissue-targeting peptides to modify exosomes can also enhance delivery efficiency. Zhang et al. used polyethylene glycol (PEG)-grafted 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG) to link CAP and exosomes loaded with siRNA targeting MMP13 to silence MMP13 in chondrocytes. Chen et al. combined cationic liposomes containing sgRNA targeting FGF18 with engineered exosomes loaded with Cas9 protein and CAP to form hybrid exosomes. These hybrid exosomes were then encapsulated in HAMA hydrogel microspheres through microfluidics and photopolymerization. The microspheres loaded with hybrid exosomes exhibit the ability to promote cartilage regeneration, reduce inflammation, and prevent ECM degradation.

Repairing Physiological Structures and Cooperating to Repair Subchondral Bone

OA is a degenerative condition that affects the entire joint. Abnormalities in the subchondral bone may precede or coincide with degeneration of cartilage. The subchondral bone and cartilage together form a functional unit, necessitating a bidirectional and synergistic approach to repair in order to restore the structural and mechanical integrity of the bone-cartilage interface. JBMSC-Exos demonstrate chondroprotective capacity through phenotype maintenance (via type II collagen promotion) and antisenescence/apoptosis effects (against IL-1β stimulation) in vitro, with lncRNA MEG-3 serving as a key functional component. Further animal experiments confirm their therapeutic potential in OA by attenuating cartilage lesions and abnormal subchondral bone changes. Li et al. developed composite bioinks by integrating decellularized porcine cartilage matrix (DCM) and bone matrix (DBM) into a GelMA hydrogel system containing oxidized hyaluronic acid (OHA) and HA-dopamine conjugate (HA-DA), alongside human adipose-derived stem cell (hADSC)-derived exosomes. The dynamic Schiff base cross-linking between OHA and HA-DA constructs a reversible covalent network, which enhanced cell adhesion through matrix–cell interaction mediation and created a hierarchical porous architecture that facilitates uniform exosome distribution and sustained release. Two kinds of bioinks were used to fabricate a bilayer scaffold by 3D printing, with a DCM hydrogel scaffold in the upper layer and a DBM hydrogel scaffold in the lower layer. The biomimetic hydrogel scaffolds were shown to significantly enhance multiple cellular functions in vitro by promoting adhesion, spreading, migration, proliferation, and multilineage differentiation toward chondrogenic and osteogenic lineages of rat BMSCs. In vivo evaluation in an osteochondral defect rat model further revealed that these scaffolds could substantially accelerate synchronous regeneration of both articular cartilage and subchondral bone tissues. Liu and team noted that both ADSC-derived exosomes stimulated by inflammation and normal ADSC-derived exosomes could promote BMSC proliferation, while miR-27b-3p loaded by the former significantly promoted M2 macrophage differentiation by targeting CSF-1. Both can promote the regeneration of condylar osteochondral defects in rabbit temporomandibular joints in vivo.

Exosomes, especially those from MSCs and specialized sources, offer a promising strategy for OA treatment (Table ). In contrast to conventional treatments for OA, including analgesic therapy and joint arthroplasty, which fail to target the pathological basis of cartilage degeneration, exosome-based therapeutics offer unprecedented opportunities to modulate key cellular and molecular processes underpinning OA pathogenesis, including cartilage repair, inflammation regulation, and tissue regeneration. Exosomes have demonstrated significant potential in promoting chondrocyte proliferation, inhibiting apoptosis, reducing inflammation, and enhancing matrix synthesis, all of which are crucial for reversing the degenerative processes characteristic of OA. Exosomes not only exhibit therapeutic effects in isolated forms but also show considerable promise when integrated with biomaterials such as hydrogels and scaffolds. These composite systems can support sustained delivery of bioactive cargos, maintain local retention, and provide a supportive microenvironment conducive to tissue regeneration. Studies utilizing engineered exosomesmodified with peptides, GFs, or siRNAshave demonstrated increased specificity for cartilage and bone tissues, overcoming some of the challenges related to tissue targeting and delivery efficiency.

4. Application of Exosomes and Engineered Exosomes in OA.

treated disease	year	exosome source	modification	signaling pathway/molecule	in vitro	in vivo	animal model	ref
osteoarthritis	2024	IPFP-MSCs	Exos were obtained after IPFP-MSCs pretreated with TNF-α	PI3K/AKT pathway	TNF-α precondition enhances the EVs secretion of IPFP-MSCs	exosomes obtained following TNF-α pretreatment can ameliorate OA pathological changes and improve gait abnormalities	medial meniscus (DMM)-induced OA mouse model
			synthesizing articular HA-based hydrogel microspheres containing chondrocyte-targeting hybrid exosomes (CAP/FGF18-hyEXO)	PI3K/AKT pathway	CAP/FGF18-hyEXO@HMs promote proliferation, migration, and ECM synthesis of OA chondrocytes	CAP/FGF18-hyEXO@HMs effectively alleviate OA progression	ACLT-induced OA rat model
		Expi293F cells	the exosome surface was modified with CAP via lipid insertion and then loaded internally with siRNA against MMP13.	PI3K/AKT pathway	CAP-Exo/siMMP13 effectively improved IL-1β-induced chondrocyte degeneration	CAP-Exo/siMMP13 groups exhibited uniform distribution of cells, organized arrangement, and dense subchondral bone	ACLT-induced OA rat model
		MSCs	Exos are incorporated into the needle tips of the core–shell MN patch, in conjunction with PDA NPs.	PI3K/Akt/mTOR Pathway	PDA@Exo has the potential to therapeutically influence chondrocyte activity and modulate macrophage polarization.	PDA@Exo treatment enhanced trabecular bone volume, maintained structural separation, and preserved cartilage surface and subchondral bone integrity	ACLT-induced OA rat model
		BMSCs	Exosomes were loaded into GMOCS hydrogels	TGFB1/Nrf2 pathway	the GMOCS-Exos hydrogel promotes chondrocyte repair and proliferation	GMOCS-Exos alleviates cartilage lesions and subchondral bone loss	rat models of OA
	2023	MSCs	the exos derived from fucoidan preconditioned MSCs were isolated from the supernatant	TRAF6 PI3K/AKT/mTOR	F-MSCs-Exo reduce inflammation and M1 polarization, while attenuating IL-1β-induced changes in anabolic and catabolic markers in chondrocytes	F-MSCs-Exo smooth the knee joint surfaces, reduce osteophytes, and effectively alleviate inflammation and ECM degradation	ACLT and medial meniscus resection induced OA rat model
	2022	platelet-rich plasma	Exos were conjugated to thermosensitive hydrogels	Smad2/3, ERK1/2 and p38 signaling pathways	Exo-Gel facilitated the proliferation, migration, and chondrogenic differentiation of mBMSCs, while also inhibiting IL-1β-induced apoptosis and degeneration in chondrocytes	Exo-Gel alleviated STOA and inhibited degeneration of cartilage of subtalar joint	mouse ankle-subtalar OA model
	2021	BMSCs	Exos and the GMOCS mixture solution formed the GMOCS-Exos under ultraviolet irradiation	NF-κB pathway	GMOCS-Exos induce the polarization of RAW264.7 cells and enhance the repair of chondrocytes damaged by IL-1β	GMOCS-Exos enhance growth plate repair, minimize bone bridge formation, regulate macrophage polarization, and promote cartilage matrix formation.	the distal femoral drill-hole growth plate injury model

Furthermore, the ability to manipulate exosome composition through preconditioning or genetic modification adds another layer of potential, improving therapeutic efficacy. For example, preconditioning exosomes with specific cytokines such as TNF-α or fucoidan can enhance their anti-inflammatory properties and support ECM preservation. When integrated with advanced biomaterial-based delivery platforms, including hydrogel-based scaffolds, 3D-printed bioinks, and nanocarrier systems, exosomes are poised to significantly improve outcomes in OA treatment, particularly for patients who cannot benefit from traditional surgical interventions such as joint replacements. The growing body of evidence supporting exosome-based therapies underscores their potential to not only alleviate symptoms of OA but also promote genuine tissue repair and regeneration. However, challenges remain in optimizing exosome production, refining delivery methods, and ensuring safety and efficacy in clinical practice. Moving forward, careful evaluation through clinical trials is necessary to fully assess the impact of exosomes on the OA progression and joint function. Ultimately, exosomes could represent a groundbreaking shift in how OA is treated, offering more personalized, less invasive, and effective options for patients.

Rheumatoid Arthritis

Selection Principle and Design Idea of Exosomes

RA is a systemic autoimmune disorder characterized by bilateral synovial inflammation and progressive cartilage/bone erosion, predominantly manifesting as the polyarticular involvement of peripheral joints. The pathological milieu of rheumatoid arthritis (RA) is characterized by chronic inflammation and aberrant proliferation of synovial tissue, resulting in the formation of a destructive pannus alongside significant dysregulation of the immune system. This dysregulation is evidenced by an imbalance in T cell subsets, notably the excessive activation of proinflammatory Th1 and Th17 cells, coupled with insufficient activity of regulatory T (Treg) cells, which possess suppressive functions. Concurrently, B cells are hyperactivated, leading to the production of autoantibodies, such as rheumatoid factor (RF) and anticitrullinated protein antibody (ACPA), and the subsequent formation of immune complexes. Furthermore, synovial fibroblasts (FLS) exhibit abnormal activation. Collectively, these factors perpetuate inflammatory responses, angiogenesis, cartilage degradation, and bone erosion. Exosomes exhibit multifaceted regulatory effects on the immune system and inflammatory processes. They can modulate overactive immune responses by regulating T cell function and suppressing B cell activation and antibody production. Furthermore, exosomes directly attenuate the inflammatory cytokine storm by significantly reducing levels of key proinflammatory mediators, such as TNF-α, IL-6, and IL-17. Additionally, exosomes target and inhibit the proliferation, migration, and invasion of fibroblast-like synoviocytes (FLS) while promoting the polarization of macrophages toward an anti-inflammatory and reparative phenotype. They also play a role in regulating angiogenesis and maintaining bone metabolism equilibrium. Collectively, these mechanisms enable exosomes to mitigate synovial inflammation, rectify immune dysregulation, and impede the progression of joint destruction , (Figure ).

5.

Pathological alterations in rheumatoid arthritis and the therapeutic application of exosomes in its management. ,, DC, dendritic cells; ICA, icariin; uPB, ultrasmall Prussian blue; TNF-α, tumor necrosis factor; IL-1β, interleukin; Th17, T helper cell 17; Treg, regulatory T cell; and ADSCs, adipose-derived mesenchymal stem cells.

Inhibition of Inflammatory Cells and the Expression of Pro-inflammatory Factors

Owing to the distinct pathological characteristics of rheumatoid arthritis, the management of synovitis and inflammation has emerged as a crucial aspect in the therapeutic approach to this condition. Yan et al. developed ADMSC-derived exosomes loaded with icariin (ADSCs-exo-ICA). This formulation enhanced the water solubility and bioavailability of ICA, inhibited the proliferation of M1-type macrophages by specifically targeting activated macrophages within synovial tissue, and reduced glycolysis through the suppression of the ERK/HIF-1α/GLUT1 signaling pathway, thereby promoting phenotypic transition from pro-inflammatory M1 to reparative M2 macrophages. In a collagen-induced arthritis (CIA) rat model, ADSCs-exo-ICA demonstrated preferential accumulation in inflamed joint tissues, exerting therapeutic effects via suppression of pro-inflammatory cytokines. Li et al. engineered a biomimetic nanodelivery system using M2-type macrophage-derived exosomes (M2 Exo) to codeliver interleukin-10-encoding plasmid DNA (IL-10 pDNA) and betamethasone sodium phosphate (BSP). This combination suppressed pro-inflammatory cytokine secretion such as IL-1β and TNF-α. Additionally, it enhanced the expression of IL-10 cytokines, thereby inducing macrophage phenotypic switching from pro-inflammatory M1 to reparative M2 phenotype. Wang et al. developed engineered exosomes based on M2-Exos, which were modified with both MMP-cleavable PEG and oligolysine. The MMP-cleavable PEG targets inflamed joints in two synergistic ways. On the one hand, owing to the concentration gradient of C–C motif chemokine ligand 2 (CCL2) from peripheral blood to inflamed joints, C–C motif chemokine receptor 2 (CCR2) on M2-Exos mediates targeted homing to inflamed joints via CCL2 binding. Additionally, PEG prolongs the circulation time of M2-Exos, thus synergizing with CCL2-mediated targeted accumulation. On the other hand, elevated MMP levels at the lesion site cause cleavage of PEG, thereby enhancing the accumulation of M2-Exos in inflamed joints. Engineered exosomes mediate anti-inflammatory effects through cationic oligolysine-dependent cfDNA scavenging and M2-Exoinduced macrophage M2 polarization.

Restoring the Homeostatic Equilibrium between Th17 and Treg Cells

The Th17/Treg balance serves as a central component of immune homeostasis in rheumatoid arthritis (RA), and its dysregulation is a fundamental driving force behind joint destruction. By precisely restoring this balance, exosomes have the potential to ameliorate the disruption of immune homeostasis and the progressive exacerbation of inflammation. Wu et al. designated the M2Exo@CuS-CitP-Rapa (M2CPR), serving as a delivery vehicle for ultrafine copper sulfide nanoparticles (CuS NPs), peptide multiepitope autoantigen (CitP), and rapamycin (Rapa). This nanocomposite targets RA-inflamed tissues, releasing CuS NPs, CitP, Rapa, and anti-inflammatory factors. CuS NPs induce cuproptosis in activated T cells, whose remnants are phagocytosed by macrophages, leading to TGF-β secretion. TGF-β and Rapa synergistically convert iDCs into tDCs. tDCs present CitP to naive T cells, promoting their differentiation into Tregs. Tregs produce TGF-β, perpetuating tDC differentiation and establishing a self-sustaining immune tolerance cycle for robust and enduring antigen-specific immune tolerance. Zhang et al. developed a nanoenzymatically modified neutrophil-derived exosome (UPB-Exo) using click chemistry to attach sub-5 nm Prussian blue NPs (uPB) to neutrophil exosome (NEs-Exo) surfaces. This uPB-Exo targets inflammatory synovitis, penetrates cartilage deeply, and ameliorates joint damage by modulating Th17/Treg cell balance.

Inhibiting the Overactivation of B Cells and the Excessive Production of Antibodies

The hyperactivation of B cells and the resultant overproduction of autoantibodies are contributing factors to joint destruction and the chronic nature of rheumatoid arthritis (RA). Exosomes have the capacity to specifically inhibit the pathogenic functions of B cells through the delivery of immunomodulatory molecules while preserving their inherent immunomodulatory capabilities. Rui et al. isolated PD-L1-overexpressing exosomes from olfactory ecto-mesenchymal stem cells (OE-MSCs), which inhibited the PI3K/AKT pathway to reduce T follicular helper (Tfh) cell polarization and mitigate RA pathology. Subsequently, the OE-MSC-Exos were encapsulated within a photo-cross-linked fibroin hydrogel. In a CIA mouse model, composite treatment substantially reduced joint swelling, lowered serum antitype II collagen (anti-CII) autoantibody levels, and provided histological protection to articular cartilage.

The growing body of research on exosome-based therapies for RA offers a highly promising avenue for addressing the complex challenges associated with this debilitating autoimmune disease. Exosomes serve as effective vehicles for delivering anti-inflammatory agents, immune modulators, and regenerative factors, thereby mitigating the inflammatory processes that underpin RA (Table ). Exosomes facilitate macrophage transition from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype, restoring immune balance and reducing destructive inflammation linked to joint damage and functional loss. The therapeutic efficacy of exosomes is further enhanced by various innovative strategies such as surface engineering, chemical modification, and the incorporation of NPs or targeting moieties. These modifications improve the targeting specificity, stability, and bioavailability of exosomes, ensuring that they effectively reach inflamed tissues and deliver their therapeutic cargo. For instance, the use of exosome formulations that codeliver immunomodulatory agents like IL-10, anti-inflammatory cytokines, or small molecules like rapamycin have demonstrated synergistic effects in reducing pro-inflammatory cytokine levels, promoting immune tolerance, and facilitating tissue repair. Additionally, incorporating exosomes with biomaterials like SF hydrogels presents a promising strategy to enhance local exosome retention and create a supportive microenvironment for joint regeneration.

5. Application of Exosomes and Engineered Exosomes in Rheumatoid Arthritis.

treated disease	year	exosome source	modification	signaling pathway/molecule	in vitro	in vivo	animal model	ref
rheumatoid arthritis	2025	M2 macrophages	Exos were loaded with CuS NPs, CitP, and Rapa to create the multifunctional nanocomplex	CuS NP	M2CPR triggers cuproptosis in activated T cells, leading to increased TGF-β secretion by macrophages via phagocytosis	the M2CPR group maintained immune tolerance and had bones structurally akin to normal.	collagen-induced arthritis (CIA) rats
				CitP
				Rapa
				TGF-β
	2024	ADSCs	exosomes loaded with ICA function as drug delivery carriers	ERK/HIF1α/GLUT1 pathway	ADSCs-EXO-ICA effectively suppressed M1 macrophage growth and facilitated their shift to the M2 phenotype by decreasing glycolysis	ADSCs-EXO-ICA diminishes cytokine levels, ameliorates synovitis, and preserves cartilage integrity, thereby mitigating the progression of arthritis	collagen-induced arthritis (CIA) rats
	2023	M2 macrophages	the exosome therapy MEX+cP was developed using MEX as a base, incorporating positively charged oligolysine and MMP-cleavable polyethylene glycol (cP) into its membrane	TLR9 pathway	MEX+cP effectively scavenged cfDNA and modulated the polarization of macrophages	MEX+cP effectively mitigated inflammatory edema and erosion of the articular surface bone	collagen-induced arthritis (CIA) mice and canine
		olfactory ecto-mesenchymal stem cell	the exos encapsulated within the silk fibroin hydrogel underwent in situ photocross-linking	PI3K/AKT pathway	Exos@SFMA demonstrates a robust capacity to recruit BMSCs and suppress T cell activation	Exos@SFMA decreased swelling in both paws, shrank draining lymph nodes, and safeguarded cartilage	collagen-induced arthritis (CIA) mice
	2022	M2 macrophages	the exos encapsulated IL-10 pDNA and BSP, forming M2 Exo/pDNA/BSP nanoparticles	IL-1β, TNF-α	M2 Exo/pDNA/BSP codelivering IL-10 pDNA and BSP facilitate macrophage polarization from M1 to M2	M2 Exo/pDNA/BSP showed relatively mild inflammatory cell infiltration and cartilage erosion	collagen-induced arthritis (CIA) rats
		neutrophil	Exos were engineered with sub-5 nm ultrasmall PBNPs using click chemistry	PI3K/AKT signaling pathway	uPB-Exo has antioxidant properties, safeguards cells from cytokine-induced apoptosis, and modulates inflammation	uPB-Exo can alleviate joint inflammation and prevent cartilage damage	collagen-induced arthritis (CIA) mice
	2021	ADSCs	Exos were isolated through the surface modification of ADSCs utilizing MGE-mediated click chemistry	Wnt, MAPK Hippo and AMPK signaling pathway	DS-EXOs enhanced M1-M2 macrophage polarization, which was hindered by miRNA blockage	DS-EXOs group had the lowest levels of cartilage erosion, neutrophil infiltration, and synovial inflammation	collagen-induced arthritis (CIA) mice

Despite encouraging preclinical outcomes, clinical translation of exosome-based RA therapies faces key challenges: optimizing exosome production, purification, and long-term stability. Elucidating precise therapeutic mechanisms and ensuring safety and efficacy consistency are critical for clinical implementation. Nevertheless, advancing exosome platforms represents a transformative step in RA management, offering targeted, personalized, and minimally invasive alternatives to conventional treatments. Continued research may establish exosomes as a cornerstone of future RA care, reducing inflammation, promoting tissue repair, and improving the patient’s quality of life.

Spinal Cord Injury

Selection Principle and Design Idea of Exosomes

SCI is recognized as a major medical challenge, often leading to severe neurological impairments, including motor and sensory dysfunction. The distinctive pathological milieu associated with spinal cord injury (SCI) is initiated by a primary insult and swiftly progresses into a multifaceted cascade comprising several stages. Initially, the acute phase is characterized by disruption of the blood-spinal cord barrier, hemorrhage, and ischemia, resulting from vascular rupture. This phase is succeeded by a pronounced inflammatory response and excitotoxicity. Subsequently, there is demyelination attributable to ongoing neuronal apoptosis and necrosis, axonal degeneration, and oligodendrocyte demise. In the chronic phase, the formation of a glial scar and the establishment of an inhibitory microenvironment significantly impede nerve regeneration and axonal remyelination.

Exosomes facilitate the delivery of bioactive molecules that mitigate the early inflammatory response by modulating the polarization of microglia and macrophages toward an anti-inflammatory phenotype, while concurrently inhibiting the release of pro-inflammatory factors. Additionally, exosomes contribute to the reduction of apoptosis and necrosis by safeguarding neurons and oligodendrocytes. When integrated with biomaterials, exosomes further enhance angiogenesis and restore the integrity of the blood-spinal cord barrier, thereby ameliorating the local ischemic microenvironment. Moreover, they inhibit the excessive activation of astrocytes and the formation of glial scars. Ultimately, exosomes promote axonal growth and remyelination by providing neurotrophic support. In summary, exosomes are engineered to impede the progression of secondary injury, protect residual neural tissue, and ultimately establish a microenvironment conducive to neural repair and functional recovery , (Figure ).

6.

Exosomes aid in spinal cord injury treatment by promoting nerve regeneration, exerting anti-inflammatory effects, and restoring barrier function. , iNOS, inducible nitric oxide synthase; IL-6, interleukin-6; IL-10, interleukin-10; TNF-α, tumor necrosis factor α; ARG-1, arginase-1; LAMP2, lysosomal associated membrane protein 2; and RGD, Arg-Gly-Asp.

Regulation of Inflammation and Protection of Nerve Cells

Following SCI, it is imperative that treatment be administered promptly to mitigate the progression of tissue damage, which includes the regulation of inflammation and the reduction of neuronal apoptosis. Immune cell influx into spinal cord tissue is driven by dysregulated microglial responses and blood-spinal barrier disruption. These cells secrete proinflammatory or immunomodulatory factors, eliciting inflammatory responses that exacerbate neuronal and axonal damage and impede the repair process. Consequently, it is imperative to modulate the polarization of microglia and macrophages toward an anti-inflammatory phenotype and to suppress the release of pro-inflammatory mediators in order to mitigate the acute inflammatory response during the initial phase.

Xiong et al. demonstrated that regulatory T cell-derived exosomes (Treg-Exos) contain miR-709, which specifically targets the NKAP gene to attenuate microglial pyroptosis and dampen neuroinflammation following SCI. Jiang et al. showed that exosomes from neurons can decrease neuroinflammation by influencing the activation of M1 microglia and A1 astrocytes via the miR-124–3p/MYH9 pathway. Zhu et al. developed a composite patch by combining PLGA-poly­(ethylene oxide) (PEO) nanofibers loaded with methylprednisolone (MP) and an HA hydrogel loaded with SC-Exos. This system promotes M2 macrophage polarization via MP/SC-Exos synergy, attenuating neuroinflammation and inhibiting neuronal apoptosis.

Improvement of the Local Microenvironment of the Injury

Inflammation triggered by SCI releases ROS. Liu et al. demonstrated that ROS can exacerbate neuroinflammation and further increase ROS levels via the MAPK-NFκB P65 signaling pathway, while exosomes derived from dental pulp stem cells can break the cycle between ROS and M1 macrophage polarization, thereby alleviating neuroinflammation. In SCI, ECM loss, like collagen and laminin depletion, and inhibitory microenvironment formation create a neurotoxic milieu Mu et al. designed an injectable fibrin glue incorporated with MSC-Exos, which can comprehensively alleviate the oxidative and inflammatory microenvironment during the acute phase of SCI and provide a matrix for neural tissue growth. Li et al. engineered an HA hydrogel incorporating the laminin-derived peptide PPFLMLLKGSTR to immobilize exosomes from human placental amniotic mesenchymal stem cells (hPAMSC-Exos). Local transplantation of this hydrogel at the injury site can replenish the ECM and reduce inflammation and oxidative stress, thereby comprehensively alleviating the local microenvironment.

Regeneration and Remyelination of Punctured Axons

Damage leads to the disruption of axonal integrity; therefore, the restoration of spinal cord function necessitates the regeneration of axonal extensions by neurons. Zhang et al. designed a dual BSP with two ends that can specifically bind to collagen and exosomes, respectively. They anchored paclitaxel-encapsulated exosomes derived from human umbilical cord mesenchymal stem cells (UCMSCs) into the collagen scaffold via BSP. The composite scaffold can recruit NSCs and enhance nerve regeneration. PTEN inhibits axon regeneration by suppressing mTOR. Shang et al. encapsulated PTEN-targeting siRNA into MSC-Exos through electroporation and subsequently incorporated these engineered exosomes into GelMA hydrogel. This exosome-functionalized hydrogel promotes neuronal regeneration via the PTEN/PI3K/AKT/mTOR signaling pathway. Qin et al. revealed that exosomal miR-34a-5p derived from EGFR⁺ NSCs promotes neurite regrowth by activating the autophagy pathway and improving microtubule stability. Phosphatase and PTEN inhibit axonal growth by downregulating the activity of mTOR. Guo et al. showed that PTEN-targeting siRNA-loaded MSC-Exos enhances axonal regeneration, attenuates neuroinflammation, and mitigates glial scarring. Intranasally administered, these engineered exosomes traverse the blood–brain barrier to specifically target spinal cord lesion sites. Ran et al. employed CP05 to conjugate neuron-targeting and growth-facilitating peptides onto autologous plasma exosomes, engineering these exosomes to enable neuron-specific targeting and axonal regeneration promotion. Zeng et al. conjugated Ile-Lys-Val-Ala-Val (IKVAV) peptide, which can acilitate the survival, proliferation, and differentiation of neural stem cells, onto the surface of M2-Exos via a click chemistry reaction. Intravenous injections of these exosomes can target the lesion site, alleviating inflammation and promoting neural tissue regeneration. Another crucial factor in axonal regeneration is the direction of extension, which is directly associated with the efficacy and quality of neurological functional recovery. Proper directional extension is fundamental for axonal regeneration to establish functional connectivity. Huang et al. designed spiral structure and gradient peptide modification (GS) scaffolds through the gradient covalent attachment of GelMA to IKVAV, subsequently forming them into a helical configuration. This helical architecture offers essential structural support and spatial accommodation, while gradient modification with the IKVAV peptide imparts directional guidance for neuronal and axonal growth. The GS scaffold notably facilitated neural extension beyond the dorsal root ganglion (DRG) via its interlamellar spaces and gradient peptide concentration. Furthermore, MSC-Exos were proficiently incorporated into the GS scaffolds to enhance motor function recovery, diminish glial scar formation, and improve nerve remyelination.

Newly developed axons exhibit considerable fragility and are prone to growth arrest or degeneration as a result of the inhibitory microenvironment present in the injured region. Prompt remyelination offers essential physical protection and metabolic support, thereby facilitating axonal stabilization and maturation. Oligodendrocytes are essential for myelination and axonal functional recovery. He et al. demonstrated that MSC-Exos rich in VGF promote oligodendrocyte development. Consequently, they developed a fibrin gel incorporated with MSC-Exos to enhance recovery from SCI. Vascularization represents a critical therapeutic strategy in SCI management. Li et al. demonstrated that hypoxia-preconditioned human umbilical vein endothelial cell (hUVEC)-derived exosomes promote angiogenesis in MSCs, facilitating a supportive environment for neural regeneration.

Physiological Structural Repair of the Blood-Spinal Cord Barrier

The primary objective, whether it involves promoting regeneration or regulating the microenvironment, is to reconstruct the fundamental physiological defense mechanism of the blood-spinal cord barrier (BSCB). The integrity of the BSCB is essential for maintaining spinal cord homeostasis. It serves a dual function: it acts as a barrier by strictly regulating the transendothelial transport of substances, thereby insulating against peripheral inflammatory factors and toxic agents, and it also provides a stable metabolic and signal transduction environment for neurons and glial cells.

Kong et al. observed that miR-2861 in Treg-Exos maintains the integrity of the BSCB by inhibiting IRAK1. Xie et al. isolated CD146⁺ CD271⁺ UCMSCs and obtained engineered exosomes modified with RGD through transfection. CD146 and CD271 are crucial for BSCB development. Intranasal delivery of RGD-modified exosomes can target endothelial cells in the lesion area of SCI, promoting BSCB repair by regulating the miR-501-5p/MLCK axis. Gao et al. demonstrated that pericyte-derived exosomal miR-210-5p maintains BSCB integrity by inhibiting the JAK1/STAT3 pathway to regulate endothelial cell lipid peroxidation and mitochondrial function. Nakazaki et al. observed that exosomes produced by MSCs were internalized by M2 macrophages at lesion sites, resulting in upregulation of TGF-β receptors and numerous BSCB-associated microvascular proteins, thereby maintaining the stabilization of BSCB. PRP-Exos isolated by Nie et al. improved BSCB integrity by regulating tight junction protein expression, thus limiting inflammatory cell infiltration. Moreover, PRP-Exos modulated M2 microglial polarization through the NF-κB pathway, reducing proinflammatory cytokine production and alleviating neuroinflammation.

Restoration of the Physiological Function of Spinal Cord Conduction

Building upon the successful reconstruction of the critical physiological architecture of the blood-spinal cord barrier (BSCB), exosome therapy further facilitates the repair of spinal cord injuries, ultimately aiming at functional recovery. This recovery is characterized by the restoration of efficient nerve signal transmission and the maintenance of electrophysiological homeostasis. Conductive hydrogels, which align with the electrical and mechanical characteristics of neural tissue, are promising candidates for SCI repair and the reconstitution of electrophysiological functions. Guan et al. developed conductive hydrogels (TP) incorporating M2 microglial exosomes (M2-ExOs) to synergistically enhance electrical signaling postspinal cord injury (SCI). The conductive matrix is composed of tannic acid-cross-linked polypyrrole (PPy), which possesses conductivity compatible with spinal cord tissue and facilitates sustained release of M2-ExOs through reversible hydrogen bonding. The TP components activated voltage-gated calcium channels, elevated intracellular Ca²⁺ levels, and negatively regulated PTEN protein via CaMKII, thereby significantly enhancing the PI3K/AKT/mTOR signaling pathway and promoting axonal regeneration. The BBB score for the TP group increased to 8 points by the eighth week, compared to ≤2 points in the SCI group. Diffusion tensor imaging (DTI) revealed the regeneration of nerve fiber tracts in the injured region, confirming the restoration of electrical signal conduction across the injury site. However, conductive hydrogels may exacerbate early inflammation following SCI. To mitigate these adverse effects, Fan et al. developed a conductive hydrogel containing BMSC-Exos, which exerts immunomodulatory effects through the NF-κB pathway and promotes neuronal and axonal regeneration.

In summary, SCI is a severe condition causing profound motor and sensory dysfunction, significantly impairing the quality of life. Exosomes have shown great potential in mitigating secondary damage after SCI by regulating oxidative stress, inflammation, and apoptosis. They also support neuroregeneration, axonal growth, and remyelination and promote angiogenesis, effectively creating a more conducive environment for spinal cord recovery. Specifically, exosomes can deliver GFs, miRNAs, and other therapeutic molecules to modulate the immune response, enhance vascularization, and replenish the ECM, thus facilitating tissue repair. Moreover, combining exosomes with advanced materials, such as conductive hydrogels, MN arrays, and gradient peptide-modified scaffolds, has further enhanced their therapeutic effects in SCI repair. These innovative carriers not only enable targeted delivery to the injury site but also provide structural support and allow for sustained release of exosomes, amplifying their healing potential (Table ). However, despite the promising outlook, several challenges remain, such as optimizing exosome purification, stability, targeting efficiency, and clinical applicability. Future research should prioritize optimizing exosome production methods, enhancing target specificity, and evaluating therapeutic efficacy while investigating synergistic combinations with other modalities to develop more effective SCI therapeutic strategies.

6. Application of Exosomes and Engineered Exosomes in Spinal Cord Injury.

treated disease	year	exosome source	modification	signaling pathway/molecule	in vitro	in vivo	animal model	ref
spinal cord injury	2024	M2 microglia	Exos were isolated and reversibly bonded to electroconductive hydrogels via hydrogen bonding	PTEN/PI3K/AKT/mTOR pathway	TPME facilitated the M2 polarization of BV2 cells and enhanced axonal outgrowth in NSCs	local injections of hydrogel-encapsulated PRP-Exos increased tight junction protein expression, reduced inflammation, and enhanced neural recovery	rat spinal cord transection model
		platelet-rich plasma	Exos were embedded in PEG/ODEX hydrogel	NF-κB signaling pathway	PRP-Exos enhance tight junction integrity by significantly reducing bEnd.3 cell permeability under hypoxic-hypoglycemic conditions	hypoxia-Exo preserves NP structure and components, thus improving IVDD	SCI mice model
	2023	EGFR+NSCs	MiR-34a-5p was enriched in exosomes	MiR-34a-5p/HDAC6 pathway	EGFR+ NSCs-derived exosomes promoted neural regrowth	EGFR+ NSCs-derived exosomes reduced lesion size and improved dysfunction	spinal cord injured mice
		pericyte	Exos carried miR-210	MiR-210/JAK1/STAT3 axis	Exosomes enhance endothelial cell barrier integrity	Exosomes promote the recovery of motor function and protect the BSCB	SCI mice model
		Treg cells	Exos carried miR-2861	MiR-2861	exosomes promoted the expression of tight junction protein in bEND. Three cells	Exosomes facilitate vascular regeneration and modulate the repair of the BSCB, consequently enhancing motor function	SCI mice model
				IRAK1
		CD146+	Exos with targeted neovascularization function were obtained through gene transfection	MiR-501-5p/MLCK axis	RGD-CD146+CD271+ UCMSC-Exos reduce OGD-treated bEnd.3 permeability	RGD-CD146+CD271+ UCMSC-Exos reduce BSCB destruction and promote motor function recovery	spinal cord contusion model
		CD271+
		UCMSCs
	2022	plasma	Exos were modified with neuron-targeting and growth-facilitating peptides	RVG	AP-EXOR&L&S rescues CSPG-mediated inhibition of neurons and promotes axon elongation	AP-EXOR&L&S enables axon regeneration and motor function recovery	contusive SCI model
				ILP
				ISP
		MSCs	Exos are encapsulated in fibrin gel	VGF	the overexpression of VGF in exosomes facilitates the formation of oligodendrocytes	the Gel-Exo composite improved motor function and electrophysiological performance and increased neural marker expression at the lesion site indicated enhanced neurogenesis.	mice spinal cord transection model
		BMSCs	Exos were integrated into dual-network electroconductive hydrogels made of photocross-linkable GM and PPy hydrogel	PTEN/PI3K/AKT/mTOR pathway.	GMPE hydrogel encourages M2 microglial polarization, enhances NSC neuronal and oligodendrocyte differentiation, inhibits astrocyte differentiation, and supports axon growth and neural synaptic network formation.	the GMPE hydrogel improved functional recovery, reduced cavitary areas, facilitated cell infiltration and tissue formation, inhibited inflammation, and boosted NSC recruitment and neural regeneration	SCI mice model
				NF-κB pathway.
	2021	MSCs	Exos are encapsulated in fibrin glue	fibrin glue	FG-Exo has anti-inflammatory properties and can safeguard neurons	the administration of FG-Exo resulted in a reduction in lesion volume, facilitated myelin regeneration, and enhanced motor function in the hind limbs	rat spinal cord transection model

Perspective and Conclusion

Exosomes, once regarded as mere cellular debris, have evolved into one of the most innovative tools in regenerative medicine, particularly in the treatment of orthopedic diseases. These naturally occurring nanovesicles are poised to revolutionize the way we approach musculoskeletal injuries, degenerative bone diseases, and tissue repair. By enhancing communication between cells, exosomes carry a diverse array of bioactive molecules, such as proteins, lipids, and nucleic acids, which are crucial for regulating cellular functions, such as differentiation, proliferation, and apoptosis. Their intrinsic ability to modulate biological pathways, particularly in bone regeneration, cartilage repair, and tendon healing, has garnered significant attention from researchers and clinicians alike.

The therapeutic potential of exosomes is particularly evident in the treatment of dense connective tissue injuries and degenerative disorders such as OA, OP, and fractures. Derived primarily from MSCs, exosomes can mimic the regenerative abilities of their parent cells, providing a highly efficient, noninvasive option compared to conventional treatments. These vesicles exhibit low immunogenicity, noncytotoxicity, and an exceptional ability to deliver bioactive molecules with high specificity to target tissues, making them attractive candidates for regenerative medicine. Preclinical studies have demonstrated that exosomes can promote tissue repair, enhance cellular regeneration, and support the restoration of function in damaged bone, cartilage, and tendon tissues. Their potential to address complex, age-related musculoskeletal disorders marks exosomes as a transformative advancement in orthopedic therapeutics.

However, despite their vast potential, the clinical application of exosome-based therapies faces several significant hurdles. Chief among these is the isolation and purification of exosomes from complex biological fluids, such as blood, synovial fluid, and bone marrow. Current isolation methods, such as ultracentrifugation, size-exclusion chromatography, and immunoaffinity-based techniques, are often labor-intensive and time-consuming and yield preparations that are heterogeneous in nature. This lack of consistency in the quantity and quality of exosomes isolated from different biological sources not only complicates the standardization of exosome-based therapies but also limits their reproducibility and therapeutic efficacy. Addressing these challenges requires the development of optimized, scalable methods for exosome isolation along with the establishment of standardized protocols to ensure consistent therapeutic outcomes across clinical settings.

Another major challenge lies in the variability of experimental protocols, including animal models, exosome sources, and administration methods. While preclinical studies have provided compelling evidence of exosome-mediated regeneration, the lack of uniformity in outcome measurements and reporting hinders the ability to draw definitive conclusions and translate these findings into clinical practice. The need for large-scale, well-controlled studies, particularly those conducted in large animal models, is paramount to validating the safety, efficacy, and long-term impact of exosome-based treatments in humans. Standardizing outcome measures and ensuring the reproducibility of results across studies are crucial steps in the process of moving exosome-based therapies from the laboratory to clinical application.

Furthermore, while exosome-based therapies hold promise in orthopedic regenerative medicine, the complexity of exosome biology presents additional challenges. A deeper understanding of the precise mechanisms through which exosomes exert their effectssuch as their interactions with specific receptors on target cells and their modulation of signaling pathwayswill be critical for optimizing their therapeutic use. Advances in exosome engineering, such as the incorporation of specific bioactive molecules, genetic modifications, or surface modifications to enhance targeting and delivery, are ongoing. However, the integration of these engineered exosomes into biomaterial scaffolds or hydrogels for controlled localized release remains an area requiring further research. Additionally, developing safe and efficient delivery systems, whether through direct injection, incorporation into implants, or systemic administration, will be crucial for optimizing exosome-based therapies.

Regulatory challenges must also be addressed as exosome-based therapeutics move toward clinical trials. Regulatory agencies such as the U.S. Food and Drug Administration (FDA) must establish clear guidelines for the approval of exosome-based therapies, ensuring that they meet the rigorous safety, efficacy, and manufacturing standards required for clinical use. The commercialization of exosome-based products will depend not only on their scientific validation but also on the development of reliable, reproducible, and cost-effective manufacturing processes.

To better apply exosomes to clinical practice, it is necessary to work together in various fields. Researchers must prioritize the development of robust, standardized, and validated methodologies for the isolation, characterization, and quantification of exosomes. A comprehensive mechanistic understanding of exosome function within specific musculoskeletal contexts, including receptor interactions and the modulation of signaling pathways, is essential. Furthermore, a rigorous evaluation of the potential biological risks associated with both native and engineered exosomes is of paramount importance. This includes a systematic assessment of unintended immune activation, tumorigenicityparticularly concerning exosomes derived from immortalized cellsand off-target effects due to nonspecific biodistribution in relevant preclinical models. Clinicians play a critical role in translating preclinical findings by designing and participating in well-controlled clinical trials. Emphasis should be placed on studies utilizing large animal models and subsequent human trials to robustly establish safety, efficacy, optimal dosing, and long-term outcomes. Defining clinically relevant end points and meticulously monitoring for potential adverse effects in patients will be essential. For the successful commercialization of exosome-based technologies, substantial investment from the industry is essential to develop cost-effective processes that comply with Good Manufacturing Practice (GMP) standards for production, engineering, and quality control. Collaboration with academic institutions and regulatory bodies is imperative to establish rigorous standards. Innovation in delivery platforms, such as the integration of exosomes into biomaterial scaffolds or hydrogels for controlled release, requires extensive development efforts. The intrinsic biological complexity of exosomes, further complicated by engineering strategies designed to enhance functionality or specificity, demands a steadfast commitment to safety assessments throughout the development pipeline. Potential risks identified during preclinical studies must be carefully monitored during clinical trials. Regulatory agencies are required to provide clear and practical frameworks for the evaluation of these novel therapeutics, necessitating comprehensive data on safety and efficacy.

In conclusion, exosomes represent one of the most exciting frontiers in the fields of regenerative medicine and orthopedic therapeutics. Their ability to modulate cellular processes, promote tissue regeneration, and deliver bioactive molecules with remarkable specificity holds promise for transforming the treatment of musculoskeletal diseases. While significant strides have been made in understanding the potential of exosomes, numerous challenges remain before they can be widely implemented in clinical practice. These include the optimization of isolation and purification techniques, the standardization of experimental protocols, the refinement of delivery strategies, and the establishment of regulatory frameworks. As the field of exosome research continues to evolve, it is imperative that these challenges be addressed with a multidisciplinary approach that combines advances in biomaterials, nanotechnology, and clinical medicine. With continued research and collaboration, engineered exosomes could soon become a cornerstone of orthopedic regenerative therapies, offering personalized, targeted, and minimally invasive treatment options for patients suffering from degenerative bone diseases and musculoskeletal injuries.

Glossary

Vocabulary

Exosomes

small extracellular vesicles secreted by cells that mediate intercellular communication through the transfer of proteins, nucleic acids, and lipids

Cargo loading

the process of incorporating therapeutic molecules, such as RNAs, proteins, or drugs, into exosomes for delivery to target cells

Modified strategies

engineering approaches applied to exosomes, including surface modification or genetic manipulation, to enhance their therapeutic potential

Biomaterials

natural or synthetic materials designed to interact with biological systems, often used to support or improve exosome stability and delivery

Delivery systems

formulations or technologies developed to transport therapeutic agents, such as exosomes, efficiently and specifically to target tissues

Tissue engineering

a multidisciplinary field combining cells, scaffolds, and bioactive molecules to restore, maintain, or improve tissue function

Musculoskeletal disorders

a diverse group of diseases and injuries affecting muscles, bones, joints, and connective tissues, often leading to impaired mobility and quality of life

§.

Z.L. and J.X. contributed equally to this work.

The authors declare no competing financial interest.