Extracellular vesicles: their challenges and benefits as potential biomarkers for musculoskeletal disorders

Abstract

Early diagnosis and timely management are critical for determining disease outcomes and prognoses. To date, certain methods for developing disease-specific biomarkers have been reported; however, strategies for musculoskeletal disease-specific biomarker development have rarely been studied. Recent studies have highlighted the potential application of extracellular vesicles (EVs) as disease-specific biomarkers. EVs encapsulate proteins, lipids, messenger RNAs, and microRNAs derived from their cellular origin; these constituents remain stable within the EVs and can traverse the blood–brain barrier. Because of these distinctive characteristics, EVs have been actively investigated as diagnostic tools for various conditions, including cancer, inflammatory diseases, and musculoskeletal disorders. Although EVs have many advantages for biomarker development, they have not yet been fully researched in the context of musculoskeletal pathologies. The current review aimed to highlight the potential of EVs in the development of disease-specific biomarkers, summarize the processes of EV biomarkers, and discuss current limitations and future perspectives of EVs as biomarkers.

Introduction

Most musculoskeletal diseases, such as osteoarthritis and avascular necrosis, are clinically diagnosed using imaging modalities, including radiography, computed tomography, and magnetic resonance imaging. An imaging referral and the diagnosis are generally based on the patient’s clinical symptoms, such as pain and abnormal gait, reported during their visit to the hospital. These clinical manifestations indicate injury/damage to the underlying structure (e.g., bone, cartilage), and at this stage, the injured structure is unlikely to heal or regenerate itself. In most cases, the damaged tissues or structures will need to be managed with interventional procedures. Thus, the diagnosis of most musculoskeletal diseases has traditionally been based on patients’ symptoms, leading to delayed diagnosis and treatment, resulting in the inability to recover/regenerate the damaged tissue or structure. Therefore, early identification and management of musculoskeletal pathologies, before tissue damage, is of utmost importance.

Extracellular vesicles (EVs) have attracted considerable attention for the development of disease-specific biomarkers. EVs are 40 to 200 nm spherical vesicles encapsulated in phospholipid bilayer membranes.^1,2 They are found in almost all body fluids, including cerebrospinal fluid, saliva, breast milk, and blood. EVs are secreted from all cell types, especially cells under specific disease conditions, and contain several bioactive molecules, such as functional proteins, lipids, and nucleic acids (messenger RNA (mRNA) and micro-RNA (miRNA)).^1,2 On the basis of these characteristics, several studies focusing on various cancers, inflammatory diseases, and cardiovascular diseases have reported the development of disease-specific biomarkers for early diagnosis (Figure 1). ¹ However, reports on EV biomarkers for musculoskeletal disorders are limited. Moreover, it is crucial to diagnose certain musculoskeletal diseases early, before disease progression, because the treatment method depends on the diagnosis time. When the disease is detected early during the cellular damage phase before the occurrence of any structural damage, the damaged tissue can be repaired and restored using regenerative medicine modalities (e.g., stem cells); however, once the disease has caused structural damage, the tissue or structure will need to be replaced using salvage methods.

Figure 1.

Extracellular vesicles have shown potential as biomarkers for both preclinical and clinical applications. Extracellular vesicles can be isolated from biofluids such as those in the respiratory and urinary systems, blood, and that in the spinal cord. They can also be isolated from cells. These extracellular vesicles can be exploited for biomarker and therapeutic purposes.

Studies on EVs in musculoskeletal disorders have been reported from various perspectives, indicating potential clinical applications. This review aims to highlight recent advancements and the clinical potential of EV-based biomarkers in musculoskeletal diseases. The authors previously identified several miRNAs that are highly associated with avascular necrosis of the femoral head and may serve as disease-specific biomarkers. ¹ Identification of EV biomarkers is significant for early diagnosis of diseases and for a comprehensive understanding of the disease itself (e.g., disease occurrence, progression, or regression). Therefore, in the current review, we aim to describe the detailed procedure for developing disease-specific biomarkers using EVs, understand the current limitations of the technique, and provide future perspectives. Given the era and emphasis on cutting-edge techniques such as EV biomarkers, the current review may serve as a cornerstone for next-generation research focused on the development of EV-based disease-specific biomarkers.

This review is guided by the Scale for the Assessment of narrative review articles. ³ Our objective is to synthesize the latest research on the role of EVs as biomarkers in musculoskeletal diseases, with a particular focus on their diagnostic and prognostic potential. Through this review, we aim to provide a cohesive understanding of the clinical utility and technological requirements needed to advance EVs as reliable biomarkers in musculoskeletal diseases.

Considerations for EV biomarker discovery

Detection of EV biomarkers in biological fluids

Genetic materials and proteins in EVs are more stable than RNA or proteins in biological fluids. However, before describing suitable EV isolation methods for the diagnosis of musculoskeletal diseases, it is important to consider the body fluids from which EVs can be sourced. Because EVs are secreted from cells, they can be isolated from cells and most biological fluids, such as saliva, urine, serum, plasma, and breast milk. Among bodily fluids lacking cells, saliva and urine samples are convenient sources because they can be obtained noninvasively. Urinary EVs are used to treat kidney diseases and urinary tumors.^4,5 Therefore, the selection of a suitable biological fluid to target a specific disease is crucial for demonstrating its potential as an EV biomarker source. In the case of musculoskeletal diseases, the extraction of cells and identification of EVs remain challenging. Therefore, changes in the blood environment can be predicted from serum and plasma, which constitute bodily fluids that circulate throughout the body and are suitable for identifying biomarkers related to a disease. ² miRNAs and proteins in serum represent potential biomarkers for detecting various diseases, including muscle or bone diseases. ⁶ Primary cell culture of diseased myoblasts or osteocytes and acquisition of the conditioned culture medium allow for the detection of biomarkers. However, the biopsy of each patient sample remains a challenging, time-consuming, and inefficient diagnostic method. Therefore, body fluids, such as blood, are suitable for diagnosing musculoskeletal diseases. Furthermore, whole blood can be separated into plasma and serum for diagnosis.

EV isolation (serum-derived EVs)

Obtaining a sufficient quantity of biological fluids for disease diagnosis remains challenging. Therefore, an efficient and simple separation method needs to be used to isolate sufficient EVs for diagnostic purposes, even from samples with small amounts. Various techniques are available for EV isolation. Classical methods involve differential and gradient-density centrifugation. EVs can be easily separated step-by-step from cellular debris and microparticles using ultracentrifugation. ⁷ However, this method requires expensive equipment, and EVs isolated at high speed and high pressure have low yield and functional problems owing to deformation. ⁸ Therefore, filters with a pore size of approximately 0.1 to 0.22 μm are used to separate EVs, which are nanoparticles ranging in size between 40 and 50 nm. Size exclusion chromatography is a size-based separation method that uses porous phases. It is suitable for separating EVs from small samples, such as plasma, and for monitoring internal miRNAs. ⁹ Tangential flow filtration in the form of cross-flow filtration can be used to separate EVs, but this method is suitable for the separation of EVs from large samples such as culture media. ¹⁰ Using commercially available reagents based on polymer-based precipitation reactions may be the easiest and most convenient method to isolate serum-derived EVs. However, several limitations need to be overcome to isolate EVs with clinical applicability. ¹¹ Moreover, it is difficult to separate pure EVs using this method because proteins with reduced solubility in the reagents (owing to polyethylene glycol) tend to precipitate and mix with the EVs. ¹² Recently, to overcome the purity issue, a non-polyethylene glycol-based precipitation technique has been developed by Invent Biotechnologies. ¹³ Meanwhile, another method was developed to separate EVs from relatively small volume samples of body fluids, such as serum or plasma, by combining two nanofilters using a lab-scale centrifugal microfluidic system. ¹⁴ Overall, various strategies for isolating EVs of sufficient quantity and high purity from biofluids are still being developed, and the methods will be optimized by characterization of the isolated EVs.

EV characterization

According to the Minimum Information for Extracellular Vesicle Research 2023 guidelines published by the International Society for Extracellular Vesicles, researchers can define the following: nomenclature, sample collection, separation, and EV characterization. ¹⁵ Quantification of the EV particle number, concentration, and size distribution can be performed using nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS). NTA and DLS can confirm the size distribution by observing the fluctuations caused by the Brownian motion of the nanoparticles through scattered light measurements. DLS can measure particles ranging from 1 nm to 6 μm but is less accurate for particle suspensions of different sizes (polydisperse suspensions). NTA can measure particles <1 μm at a wavelength of 30 nm. Visual verification is achieved by capturing light scattering-based measurements with a camera, and the size distribution, average size, and concentration of particles can be calculated.^16,17 The external morphology of EVs can be visualized using transmission electron microscopy (TEM) and cryo-TEM. TEM and transmission scanning electron microscopy are used to detect and characterize EVs. TEM analysis requires the fixation and staining of EVs; however, these processes can be avoided in cryo-TEM. Therefore, cryo-TEM imaging allows for the characterization of EVs under conditions closest to their native state by directly applying the sample to an EM grid and visualizing it. ¹⁸ The internal or surface proteins of EVs can be analyzed using western blotting, enzyme-linked immunosorbent assays, and flow cytometry. ¹⁹ These methods generally use the tetraspanins CD9, CD63, and CD81, flotillin 1 and 2, which are proteins that make up the surface membrane of EVs, and ALIX, TSG101, clatherin, and ubiquitin, which are proteins involved in EV biogenesis. ²⁰ Additionally, heat shock protein (HSP)70 and HSP90 are used as protein biomarkers. EV markers may appear different depending on their source (disease type) and isolation method. ²¹

miRNAs in EVs for biomarker discovery

Numerous reports have shown that the miRNA profiles of EVs isolated from the body fluids of patients differ from those of healthy individuals. Through small RNA analysis, a list of miRNAs that are candidate indicators specific to musculoskeletal diseases can be obtained. miRNAs are small non-coding RNA molecules that are primarily involved in regulating gene expression. ²² Therefore, they can be used as diagnostic markers for cancer and diseases to identify gene regulation targeted by miRNAs and analyze related mechanisms. The feasibility of miRNA expression analysis in body fluids, including blood, has increased, and this method is helpful to characterize disease states. miRNAs are being studied for the early diagnosis of cancers, including breast, lung, and colorectal cancers, and are being identified in various other diseases such as cardiovascular diseases, sepsis, nervous system disorders, diabetes mellitus, and rheumatoid arthritis.^{23 –25}

Identification and target prediction of miRNAs have emerged as novel strategies in the field of diagnostic biomarkers for diseases. Recent advances in next-generation sequencing technology have provided improved predictions because of the improved accuracy and reliability obtained by experimentally validating miRNAs in EVs. The workflow of standardized miRNA analysis consists of (i) downloading datasets using miRNA sequencing and microarray Gene Expression Omnibus databases, (ii) searching existing literature and compiling existing information using PubMed, (iii) use of a short lead aligner, (iv) identification and characterization of miRNAs, (v) target mRNA prediction, and (vi) functional and downstream analyses. ²⁶ Various tools with high sensitivity and specificity have been developed for miRNA validation based on quantitative reverse-transcriptase polymerase chain reaction and TaqMan miRNA assays that incorporate target-specific stem-loop reverse transcription.^27,28

Proteins in EVs for biomarker discovery

EV proteins can be analyzed to identify biomarkers for different types of musculoskeletal diseases. Proteins specific to the early stages of disease development must be identified among the numerous proteins and verified for reproducibility and correlation with the disease. Liquid chromatography (LC)-mass spectrometry (MS)-based proteomics analysis is the most widely used technique for identifying new disease biomarkers in the discovery phase. ²⁹ This technology is suitable for the discovery phase because it can precisely identify specific and abundant proteins in a limited group of patient samples. ³⁰ The workflow to identify biomarker candidates using mass spectrometry involves the following steps: (i) protein digestion; (ii) sample desalting; (iii) data acquisition using LC-MS/MS equipment; (iv) peptide identification; (v) quality control; (vi) statistical analyses; (vii) data analysis and validation. Predicting or diagnosing a disease using a single marker remains challenging. ³⁰ Therefore, multiple protein diagnostic analyses are essential to ensure the accuracy of protein biomarkers associated with musculoskeletal diseases.

EVs as musculoskeletal disease biomarkers

Recent studies have explored the role of EVs in musculoskeletal diseases, revealing specific proteins, miRNAs, and other molecular components in EVs that are associated with disease processes such as inflammation and tissue degeneration. For instance, in osteoarthritis, ³¹ EVs have been shown to carry miRNAs and inflammatory markers that reflect cartilage degradation stages, potentially providing early diagnostic cues before radiographic evidence is apparent. Similarly, in rheumatoid arthritis, ³² EVs containing immunomodulatory molecules have been correlated with disease activity, suggesting their use in monitoring treatment responses and inflammation levels in rheumatoid arthritis patients. In femoral head avascular necrosis, ¹ EVs have been identified to carry markers linked to joint and spine inflammation, which could help to track disease progression at early stages where imaging may not yet show structural changes. Moreover, new approaches to identify disease-specific biomarkers using less invasive or different approaches have been developed. A diagnostic approach to develop stage-specific biomarkers using EVs from joint fluid from osteoarthritis patients has been studied. ³³ In addition, marker development with less invasive specimens such as saliva and hair has been attempted and has shown potential. ³⁴ These disease-specific EV profiles indicate promising applications of EVs as diagnostic and prognostic biomarkers in musculoskeletal diseases, enhancing the precision of current diagnostic approaches. On the basis of the relevant research that exists, the diagnostic efficacy and accuracy for musculoskeletal diseases using EVs has been promising at the experimental level, but mass production for diagnosis using EVs is challenging because miRNA-based techniques need to be improved to approach the level of over-the-counter products. ³⁵

Clinical applications in musculoskeletal disease

We have expanded our discussion on the application of EVs in musculoskeletal diseases, integrating recent studies that highlight their utility in rheumatoid arthritis and ankylosing spondylitis in addition to osteoarthritis and avascular necrosis. Specific proteins, miRNAs, and inflammatory markers found in EVs have shown correlations with disease progression and severity in these conditions. For example, in rheumatoid arthritis, ³² EVs containing immunomodulatory components correlate with disease activity, providing potential biomarkers for monitoring inflammation and the treatment response. Similarly, in ankylosing spondylitis, ³⁶ specific EV-associated proteins linked to inflammation offer insights into early-stage disease progression, even before significant radiographic changes occur. These additions provide a comprehensive overview of EV biomarkers across a broader range of musculoskeletal diseases, aligning the manuscript more closely with the title’s emphasis on musculoskeletal applications.

Limitations of EV-based biomarker development

In the field of biomarker development, EVs have emerged as promising candidates owing to their role in intercellular communication and their ability to transport a variety of bioactive molecules, including proteins, lipids, and nucleic acids. ³⁷ EVs are involved in numerous physiological and pathological processes, making them valuable for disease diagnosis, prognosis, and therapy. ³⁸ However, despite their potential, several limitations and technical challenges hinder the effective utilization of EVs in clinical applications.

Standardization of isolation and characterization protocols

Current isolation techniques, such as ultracentrifugation,^{39 –41} tangential flow filtration,^{42 –44} size-exclusion chromatography,^44,45 and immunoaffinity capture^40,46 often result in contamination of samples with non-EV particles, such as protein aggregates, lipoproteins, and other extracellular components.^47,48 Such contaminants compromise the specificity and accuracy of downstream analyses. Furthermore, the variability in these methods results in differences in the yield, purity, and composition of isolated EVs, making it difficult to compare results across studies.^49,50 This inconsistency can hamper the reproducibility and reliability of EV-based biomarkers.

Heterogeneity and variability of EVs

EVs are heterogeneous in nature and comprise various subpopulations that differ in size, origin, and cargo.^51,52 The content and properties of EVs are influenced by various environmental factors, including stress conditions, disease states, and treatment regimens.^53,54 This variability needs to be carefully considered and controlled in biomarker studies.^55,56 The heterogeneity poses a significant challenge in identifying disease-specific biomarkers because the specific subpopulation responsible for biomarker activity needs to be accurately identified and isolated. The presence of various EV types in biological samples complicates data analysis and interpretation. Biological variability, such as differences in EV content owing to patient heterogeneity (e.g., age, sex, and disease state) and technical variability, including differences in sample handling and processing, significantly affects EV analyses.^53,57 This variability complicates the identification of reliable and reproducible biomarkers. Standardized preanalytical and analytical protocols are required to minimize these variations.

Limited understanding of the biogenesis and functions of EVs

Although significant progress has been made in the study of EVs, the mechanisms underlying the biogenesis, release, and uptake of EVs are not fully understood. The limited understanding of these processes hinders the effective manipulation of EVs for biomarker discovery and therapeutic purposes. A deeper understanding of EV biology is essential for the advancement of EV-based biomarker development.^{58 –60}

Challenges in cargo analysis

Analysis of EV cargos, including proteins, lipids, and nucleic acids, requires advanced and often complex technologies such as MS and next-generation sequencing.^{61 –63} The sensitivity and specificity of analyses are crucial for identifying potential biomarkers but are limited by current technological capabilities. Therefore, improved methods for high-throughput and high-sensitivity analyses are needed.^64,65

Regulatory and clinical implementation hurdles

Translating EV-based biomarkers into clinical practice involves the navigation of complex regulatory pathways. Ensuring the clinical efficacy, safety, and reproducibility of EV-based diagnostics and therapeutics is challenging and requires extensive clinical validation.^66,67 Regulatory guidelines specific to EV-based products are lacking, which can delay their clinical application. ⁶⁸ Additionally, the translation of EV-based biomarkers from the laboratory to the clinic involves scaling up the isolation and analysis processes to handle large sample volumes. Scalability requires the development of automated and high-throughput technologies that can efficiently and reproducibly process clinical samples.

Storage and stability issues

The stability of EVs during storage and the effect of storage conditions on EV integrity and bioactivity have not yet been determined, rendering this a key area for further research.^{69 –71} This uncertainty can affect the reliability of EV-based biomarkers when stored for extended periods or transported between locations. The development of standardized storage protocols is essential to maintaining the integrity of EVs.

Future perspectives on developing EV-based biomarkers

The use of EVs as biomarkers for specific diseases holds great promise owing to several emerging trends and advancements in the field (Figure 1). The following points highlight key areas of focus for future research and development.

Advanced isolation and characterization techniques

The development of standardized and efficient isolation techniques is crucial for advancing EV research. Innovations in microfluidics, nanotechnology, and immunoaffinity capture are expected to enhance EV purity and yield.^{72 –74} Improved characterization methods, including high-resolution imaging and single-vesicle analysis, will allow for a more detailed understanding of EV heterogeneity and its specific roles in diseases.^75,76 For example, recent studies have shown the necessity of combining multiple approaches to accurately diagnose or predict diseases such as breast cancer, highlighting the improved accuracy of multi-omic biomarker signatures over single-omic biomarkers. ⁷⁷

Integration with omics technologies

The integration of multi-omics approaches, such as genomics, proteomics, metabolomics, and lipidomics, will provide comprehensive insights into the molecular cargo of EVs.^{78 –80} High-throughput technologies and advanced bioinformatics will enable the identification of novel biomarkers and the elucidation of complex disease mechanisms. This holistic approach will facilitate the discovery of robust and clinically relevant EV-based biomarker signatures. In the context of neurodegenerative diseases such as Alzheimer’s disease, multi-omic approaches may help uncover the dual roles of EVs in disease progression and therapy. ⁸¹

Clinical validation and standardization

Rigorous clinical validation of EV-based biomarkers is essential before their adoption in clinical practice.^82,83 Large-scale studies and longitudinal cohorts are required to confirm the diagnostic and prognostic value of EV biomarkers. Standardization of protocols for EV isolation, characterization, and analysis will ensure the reproducibility and comparability of results across different laboratories and studies.^{84 –86}

Single-vesicle analysis

Advances in single-vesicle analysis techniques such as super-resolution microscopy and flow cytometry will enable researchers to study the heterogeneity of EV populations in greater detail. This will help to identify specific EV subpopulations associated with particular disease states, thereby improving the specificity and sensitivity of EV-based biomarkers.^75,87,88

Functional studies and mechanistic insights

Understanding the functional role of EVs in disease pathogenesis is critical for their application as biomarkers and therapeutics. Future studies should focus on elucidating the mechanisms by which EVs mediate intercellular communication, modulate immune responses, and influence disease progression. Functional studies using advanced in vitro and in vivo models will provide valuable insights into the biological relevance of EVs.^{89 –91}

Ethical considerations

Addressing regulatory and ethical challenges is essential for the successful clinical implementation of EV-based diagnostics and therapeutics. Clear regulatory pathways and guidelines must be established to ensure the safety, efficacy, and quality of EV-based products. Ethical considerations, including patient consent and data privacy, must be thoroughly addressed in clinical studies.^15,92

Personalized medicine and precision health

EV-based biomarkers have the potential to revolutionize personalized medicine by enabling the development of tailored diagnostic and therapeutic strategies. The ability of EVs to reflect dynamic changes in disease states and treatment responses will facilitate the creation of precision health approaches that improve patient outcomes and reduce adverse effects.^93,94 Beyond their use as biomarkers, EVs are being explored as therapeutic agents because of their ability to deliver bioactive molecules to target cells. Engineering EVs for enhanced targeting and therapeutic efficacy is a promising area of research.^{95 –97} The development of EV-based therapeutics will require overcoming challenges related to large-scale production, stability, and delivery.

Conclusion

The future of EV-based biomarker research is promising, with numerous opportunities for technological and clinical advancement. By encapsulating unique bioactive molecules reflective of disease-specific processes, EVs can supplement existing diagnostic tools for conditions such as osteoarthritis, rheumatoid arthritis, and ankylosing spondylitis, offering insights into disease status and therapeutic responses. Future research should focus on standardizing EV isolation and analysis protocols to ensure reproducibility and exploring EV biomarkers in larger, disease-specific cohorts to validate their clinical applicability in musculoskeletal diagnosis and management.

Search strategy

To ensure a comprehensive review of the existing literature, we performed a structured search strategy across databases including PubMed, Scopus, and Web of Science. The key search terms used included “extracellular vesicles,” “EVs,” “biomarkers,” “musculoskeletal diseases,” “osteoarthritis,” “rheumatoid arthritis,” and “ankylosing spondylitis.” This approach allowed us to capture a thorough range of recent studies, offering a broad view of EV applications in the musculoskeletal field.